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Chemical modification of silicone elastomer for controlled biological interactions on microtextured surfaces

University of Florida Institutional Repository

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CHEMICAL MODIFICATON OF SILICONE ELASTOMER FOR CONTROLLED BIOLOGICAL INTERACTIONS ON MICROTEXTURED SURFACES By AMY LOUISE GIBSON A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2002

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Copyright 2002 by AMY LOUISE GIBSON

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This thesis is dedicated to my loving parents David and Betty Gibson for their continuous love and support throughout my educational career.

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iv ACKNOWLEDGMENTS I would, first of all, like to acknowledge my advisor and committee chairman, Dr. Anthony Brennan, for the confidence, motivation, and valuable advice that he has provided me through my graduate years at the University of Florida. My gratitude also goes to my other committee members, Dr. Ronald Baney and Dr. Christopher Batich, who have provided me with insightful direction and inspiration, especially with their silicone chemistry and interfacial behavior expertise. Of course, the help and advice from my fellow group members through this strenuous process cannot go unnoted. I would like to thank the past group members, Jeanne Macdonald, Jeremy Mehlem, Wade Wilkerson, and Chuck Seegert, who provided me with the initial direction and enthusiasm for polymer research. My deepest gratitude and appreciation go to the group members who have seen me through this entire process: Clay Bohn, Leslie Wilson, Adam Feinberg, Brian Hatcher, and Nikhil Kothurkar. Without their support, expertise, and friendship this whole process would not have been possible. The recent addition of Rob Sparkman, Thomas Estes, Michele Carmen, and Kiran Karve to the research laboratories has been instrumental to my research and I give them my thanks for all of their hard work and dedication. Special thanks also go to the members of Dr.Eugene Goldberg’s group, past and present, who have given me knowledgeable advice and assistance in my research and life through the years.

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v Assistance was provided by Dr. Maureen Callow, Dr. Jim Callow, and Dr. John Finley at the University of Birmingham, UK, by performing all of the Enteromorpha spore settlement and release assays. All of their hard work and flexibility were much appreciated and provided me with the opportunity for a significant collaboration. My biggest thanks go to my family and friends whose emotional support and love have driven me through my academic years. My close friends, Athena West and Ginny Miliusis, deserve much thanks and appreciation for keeping my life exciting and for pushing me through the hard times. My gratitude and love also go to Brett Almond, who gave me the confidence and motivation to overcome any hurdle that I reached. Lastly, I am completely and forever indebted to my family who has given me nothing but love and support. They should be credited with the success that I have had through my life and educational path.

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vi TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF TABLES.............................................................................................................ix LIST OF FIGURES.............................................................................................................x ABSTRACT....................................................................................................................... xv CHAPTER 1 INTRODUCTION.........................................................................................................1 2 BACKGROUND...........................................................................................................5 Biofouling..................................................................................................................... 5 Adhesion....................................................................................................................... 6 Mechanics...............................................................................................................6 Coupling Agents.....................................................................................................8 Roughness Factor....................................................................................................9 Silicones...................................................................................................................... ..9 Introduction.............................................................................................................9 Mechanical Properties...........................................................................................10 Surface Energy/Chemistry....................................................................................13 Enteromorpha Algae Spores.......................................................................................16 Quaternary Ammonium Salts.....................................................................................18 Introduction...........................................................................................................18 Biocidal Mechanism.............................................................................................18 3 SILICONE ELASTOMER CHEMICAL MODIFICATION AND CHARACTERIZATION.............................................................................................20 Introduction.................................................................................................................20 Materials.....................................................................................................................2 1 Silicone Elastomer................................................................................................21 Nonfunctional Additives.......................................................................................22 Surface Energy Liquids.........................................................................................22 Methods.......................................................................................................................2 4

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vii Sample Preparation...............................................................................................24 Mechanical Properties...........................................................................................26 Surface Energetics................................................................................................27 Results and Discussion...............................................................................................29 Mechanical Properties...........................................................................................29 Crosshead displacement measurements..........................................................29 Laser extensometer measurements.................................................................32 Aged silicone measurements...........................................................................38 Soaked silicone measurements.......................................................................41 Surface Energetics................................................................................................42 Goniometer.....................................................................................................42 Digital capture.................................................................................................46 4 ENTEROMORPHA SPORE SETTLEMENT/RELEASE ASSAY.............................51 Introduction.................................................................................................................51 Materials.....................................................................................................................5 1 Silicone Elastomer Substrate................................................................................51 Epoxy....................................................................................................................52 Glass Slide Preparation.........................................................................................52 Biological Algae Material.....................................................................................53 Methods.......................................................................................................................5 4 Unpatterned Elastomer Samples...........................................................................54 Patterned Elastomer Samples................................................................................54 Adhesion Fidelity..................................................................................................59 Topography Fidelity..............................................................................................60 Spore Settlement Assay........................................................................................60 Spore Removal Assay...........................................................................................61 Results and Discussion...............................................................................................61 Silicone Elastomer Fidelity...................................................................................61 Spore Settlement Assay........................................................................................64 Spore Removal Assay...........................................................................................69 5 QUATERNARY AMMONIUM SALT CHEMICAL MODIFICATIONS................75 Introduction.................................................................................................................75 Materials.....................................................................................................................7 6 Biocide..................................................................................................................76 Silicone Elastomer Substrate................................................................................76 Polysulfone Substrate............................................................................................77 Methods.......................................................................................................................7 9 Glass Slide Grafting..............................................................................................79 Silicone Grafting...................................................................................................79 Polysulfone grafting..............................................................................................80 SEM/EDS..............................................................................................................82

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viii FTIR......................................................................................................................82 Contact Angle.......................................................................................................82 Results and Discussion...............................................................................................83 Glass Slide Grafting..............................................................................................83 Silicone Elastomer Grafting..................................................................................89 Polysulfone Grafting.............................................................................................91 6 CONCLUSIONS AND FUTURE WORK..................................................................98 Conclusions.................................................................................................................98 Future Work..............................................................................................................103 Surface energetics...............................................................................................103 Topography.........................................................................................................104 Spores..................................................................................................................104 Quaternary Ammonium Salt Grafting................................................................104 LIST OF REFERENCES.................................................................................................106 BIOGRAPHICAL SKETCH...........................................................................................114

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ix LIST OF TABLES Table Page 3.1 Typical properties of Dow Corning’s Silastic T2 addition cured silicone elastomer. (From Dow Corning’s product information sheets).....................................................22 3.2 Chemical structures and properties of the nonfunctional silicone oil additives from Gelest, Inc. (Information obtained from Gelest, Inc. catalog).....................................23 3.3 List of chemicals used for contact angle measurements with their corresponding liquid surface tensions..................................................................................................23 3.4 Effect of cure time at 80C for 5wt% 50 cSt silicone oil modified silicone elastomer. (n = 10).......................................................................................................32 3.5 Contact angle measurements and calculated critical surface tension values (modified Zisman plot) from a goniometer of a few silicone elastomer formulations.46 3.6 The effect of cure surface on the contact angles and calculated critical surface (from modified Zisman plot) on an unmodified silicone elastomer.......................................47 3.7 Average contact angle measurements and calculated critical surface tension (modified Zisman plot) for various silicone elastomer formulations from the digital capture system...............................................................................................................48 4.1 Measured contact angles and calculated surface energies examining the effect of storage and shipment of unmodified silicone elastomer samples in polyethylene bags........................................................................................................................... ....64 5.1 Typical material properties for poly( ether sulfone) as provided by the distributor, Amoco Products............................................................................................................78 5.2 Density and molecular weight values for various chemicals and polymers necessary for the molar ratio calculations in the binding of the quaternary ammonium salt to the polysulfone backbone.............................................................................................78

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x LIST OF FIGURES Figure Page 2.1 Chemical species on the surface of fumed silica and trimethylsilylated silica particles...................................................................................................................... ...12 2.2 The chemical reaction for the trimethylsilylation of a fumed silica particle by addition of hexamethyldisilazane.................................................................................12 2.3 Setup and labeled interfaces/contact angle for both the (A) sessile drop technique and (B) captive air bubble technique............................................................................13 2.4 Energy balance approach at the point of triple phase (liquid-solid-vapor) intersection for the derivation of Young’s equation. The forces are labeled previously in Figure 2.3................................................................................................14 3.1 Dow Corning’s Silastic T2 silicone elastomer crosslinking reaction.........................21 3.2 Mold setup for preparation of silicone elastomer films (left) and silicone elastomer coated glass s lides (right)..............................................................................................25 3.3 Digital picture of the microscope goniometer setup (left) and the digital capture setup (right) for contact angle measurements...............................................................28 3.4 Computer screen image capture of the ImageTool software for measuring contact angles from digital images of the liquid droplets. The image is of a 2L droplet of water on an unmodified silic one elastomer substrate...................................................29 3.5 Effect of nonfunctional additives at different amounts on the elastic modulus (calculated between 20-50% strain) for a silicone elastomer. Strain measurements were made from crosshead displacement.....................................................................30 3.6 Stress-strain curve for unmodified silicone elastomer with strain measurements made from crosshead displacement. Notice the 2 different linear segments, which correspond to different moduli values at low strain versus high strain. Performed at room temperature with a 1in. gage length and 2 in/min crosshead displacement........31

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xi 3.7 Stress-strain curve for an unmodified silicone elastomer with strain measurements made from a laser extensometer. Two different slope regions are evident between high strain and low strain corresponding to 2 different moduli values........................34 3.8 Comparison of elastic moduli values between the different freshly cured silicone elastomer formulations from both the low strain and high strain regions. All of the strain measurements were collect ed from a laser extensometer...................................37 3.9 Comparison of elastic moduli values between the different freshly cured silicone elastomer formulations from the two sources of strain values, the laser extensometer and crosshead displacement. All modulus values are from the low strain region.......38 3.10 Comparison of elastic moduli values between the different 128-day aged silicone elastomer formulations from both the low strain and high strain regions. All of the strain measurements were collect ed from a laser extensometer...................................39 3.11 Comparison of the low strain region elastic moduli values between the different silicone elastomer formulations at two different resting times after cure, 1 day before testing (fresh) and 128 days before testing (aged).............................................40 3.12 Comparison of the high strain region elastic moduli values between the different silicone elastomer formulations at two different resting times after cure, 1 day before testing (fresh) and 128 days before testing (aged).............................................41 3.13 Comparison of the effect of environment (air, DI water, and seawater) on elastic moduli at high strain region for the various freshly cured silicone elastomer formulations..................................................................................................................4 3 3.14 A modified Zisman plot for an unmodified silicone elastomer from digital capture contact angle measurements. The equation for the linear regression and respective regression correlation are provided on the graph.........................................................45 3.15 Digital images of each of the 5 liquids used to measure contact angles and subsequently calculate surface energy (modified Zisman plot) on an unmodified silicone el astomer.........................................................................................................48 3.16 Digital image of a DMF droplet on a visibly oily surface of a 5wt% 5000 cSt silicone elastomer sample (left). The deformation of the surface caused by the liquid droplet was observable. This relates to Rusanov’s model for contact angle induced surface deformation (right). (adapted from Andrade)50..................................50 4.1 Epoxy cure reaction. Reaction at each of the NH’s is possible, which leads to a crosslinked matrix.........................................................................................................53 4.2 Placement of topography on the etched silicon wafers. Each large quadrant of patterns had 3 smaller rectangular areas, which corresponded to the 3 different valley/flat-area widths, 5m, 10m, 20m...................................................................55

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xii 4.3 Replication process from patterned silicon wafer to final patterned silicone elastomer coated glass slides........................................................................................56 4.4 Topography layout on the silicone elastomer coated glass slides as compared to the direction of hydrodynamic flow....................................................................................57 4.5 Final mold setup for reproduction of a patterned silicone elastomer coated glass slide from an epoxy master mold..................................................................................59 4.6 SEM and WLIP images of an epoxy mold and silicone elastomer coated glass slide. A) SEM at 800X magnification of patterned epoxy with the negative topographical features (10,000m length ridges at the tran sition point from 5m wide ridges to 10m wide ridges) replicated from a silic one well. B) SEM (350X magnification) of silicone elastomer with 10,000m length ridges at the transition from 5m wide valleys to 10m wide valleys that was replicated off of an epoxy mold from the last step in the replication process. C) WLIP image of the 10,000m length ridges with 5m valleys from the silicon wafer. D) WLIP image of the 60m length ridges with 5m wide ridges from an epoxy replicate............................................................62 4.7 The settlement of Enteromorpha spores on the different unpatterned silicone elastomer fo rmulations..................................................................................................65 4.8 The settlement of Enteromorpha spores on 5m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features................................................................................................................... .66 4.9 The settlement of Enteromorpha spores on 10m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features................................................................................................................... .66 4.10 The settlement of Enteromorpha spores on 20m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features................................................................................................................... .67 4.11 Fluorescent microscope images of Enteromorpha spores settled on 3 different 5m deep patterned areas on unmodified silicone elastomer. (A) 5m wide ridges with 5m wide valleys. (B) 5m wide ridges with 10m wide valleys. (C) 5m wide pillars with 5m spacing. (Images courtesy of Dr. Maureen Callow).........................68 4.12 Spore density measurements before and after immersion in a flow cell for the flat regions between the 5m deep patterns of the different elastomer formulations.........70 4.13 Spore density measurements before and after immersion in a flow cell for the 5m deep and wide valley pattern set parallel to flow direction of the different elastomer formulations..................................................................................................................7 0 4.14 Comparison of the percent spore removal for the different 5m deep pattern sets and the different silicone elastomer formulations.........................................................72

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xiii 4.15 Enteromorpha spore settlement densities on the flat areas between the patterns and on the pillars for the different elastomer formulation samples with either 1.5m or 5m deep pattern features.............................................................................................72 4.16 Percent Enteromorpha spore removal on the flat areas between the patterns and on the pillars for the different elastomer formulation samples with either 1.5m or 5m deep pattern features after exposure to hydrodynamic flow.........................................74 5.1 Chemical structures of Dow Corn ing’s 5700 Antimicrobial Agent (top) and a trifunctional alkoxy silane (bottom) with same length unreactive alkyl chain.............76 5.2 Chemical structure of UdelTM poly(ether sulfone), which was used as a more durable substrate as compared to silicone elastomer for chemical modifications........77 5.3 Single frames from a digital video of th e placement of an air droplet at the untreated glass (at the top of each image) and water interface with a microliter pipet. This setup is the captive air bubble contact angle technique. Images from A) to D) are in sequential tim e order.....................................................................................................85 5.4 Single frames from a digital video of the attempt to place of an air droplet at the quaternary ammonium surface grafted glass (at the top of each image) and water interface with a microliter pipet. A) Deformation of the air bubble is observed as it is pressed against the modified glass surface. B) The same air bubble transforms completely back to a sphere after deformation observed at modified glass surface.....86 5.5 Single frames from a digital video of the placement of an air droplet at the quaternary ammonium surface grafted glass (at the top of each image)and water interface with a microliter pipet. This air bubble only moved a short distance until attaching to surface. Images from A) to D) are in sequential time order....................87 5.6 Single frames from a digital video of the placement of two air droplets (which split upon release from the micropipet from a single bubble) at the quaternary ammonium surface grafted glass (at the top of each image) and water interface with a microliter pipet. These air bubbles glided completely across the camera view on the modified glass surface until they reached the side of the water holder. Images from A) to D) are in sequential time order...................................................................88 5.7 Single frames from a digital video of the placement of an air droplet at the octadecyltrimethoxysilane treated glass–water interface with a microliter pipet. Images from A) to D) are in sequential time order. No movement of the air droplet is observed.................................................................................................................... 90 5.8 Digital image of the water-air-silicone elastomer intersection by two contact angle methods: A) sessile drop and B) captive air bubble.....................................................91 5.9 Chemical structures of the modified polysulfone after each chemical reaction. The final result (bottom structure) is of the quaternary ammonium salt bound to the polysulfone.................................................................................................................... 92

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xiv 5.10 FTIR spectras of the different steps in the binding of quaternary ammonium salt to polysulfone. All films were neat on NaCl crystals. A) polysulfone (PSF). B) sulfonated polysulfone (SPSF). C) GPS s ilanated SPSF. D) Quaternary ammonium salt grafted to the silanated SPSF.................................................................................93 5.11 SEM images of glycidoxypropyltrimethoxysilane silanated SPSF by solution reaction. A) 400X magnificati on. B) 3300X magnification.......................................95 5.12 EDS graph and identifications of chemical atoms present on the surface....................96 5.13 Contact angle images of 2L droplets of water on A) sulfonated polysulfone (SPSF) and B) quaternary ammonium salt grafted onto silanated SPSF..................................97

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xv Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science CHEMICAL MODIFICATION OF SILI CONE ELASTOMER FOR CONTROLLED BIOLOGICAL INTERACTIONS ON MICROTEXTURED SURFACES By Amy Louise Gibson December 2002 Chairman: Dr. Anthony Brennan Major Department: Materials Science and Engineering The ability to control a biointerface in terms of biological growth and adhesion to a material’s surface is beneficial to many fields, including marine coatings and biomedical devices. By examining some of the individual factors that influence these biointerfaces, a step towards developing a sy stematic model for biological interactions with materials can be established. As a result, a material could be engineered to elicit a specific biological response. In the case of anti-fouling coatings for marine applications, an engineered surface to prevent biofouling is desired. In particular, silicone elastomers have been heavily studied recently for their foul-release properties, which means that only a small force is required to remove adhered biological organisms. In order to increase effectiveness of these elastomer releas e coatings, the factors of surface lubricity and topography are examined in this study as a function of biological growth and adhesion. The lubricity of the silicone elastomer surface was controlled by the

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xvi incorporation of nonfunctional poly(dimethylsiloxane) linear oils. This chemical modification allowed the surface chemistry and bulk mechanical properties to stay relatively constant with only the lubricity as a variable. These design criteria for the surface lubricity factor were proven to be effective through meticulous testing. An increase in surface lubricity was expected to increase the removal of organisms under a constant force. When Enteromorpha algae spores were examined for settlement/removal characteristics with a hydrodynamic flow cell, however, the predicted trend for surface lubricity was only observed with the excessively oily surfaces. The factor of surface topography was examined through engineered microtextured silicone elastomer surfaces of 5m wide pillars or ridges separated by 5,10 or 20m wide valleys/spaces. The factor of topographical depth was also inspected with both 5 and 1.5m deep features. With the Enteromorpha spores, the release from the microtextured, unmodified silicone elastomer surfaces was significant for the pillar features and the larger depth. The interaction between the coupling of these two factors on settlement and adhesion was also in the experimental design. The ridges had less release of spores for all elastomer formulations. There were definite topographical settlement cues as the spores preferentially adhered in the valleys, as opposed to on top of the ridges, and around the pillars. Through chemically functionalizing the surface with a quaternary ammonium antimicrobial salt, the potential for preventing the growth of biological organisms onto the surface is a prospect. Chemical modifications for binding a quaternary ammonium salt to glass, silicone elastomer, and polysulfone are presented and analyzed for their fidelity and interfacial properties. This entire study presented significant aspects and directions for designing biointerfaces for the purpose of controlling biological interactions.

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1 CHAPTER 1 INTRODUCTION The ability to engineer a surface in order to control or prevent biological adhesion is of great importance. Whenever a man-made structure or device is placed into a biological environment, there is an immediate settlement cascade that initiates on the surface. In the field of anti-fouling coatings for ship hulls, biological adhesion to the surface is not desired because an increase of biofouling on the hull causes an increase in the hydrodynamic drag and thus an increase in fuel consumption and a decrease in optimum speeds.1 Even a 100m thick biological growth can cause a 6% increase in fuel consumption.2 It is also costly to dry dock the large ships in order to clean off the biofouling or reapply the anti-fouling coating, so increasing the time in between dry docking by increasing coating efficiency would be cost beneficial. In the biomedical field, the contact guidance of cells, cellular response in a directional orientation, can allow the desired biological response to be achieved on the surface of a medical device or implant. One particular area of need is with tissue grafts where the mechanical stability in particular directions is desired. The ex act cues which allow for control of biological interactions with synthetic materials is the key for engineering surfaces. The contact guidance of cells has been shown to be directly influenced by the chemistry and topography of a material’s surface.3-5 Cellular alignment and growth can either be enhanced or inhibited depending on topographical dimensions. The mechanical stability of cells has been shown to increase when settled within valley patterns due to the

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2 increase in adhesion contact area.6-8 However, if the biological organisms are unable to settle within the valley topographies, then the adhesion contact area should decrease and thus the mechanical stability of the organism should also decrease. Cells and other organism have also shown specificity to various surface chemistries.9, 10 Hydrophobic surface monolayers have shown larger cell adherence then with hydrophilic surfaces.11 The friction at an interface also has been observed to have a major impact on the adhesion between two substrates.12, 13 As a result, the adhesion of a biological organism should decrease as a surface becomes more lubricious. The purpose of this study was to examine the role of lubricity and topographical surface modifications on biological settlement cues and adhesion forces. The surface grafting of a chemical biocide, quaternary ammonium salt, was also introduced as a chemical modification to prevent biological adhesion. In particular, the settlement and adhesion of marine algae Enteromorpha zoospores were examined. This biological organism is a major nuisance in the biofouling of ship hulls and thus its cues are an important role to examine. The substrate chosen for these modifications was a silicone elastomer because of its foul-release properties. One specific aim of this study was to characterize the chemical modifications of the silicone elastomer in order to verify that only the surface lubricity was variable. In particular, the elastic modulus and surface energy was characterized for the different formulations since these two variables have been shown to influence adhesion.14 With the addition of nonfunctional silicone oils, the lubricity of the surface was predicted to change, but the elastic modulus and surface energy values should have stayed relatively

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3 constant. This was expected due to similar molecular chemistries between the elastomer and additives and the nonfunctionality of the silicone oil additives. Another specific aim was to examine the effects of the lubricity and topographical surface modifications on settlement and relative adhesion with Enteromorpha spore assays. An increase in surface lubricity was predicted to decrease adhesion, thus increase the number of spores removed after subjected to a hydrodynamic flow, which had a 55Pa wall shear stress. The amount of removal was quantified as the difference of the spore densities on the surface from before and after exposure to a hydrodynamic flow. The spore adhesion to the surface should decrease as the force required to remove the organism decreases or the amount removed under a constant force increases. The addition of pillar topographies on the surface was expected to disrupt the standard settlement by providing an increased adhesion surface area. However, having a high density of pillars over the surface should ultimately disrupt the settlement so that the spores never obtain stability and could thus be more easily removed. The micropatterned valleys were predicted to enhance settlement in a directional manner as the spore should settle in the valleys where they have the highest adhesion contact area. However, again when the width of the valleys was smaller than the spore, there should be a decrease in the stability of the spore’s adhesion and thus easier removal from the surface. There was also predicted to be an increase in removal when the valleys were placed in the parallel direction with flow, as a laminar flow would then be localized down the valleys and spores would be subjected to a slightly hi gher shear force. Coupling both topographical and lubricity factors together should enha nce the control of biological adhesion to engineered surfaces.

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4 The third specific aim of this study was to chemically bind a biocide to the surface in order to prevent biological adhesion. A quaternary ammonium salt, which has antibacterial properties, was grafted to the surface to prevent biofilm formation. The initial step in any biological-material interaction cascade is the formation of a biofilm.1517 Reducing this initial step should decrease the biological settlement and adhesion of other organisms. The polymer substrates chosen for chemical modification were silicone elastomer and polysulfone. The polysulfone was investigated due to its higher durability as compared to the elastomer and its history of naturally reducing biofouling as a dialysis membrane.18, 19 This chemical surface modification allows for an anti-fouling coating for either marine structures or medical devices without the leaching of toxic chemicals into the biological environment.

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5 CHAPTER 2 BACKGROUND Biofouling In the recent past, tributyltin (TBT) based paint coatings have been the standard for use on the hulls of ships and other immersed man-made structures to prevent biofouling. However, this anti-fouling (AF) coating releases an environmentally toxic biocide chemical into the water. Some toxicity readings around the water layer of a ship’s hull were classified as safe, but one thing was not considered. When the ship is docked in a bay or other semi-closed body of water for a long period of time, the time released toxic material continuously rises in quantity and concentration in the surrounding waters. In the 1970s, the effects of TBT in the waters were observed in abnormal shell and reproduction of oysters.20 Since then abnormalities have been reported in other non-target organisms such as marine vertebrates and invertebrates. As a result, a ban was placed restricting the application of a TBT coating on small vessels in 1982.20, 21 The goals of the US Navy, following the passing of the US Organotin Antifouling Paint Control Act in 1988 and the ban set by the International Maritime Organization, are to have the application of all TBT coatings universally banned by 2003 and the use of them banned by 2008.22 Thus a replacement coating is necessary. Many of the coatings have been switched to copper based paints, which perform under the same mechanism, but these are claimed to be less toxic to non-target organisms.23 However, these copper based coatings are also showing signs of accumulation and toxicity and their long-term effects are currently unknown.21 Currently, significant research has focused on

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6 silicone elastomers as foul-release coatings (FR), from which either movement of the ship or effortless cleaning can easily remove organisms. However, the elastomer lacks durability as compared to the other coatings, which is a critical component for ship hulls. Thus there are also steady attempts to alter this mechanical property, while keeping the elastomer’s release properties. Adhesion Mechanics In order to control adhesion of biological organisms to a substrate, some fundamental fracture mechanics have to be considered. The term adhesion is defined as the force required to separate two surfaces whereas, interfacial surface energy is the work that is required to separate the surfaces. Currently, there are four methods classified for obtaining adhesion: chemical bonding, el ectrostatic interactions, mechanical interlocking, and diffusion.14 Adhesion of biological organisms is complicated not just because one mode is followed, but a combination of modes. Thus a surface must be engineered to be favorable in preventing adhesion by multiple mechanisms. Currently, silicone elastomers are being studied for their combination of reducing adhesion by the smooth residual silicone oil layer on the surface preventing adsorption, the formation of only weak chemical bonds, and the high compliance aiding in removal. 14 For prediction of the force required to break an adhesive from a silicone elastomer substrate, basic fracture mechanics should be examined. Griffith formulated an equation (eqn. 2.1) for the critical stress ( c) required to propagate a crack in a plate c = EGc / a(1 – 2) (2.1) from an uniaxial direction, where E, Gc, a, are the elastic modulus, Griffith’s critical fracture energy per area, half the crack length, and Poisson’s ratio, respectively.24 He

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7 then applied this equation to th e stress over a set crack area (A= a2), known as the critical pull-off force (Pc) (eqn 2.2). Pc = EGca3 / (1 – 2) (2.2) Quite a few years later, Kendall followed Griff ith’s fracture analysis to model adhesion to elastomer substrates.25, 26 He used a circular disc to design the force for removal from the elastomer. He then derived the critical pull off force, assuming a thin elastomer film and the radius of the disc being smaller than the size of the elastomer film, as Pc = a2 (2GcK/ t)1/2 (2.3) where Gc, a, t, and K are the critical fracture energy, radius of the contact area, elastomer film thickness, and bulk modulus (K= E /3(1 2 )). These equations show that there is a proportional relationship between the critical pull-off force and (EGc)1/2, which are material properties.14 In this case, fracture energy is directly related to the work of adhesion, which is then equal to double the critical surface tension ( c) of the elastomer.27, 28 As a result, the adhesion correlates with (2E c)1/2. The elastic modulus and surface energy are all parameters that can be engineered with a material. Another term that is used considerably in the literature is the compliance of a material, defined as the inverse of the elastic modulus, which measures its softness. These fracture mechanic equations assume that the applied force is in the normal direction to the elastomer film. However, when the force is applied at an angle, as would be the case when a ship is moving or in a laboratory hydrodynamic flow cell, there is the additional component of interfacial slippage that has to be considered. According to Newby et al ., there is evidence that the adhesion of a viscoelastic adhesive on silicone elastomers is controlled heavily by interfacial slippage rather than thermodynamics.29 When peeling a viscoelastic adhesive, an extension deformation occurs behind and

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8 contraction occurs in front of the moving crack tip.30 If slippage is allowed to occur by the substrate, then the work required to move the crack tip is lowered, which results in a lower adhesion strength. The more mobile chains are on the substrate surface, the lower the friction observed. When force is applied in the normal direction the release mode is peeling from nucleated voids within the contact area, but when the force is directed at an angle, failure occurs by a fingering process (like webbed fingers) as described by Newby et al. which starts at the edges and moves inward.29, 31 These finger adhesion release deformations were also shown to increase in length (i.e., amplitude) slightly as the modulus or the thickness of the elastomer coating increased and significantly as the rigidity of the adhesive increased.32 Coupling Agents Sometimes coupling agents of double or more functionality are used to promote adhesion through a chemical bond between two molecules or materials. If the coupling agent only has a single functionality, then it can be used as an end-capping agent. The most common types of coupling agents between polymers are organofunctional silane based molecules.33 Typically the four end groups are 1 vinyl or other organic functional group (R) and 3 alkoxy groups (OR1) so that the general formula is RSi(OR1)3. The alkoxy groups first hydrolize into silanols relatively quickly and then slowly undergo a condensation reaction to make a siloxane chain when in water. The reaction rates are dependent on the pH of the solution. The longer the alkoxy side chains are, the slower they hydrolyze. Solubility of the silane molecules decreases as the condensation reaction progresses and thus as the solution becomes hazy, the coupling agent becomes unusable.33 When the silane is added to an aqueous alcohol solution, the coupling agent

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9 has a longer work time as an equilibrium reaction is reached between the hydrolysis of the alkoxy groups to silanol and the reesterification of the silanols by the alcohols. Roughness Factor With the fracture mechanisms discussed previously, the surfaces were assumed to be completely smooth. However, even the smoothest substrate has molecular roughness on the surface. As a result, the factor of surface roughness, random or engineered, on adhesion strength needs to be examined. The biological adhesive usually starts as a liquid and then either stays liquid or cures. Depending on the viscosity of the liquid, the adhesive might not fill in all of the small crev ices because of its inability to displace the air pockets.34 When solidification of the adhesive occurs, there are stress concentrations that occur at the focal points of the roughness. The stress at these focal points is much higher than the applied force, so less applied force is needed to fracture the adhesion. If the voids are relatively close together then the fracture crack can propagate even easier.34 The unfilled crevices can be on the molecular level or micron scale depending on the size of the biological organism. Silicones Introduction Poly(dimethylsiloxane) (abrev. PDMS) elastomers are widely used in coatings and medical devices because of its relative biocompatibility, high resistance to chemical attack, and elastomeric properties. This material also has a high oxygen permeability, which promotes its use in contact lenses and membrane oxygenators.35 An additional benefit with PDMS is its low toxicity to the environment. The polysiloxane chains can hydrolyze to dimethylsilanediol oligomers over time. When these silanediols are in a microbial or UV light environment, degradation into CO2 and SiO2 (silica) occurs, which

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10 are all naturally occurring materials.36-38 In the literature, there have been discrete references to the possible side product Me3SiOH, which is known to be a nervous system depressant, introduced upon degradation, but there has been little published followup.39,40 There are a few mineral catalysts that seem to increase the degradation rate slightly.36 However, this degradation process is very slow for PDMS chains and even slower in silicone elastomers. Many different marine and soil environments have been examined for silicone toxicity and little adverse effects on growth or reproduction in aquatic animals have been discovered.41, 42 Polydimethylsiloxane (PDMS) chains have a silicon-oxygen backbone, which has a longer bond length of 1.63 than carbon bonds in many of the standard organic polymers.43 This property presents large bond rotation and thus large chain mobility and restructuring ability. As a result, the nonpolar and polar groups can reorient on the surface to their most favorable position depending on its environment. 44 PDMS at its lowest energy state is in the all-trans conformation due to this arrangement having the most favorable van der Waals interactions where the methyl groups are separated by four bonds.45 This ability to recovery to its lowest energy state is beneficial when trying to predict the material properties when exposed to diverse environments. Mechanical Properties Elastomers have extremely low tensile values without reinforcement (~0.35MPa) which limits their applications.39, 46 In order to increase the mechanical properties and tear strengths of elastomers, reinforcement particles are incorporated into the bulk. In particular, silica particles are most widely used for reinforcement in silicone elastomers because they have shown to provide the highest change in mechanical properties.47 Three factors of the silica particles influence the resulting mechanical properties from

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11 reinforcement: size, structure (pore volume), and surface interaction.48, 49 There are 3 interactions that have to be considered when introducing silica particles: chemical fillerelastomer bonds, physical interference in the elastomer, and filler-filler interactions.47 With fumed silica, there are hydrophobic and hydrophilic portions on the surface, as shown in Figure 2.1. The isolated hydroxyl groups introduce the hydrophilic portions and the hydroxyl groups that have reacted again with the silica surface introduce a nonpolar, hydrophobic portion.48 In some cases, the silica particles are made unreactive by the introduction of hexamethyldisilazane, which reacts to produce trimethylsilylated silica surfaces, as shown in the reaction in Figure 2.2. The fumed silica increases the crosslink density of the elastomer network and thus has been shown by Boonstra et al to increase elastic modulus at all strain ranges, but decrease elongation as compared to trimethylated particles.48 During synthesis of the particles, agglomeration also occurs. The packing factor of these agglomerates cha nges the sizes of pores within the particle clusters. Agglomeration also introduces polymer chains that can be occluded within the irregularities of the clusters and thus be secluded from distribution of stresses within the elastomer network. Baker et al have shown that the smallest fillers produce the highest reinforcement.47 The elastic modulus and tensile strength have also been shown by Polmanteer and Lentz to increase as the filler structure, measured by pore size, increases for silicone elastomers.49 With the multiple factors involved in particle reinforcement, declaration of trends of just one single factor alone is difficult.

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12 Figure 2.1: Chemical species on the surface of fumed silica and trimethylsilylated silica particles. Figure 2.2: The chemical reaction for the trimethylsilylation of a fumed silica particle by addition of hexamethyldisilazane. Si HO Si OH Si Si OFumed SilicaTrimethylsilylated Silica Si Si O Si OHHydrophilic Hydrophobic Si Si O Si Si O Si O (CH3)3Si Si O (CH3)3Si Si O (CH3)3Si Si HO Si OH Si Si OFumed SilicaTrimethylsilylated Silica Si Si O Si OHHydrophilic Hydrophobic Si Si O Si Si O Si O (CH3)3Si Si O (CH3)3Si Si O (CH3)3Si H3CSiN H SiCH3CH3CH3CH3CH32 H2O 2H3C CH3CH3SiOH NH3 Si Si Si OH OH OH CH3CH3CH3Si HO Si Si Si O OH OH CH3CH3CH3SiH2OHexamethyldisilazaneTrimethylsilanol Fumed SilicaTrimethylsilanolTrimethylsilylated Silica H3CSiN H SiCH3CH3CH3CH3CH32 H2O 2H3C CH3CH3SiOH NH3 Si Si Si OH OH OH CH3CH3CH3Si HO Si Si Si O OH OH CH3CH3CH3SiH2OHexamethyldisilazaneTrimethylsilanol Fumed SilicaTrimethylsilanolTrimethylsilylated Silica H3CSiN H SiCH3CH3CH3CH3CH32 H2O 2H3C CH3CH3SiOH NH3 Si Si Si OH OH OH CH3CH3CH3Si HO Si Si Si O OH OH CH3CH3CH3SiH2OHexamethyldisilazaneTrimethylsilanol Fumed SilicaTrimethylsilanolTrimethylsilylated Silica

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13 Surface Energy/Chemistry The surface energetics of silicone elastomer is very complicated since it is such a low surface energy material with highly mobile methyl groups at the surface being its lowest energy state. A basic method for examining the surface energetics of a solid substrate is with static contact angles, either through sessile drop technique or captive air bubble technique. The diagrams in Figure 2.3 show the general setup and variables for the two contact angle measurement techniques. Measurement by contact angle provides information about the outer angstroms of the surface and is a result of adhesive forces between the two phases and cohesive forces within the liquid.50, 51 This is one of the most sensitive techniques for examining the actual surface and it is relatively simple to perform.50 However, analysis of the contact angl es can be confusing because of the many assumptions and factors that have to be considered. Both static techniques follow Young’s interfacial equilibrium equation, sv – sl = lv cos (2.1) where sv, sl, and lv are the surface tensions for the surface-vapor, surface-liquid, and liquid-vapor interfaces, respectively, and is the contact angle. This is derived from an Figure 2.3: Setup and labeled interfaces/contact angle for both the (A) sessile drop technique and (B) captive air bubble technique. LVSolid Liquid VaporSLSV Substrate Liquid Air LVSL SVAB LVSolid Liquid VaporSLSV LVSolid Liquid VaporSLSV Substrate Liquid Air LVSL SV Substrate Liquid Air LVSL SVAB

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14 energy balance approach at the sessile drop intersection point, shown in Figure 2.4, with a Helmholtz free energy (F) equation (eqn. 2.2) over a small distance, dx. (dF)T,V,n = svdx – sldx – lvdx cos (2.2) At equilibrium, dF = 0, and Young’s equation is the resultant.50 The experimental technique involves measuring the contact angles ( ) of different liquids, of various surface tensions ( lv), on the desired substrate. However, there are still two unknowns in Young’s equation, sv and sl. As a result, W.A. Zisman proposed a plot of cos versus lv which he found to be empirically linear.52 Theoretically, then at = 0, in other words cos = 1, the liquid completely wets the substrate surface. At this point, Zisman claims that sl = 0 and, as a result, the surface tension of the solid vapor interface can be calculated from a linear regression on the Zisman plot. This is then termed the critical surface tension ( c) of the solid. For a homogenous solid, the critical surface tension is the same as the surface free energy assuming that there is no other forced elastic strains on the solid and no solvent adsorption.53, 54 Figure 2.4: Energy balance approach at the point of triple phase (liquid-solid-vapor) intersection for the derivation of Young’s equation. The forces are labeled previously in Figure 2.3. dx cos dx Solid Liquid Vapor dx cos dx Solid Liquid Vapor

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15 There are many assumptions that coincide with this technique for surface analysis.50, 51 First, the droplet (or air bubble) is assumed to be small enough that there is minimal gravitational effect and thus little distortion of the droplet shape.51 A major assumption when examining a thermodynamic system is that everything is at equilibrium.50 With the factors of solvent volume, due to evaporation, and surface rearrangement, equilibrium is hard to obtain and hold for contact angle measurement. This also implies that time and temperature are factors which can alter the contact angle. Some other basic assumptions are that the surface is rigid (i.e., nondeformable), smooth, homogeneous, does not reorient, and that the solvents have exact, known surface tensions that do not swell the surface. These assumptions are obviously not all met in many scenarios, but care can be taken in interpretation of measurements if the limitations are defined. Many factors can not be controlled easily which led to R.E. Johnson and R.H. Dettre’s claim about contact angle measurements that “the limitation on accuracy is not the measuring technique, but rather the reproducibility of the surface being studied”.51 When one or more of these assumptions are not met, a contact angle hysteresis is observed. This is defined as the difference of the contact angle advancing over a solid-air interface and the contact angle receding back over the solid-liquid interface. Silicone elastomers are known to have large contact angle hysteresis due to their highly mobile backbone chain and thus surface rearrangement. When a static surface energy method is performed, the measured contact angle is observed between the advancing and receding angles from a dynamic method, but closest to the advancing angle.53 One of the large factors that influence contact angle measurements and introduces a hysteresis is surface roughness. Wenzel first attempted to define a relationship between the measured contact angle and the surface roughness with equation 2.3 where ’ is the

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16 cos ’ = r cos o (2.3) observable contact angle on the rough surface and o is the intrinsic contact angle. The roughness ratio, r, is defined as r = A / A’ (2.4) where A’ is the apparent surface area and A is the true surface area taking the roughness into account.50, 55 This means that the ratio, r, is always greater than 1. According to Johnson and Dettre, the trends observed from many studies were that as the roughness increased, the advancing angle increased and the receding angle decreased. 55 This means that when static conditions are examined, as the roughness increases, the contact angle increases and thus the critical surface tension calculated increases. However, this theory does not consider the size, the shape, and the exact location that the droplet edge falls in reference to the rough features. These are all factors that are still undefined in their relationship to measured contact angles. This roughness influence on the spreading of liquids needs to be considered when examining the settlement of biological organisms, which initially excrete a liquid cement to increase adhesion on engineered topographies. Enteromorpha Algae Spores There are many different types of seaweeds that are present in waters, including brown, red, and green algae. All types are unicellular at some point in their life cycle in the form of spores or zygotes.56 One type of green algae that is of particular interest is the Enteromorpha species genus, which is the most common and taxing algae fouling on ship hulls and other submerged marine structures.57, 58 This type of algae, from the Ulvaceae family, is of high nuisance because it is tolerant to a wide variation of water salinities and can thus travel and survive with ships to many freshwater and ocean environments.59 The Enteromorpha reproduce by the release of quadriflagellate, pear-

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17 shaped zoospores (7-10m long), which attach to substrates.60 The cells then divide and grow into a tubular type organization.59, 61 The mechanism behind the settlement and adhesion of these spores to a substrate is of interest when trying to design an anti-fouling coating. As motile spores, they lack a cell wall and search for a substrate on which to colonize. Once a spore identifies an acceptable surface, a glycoprotein is secreted in order to form an adhesion.60 The spore is then settled and a cell wall is developed and eventually a new algae plant is produced. When performing algae laboratory studies, there are always considerations that have to be recognized during interpretation of results, mostly due to the culture having more ideal, uniform conditions than in the environment. One important factor is that there is no longer competition or grazing from other biological organisms in a laboratory environment.56 This is just one reason out of many for large standard deviations that are encountered on biological organism studies. There are many known cues that are involved in spore settlement, such as negative phototaxis (light), thigmotaxi s (roughness), and chemotaxis (chemical). 10, 62 All of these factors have been shown to cause spores to not evenly distribute on a surface. The adhesion of the spore to a substrate can also be influenced by these factors with the spreading and bonding of the chemical secretion.61 All of the rough surfaces examined previously were random and an increase in settlement was assumed to be due to an increase in surface area contact. However, if a surface was engineered with regular patterns at defined aspect ratios perhaps the contact area could be disrupted or decreased. This is the basis for the topographical studies presented here.

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18 Quaternary Ammonium Salts Introduction Recently, the antimicrobial action of su rfaces has come into popularity with disinfectants for textiles, medical devices, marine coatings, and food manufacturing.63-66 Quaternary ammonium salts are one of the most popular types of antimicrobial agents. At first, this biocide was incorporated into the bulk resin and allowed to leach over time in order to obtain a continually active surface. However, when attempted by researchers with silicone elastomer, because of the favor able surface energetics with the methyl side groups at the surface, the quaternary ammonium was unable to diffuse to the surface, which was visible with simple contact angle measurements.67 This led to the motivation of binding (or immobilizing) the quaternary ammonium to the surface so there was no leaching or toxic effects to the environment and an increased productive time.68, 69 There are other factors that influence the activity level of the quaternary ammonium salt, such as the accessibility of the active centers (directly related to the length of the alkyl chains), the concentration on a surface, the chemistry of the spacers along the surface (to change concentration), and the counter ion.70 Biocidal Mechanism Quaternary ammonium compounds interact with bacterial cells, but not by chemically reacting. The extreme polarity and long hydrophobic alkyl chains interact with the phospholipid bilayer of the cytoplasmic membranes to cause a phase separation of the hydrophobic and polar regions.71 As a result, weakness and leakage of the cytoplasmic membrane is caused by these compounds, which leads to a disruption of the permeability and instability of the membrane. The absorption of this biocide is enhanced by the cells negative charge.72 First the long alkyl groups face away from the cell in

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19 order to introduce the cationic species. This causes the disruption of the cytoplasmic membrane and results in displacement of potassium and magnesium ions, which contribute to the membranes stability. The incr ease in permeability that results allows the long alkyl chain to enter the cytoplasma and cause precipitation of the nucleic acids and proteins.72 Many researchers have discovered that these cationic biocides are not as effective for gram-negative bacteria as compared to gram-positive bacteria. This has phenomena has been contributed to the presence of an outer membrane on only the gramnegative bacteria. This outer membrane incr eases the impermeability of the antimicrobial agents, even though some are still successful.73, 74 As a result, binding the quaternary ammonium salt to a surface should prevent or reduce the initial stage of biological adhesion, biofilm formation from bacteria and diatoms, and ultimately reduce the later stages of biological growth.

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20 CHAPTER 3 SILICONE ELASTOMER CHEMICAL MODIFICATION AND CHARACTERIZATION Introduction A factor of biological adhesion that should be examined is friction/lubricity of a surface. However, the substrate materials must be fully characterized before analysis of growth data can be performed. Currently, the substrate of silicone elastomer is being studied for its foul-release properties in marine coatings and for medical devices.75 Thus this substrate allows for application in many different fields. In order to engineer the elastomer substrates with lubricity as the predominant variable, various nonfunctional silicone oil additives were introduced into the bulk of the elastomer creating a lubricious surface. Due to surface energetics, the lo wer surface energy silicone oils migrate and coat the elastomer surface.76 As a result of similar chemical structures and nonfunctionality between the silicone oils and elastomer, the elastic modulus and surface energetics should stay relatively the same. However, this idea needed to be proven through experimentation. For characterization of these chemical modifications, the mechanical tensile properties, specifically Young’s modulus, and the static surface energetics were examined.

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21 Materials Silicone Elastomer A silicone elastomer substrate was used as a basis for a chemically and topographically engineered material in order to examine the biological response to these modifications. In particular, the silicone elastomer substrate was Dow Corning’s Silastic T2, which is a hydrosilylation, platinum catalyzed poly(dimethylsiloxane) elastomer. The two-part, vinyl-functionalized, addition cure reaction is shown in Figure 3.1. There are trimethylated silica particles also present in Dow Corning’s formulation in order to increase the material’s mechanical stability. This specific silicone was chosen for its transparency, low shrinkage, and few unknown/undisclosed additives. Thus it allowed modification of this material to be controlled and engineered topographies be reproduced with high fidelity. This resin was mixed in a 10:1 weight ratio of base resin to curing agent, as directed by Dow Corning. The typical properties of this elastomer prepared with this ratio are provided by the manufacturer and are shown in Table 3.1. This material, in its original form, is referred to as “unmodified” elastomer. Figure 3.1: Dow Corning’s Silastic T2 silicone elastomer crosslinking reaction. Silastic T2 Base ResinSilastic T2 Curing AgentSilicone Elastomer Pt H2CC H Si CH3CH3OSiO CH3CH3SiCH2CH2Si Si CH3CH3C H CH2n+ PDMS H3CSi H CH3OSiO CH3CH3Si CH3CH3CH3xySilastic T2 Base ResinSilastic T2 Curing AgentSilicone Elastomer Pt H2CC H Si CH3CH3OSiO CH3CH3SiCH2CH2Si Si CH3CH3C H CH2n+ PDMS H3CSi H CH3OSiO CH3CH3Si CH3CH3CH3xy

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22 Table 3.1: Typical properties of Dow Corning’s Silastic T2 addition cured silicone elastomer. (From Dow Corning’s product information sheets) Nonfunctional Additives The role of nonfunctional silicone oils added to the bulk silicone elastomer was to maintain similar chemistry and mechanical properties, but have a varying lubricious surface. All the silicone oils used were either nonfunctional, trimethylsiloxy terminated poly(dimethylsiloxane) (PDMS) linear chai ns, of varying chain length, or a bulky, nonfunctional silane molecule. All the nonfunctional additives were purchased from Gelest, Inc and their chemical structure, viscosity, and molecular weight are shown in Table 3.2. All of the additives discussed were nonfunctional, thus none of them reacted into the vinyl-functionalized bulk elastomer and the elastomer crosslink density was maintained. Energetics drives these low surface energy silicone oils to migrate to the surface, thus allowing a controlled release and complete surface coverage. Surface Energy Liquids In order to calculate surface energy from contact angles, a variety of liquids of varying surface tensions must be used. In these experiments, 5 different liquids, all < 0.1 % Linear Shrinkage 800 psi (0.55 MPa) Tensile Strength 1.12 Mixed (10:1 weight) Specific Gravity 550 cp Viscosity of Curing Agent 120 ppi (Die B) Tear Strength 300 % Elongation After Cure (24hrs at 25C): 50,000 cp Viscosity of Base Resin Translucent/Transparent Base/Curing Agent Color Result Test < 0.1 % Linear Shrinkage 800 psi (0.55 MPa) Tensile Strength 1.12 Mixed (10:1 weight) Specific Gravity 550 cp Viscosity of Curing Agent 120 ppi (Die B) Tear Strength 300 % Elongation After Cure (24hrs at 25C): 50,000 cp Viscosity of Base Resin Translucent/Transparent Base/Curing Agent Color Result Test

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23 Table 3.2: Chemical structures and properties of the nonfunctional silicone oil additives from Gelest, Inc. (Information obtained from Gelest, Inc. catalog) Table 3.3: List of chemicals used for contact angle measurements with their corresponding liquid surface tensions. 0.868 0.960 0.971 0.973 0.978Sp. Gravity-384 ~3 Tetrakis (trimethylsiloxy)silane 49 231 664 4154 3,800 17,300 49,400 308,000 50 500 5000 1,000,000 Poly(dimethylsiloxane), trimethylsiloxy terminatedn MW (g/mol) Viscosity (cSt) Structure Name 0.868 0.960 0.971 0.973 0.978Sp. Gravity-384 ~3 Tetrakis (trimethylsiloxy)silane 49 231 664 4154 3,800 17,300 49,400 308,000 50 500 5000 1,000,000 Poly(dimethylsiloxane), trimethylsiloxy terminatedn MW (g/mol) Viscosity (cSt) Structure Name SiO CH3CH3Si CH3CH3CH3 Si CH3CH3CH3 n O Si OSi(CH3)3OSi(CH3)3(CH3)3SiO (CH3)3SiO 47.7 Ethylene Glycol 23.8 1-Propanol 29.3 Acetonitrile (ACN) 37.1 N,N-Dimethylformamide (DMF) 50.7 Diiodomethane (methylene iodide) 72.8 HPLC grade Water Surface Tension (mN/m) Chemical Name 47.7 Ethylene Glycol 23.8 1-Propanol 29.3 Acetonitrile (ACN) 37.1 N,N-Dimethylformamide (DMF) 50.7 Diiodomethane (methylene iodide) 72.8 HPLC grade Water Surface Tension (mN/m) Chemical Name

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24 obtained from Sigma Aldrich, were chosen with varying surface tensions, as shown in Table 3.3. These liquids included, HPLC grade water, diiodomethane (methylene iodide), N,N-dimethylformamide (DMF), acetonitrile (ACN), and 1-propanol. During later experiments, the diiodomethane liquid was replaced with ethylene glycol as the liquid with the surface tension in the range of 50 mN/m. Methods Sample Preparation The unmodified silicone elastomer was always prepared according to Dow Corning’s recommendations. The base resin was weighed out as 100 parts to 10 parts of the curing agent. Both were mixed thoroughly together in a polypropylene tri-cornered beaker with a metal scoopula for approximately 5 minutes. The mixture was then degassed in a vacuum desicator for approximately 30 minutes in order to get rid of the many trapped air bubbles. Not all of the air bubbles disappear, but the elastomer selfreleases the residual small air bubbles within the first 30 minutes of cure. If the addition of silicone oils was desired then they were added into the elastomer mixture at a 5, 10, or 20 weight % to the already mixed base and curing agent weight and subsequently degassed in the same manner as the unmodified elastomer. The approximate work time for the elastomer mixture was 1 hour. The elastomer was cured at either room temperature for 24 hours or at 80C for 2 hours depending on experimental conditions. For the preparation of free-standing films, the silicone elastomer was poured onto a clean glass plate (7in x 7in x 3/8in) with the setup as shown in Figure 3.2. All glass plates used in these experiments were cleaned with acetone, then ethanol, and lastly wiped with hexamethyldisilazane (HMDS) (from Gelest, Inc) to cap any potential

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25 Figure 3.2: Mold setup for preparation of silicone elastomer films (left) and silicone elastomer coated glass slides (right). silanol groups on the glass surface that would make the silicone elastomer adhere during curing. On the bottom glass plate, there were 4 spacers of specific thickness, depending on the thickness of the film desired, placed with double-sided tape. After pouring the elastomer onto the bottom glass plate, the top glass plate, which was covered with a thin, amorphous poly(ethylene terepthalate) (PET) sheet to ease film removal, was slowly laid on top of the silicone elastomer, allowing it to rest on the spacers. A 5 lb weight was needed on top of the mold to press the top glass plate completely down onto the spacers. This mold setup provided smooth elastomer films of consistent thickness that would be used for tensile mechanical testing. For the preparation of silicone elastomer coated glass slides, the general mold discussed above (Figure 3.2) was used. However, clean glass slides were placed on the bottom glass plate with coupling agent treated side up just prior to pouring the elastomer mixture. The slides were borosilicate glass of 75mm x 25mm dimensions and 1mm thick (Fisher Brand). Before use, the slides were burned with a clean flame (blue color) from a bunsen burner. Then they were wiped with a 1 Normal HCL solution. Lastly they were Top Glass Plate PET sheet Metal Spacers Bottom Glass Plate Glass Slides Top Glass Plate PET sheet Metal Spacers Bottom Glass Plate Glass Slides

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26 treated with a 0.25 vol% allyltriethoxysilane (Gelest, Inc) coupling agent solution for 3 minutes on one side, rinsed with ethanol, and then dried in an oven at 120C for 10 minutes. This coupling agent solution was prepared by first mixing 30 mL of ethanol with 1.5 mL DI water. The pH of the solution was then dropped down to approximately 4.5 by adding Glacial Acetic Acid (Aldrich, Inc). The amount of acid added usually was 2 drops from a transfer pipet. After stirring for 3 minutes, 0.085 mL of allyltriethoxysilane (ATS) was added and allowed to mix for an additional 5 minutes before application to the glass slides. Another difference in the mold setup from the film preparation was that the top glass plate was not covered with a PET sheet. This allows the ultra smoothness of the glass to transfer to the elastomer surface on the glass slides. These glass slides were used as a support mechanism for the elastomer film for surface energy measurements and later flow cell experiments. Mechanical Properties Mechanical properties were examined of all the silicone elastomer formulations in order to detect any significant changes in the bulk properties due to the silicone oil additives. All the tensile mechanical testing experiments were performed on 1 mm thick dogbones cut with an ASTM D1822-68 Type L die from silicone elastomer films. An Instron model 1122 load tower with 90psi pneumatic grips, a 200lb calibrated load cell, and Testworks 4.0 software was used for data collection and analysis of all experiments. The tests were either performed with crosshead displacement or MTS high elongation laser extensometer strain measurements, depending on the experimental protocol. The age of the samples, either 1 day or 128 days old after cure, also depended on the experimental design. Strain measurements were based on a 2 in/min crosshead displacement and a 1 inch crosshead gauge length. All experiments conformed to ASTM

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27 D412-97 at room temperature. For the extremely oily surfaced materials, a kimwipe was wrapped around the gripped portion of the dogbones for better hold in the pneumatic grips. For the crosshead displacement and the 1-day-old laser extensometer measurements, 10 specimens were run for each formulation. For the other laser extensometer measurements, only 2-3 specimens were available for each experiment. Some of the mechanical properties experiments involved soaking the dogbones in simulated seawater or DI water. The imitation seawater was mixed from InstantOcean salts to a 1.0215 specific gravity, which was measured with a hydrometer. The specimens were soaked in 15mL polypropylene centrifuge tubes of the desired liquid environment for 3 hours at 80C. This time and temperature were chosen in accordance with coupling agent adhesion test where 2 hours in boiling water was found to be equivalent to 30 days immersion at room temperature.33 Placing the filled centrifuge tubes in a heated water bath provided a constant heating environment. The liquid-filled tubes were allowed to equilibrate to temperature before the elastomer dogbone specimens were added. After the 3 hours, each silicone elastomer specimen was removed from the liquid and allowed to equilibrate to room temperature for 5 minutes before being mechanically tested. Surface Energetics Various silicone elastomer formulations were examined with contact angle measurements and calculated surface energies in order to detect any significant change in the surface energetics due to the additives. A static sessile drop technique was performed on silicone elastomer coated glass slides with both a goniometer and a digital capture system. A variety of liquids, shown previously in Table 3.3, in 2 L droplets were gently placed on the solid substrate with a micropipet. With the goniometer, shown in Figure 3.3, the contact angles were read from the horizontal microscope lens piece. From the

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28 digital capture setup, also shown in Figure 3.3, an image of a droplet on the surface was taken from a web cam (3Com Homeconnect Web Cam) through the Viviewer software on a computer. The lighting and contrast were adjusted with the software before image was captured. The stages were aligned with a circular level and the sample was centered and focused with a 3-dimensional micro-manipulator also before image capture. After initial setup, the images were captured quickly after the liquids contacted the surface and reached equilibrium in order to minimize interaction, evaporation, and contamination. After all the images were collected, the UTHSCSA ImageTool 2.0 software was used to measure the contact angles. The desired angle was drawn on the software, and then the computer measured and reported the angle in a spreadsheet, as shown in Figure 3.4. From all of these contact angles, the critical surface tension of the solid could be determined through the modified Zisman plot (to be discussed later in the chapter). The angle on both sides of the droplets was measured and each liquid was repeated 3-5 times depending on the experiment. Figure 3.3: Digital picture of the microsc ope goniometer setup (left) and the digital capture setup (right) for contact angle measurements.

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29 Figure 3.4: Computer screen image capture of the ImageTool software for measuring contact angles from digital images of the liqui d droplets. The image is of a 2L droplet of water on an unmodified silicone elastomer substrate. Results and Discussion Mechanical Properties Crosshead displacement measurements In order to examine the effect of additives on the bulk silicone elastomer, tensile mechanical tests were performed. Initially, the nonfunctional additive formulations of the silicone elastomer were examined with the crosshead displacement strain measurements after curing at room temperature for 24 hours. The calculated elastic modulus (E) results are shown in a graph in Figure 3.5. The software automatically calculated the elastic modulus at the most linear portion of the graph as: E = / (1)

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30 where was the change in stress and was the change in strain of two points in the linear region of the stress-strain curve. However, upon examination of the stress-strain curves for all of the silicone elastomer formulations, it was evident that there were two linear regions on each graph, as shown in Figure 3.6. The kinetics for the increase in the line slope at high elongations is examined more closely with the laser extensometer data (the next section). All of the data in Figure 3.5 were recalculated so that the elastic modulus was measured in the first linear portion at lower strain (20-50% strain). This linear portion was chosen originally because of the low strain that a biological environment would impose on a material. However, in later studies, the high strain portion was also examined because of the high shear environment introduced to the material as a foul-release coating. In Figure 3.5, the unmodified silicone (0wt% additive) was only examined once, but compared next to each of the other sample sets. The unmodified silicone had a noticeably higher elastic modulus, 1.4 MPa, than most of the nonfunctional additive formulations. It was not statistically different, though, when compared to the 5wt% 500cSt and 5000cSt formulations in this experiment because of their large standard deviations. The 20wt% silicone oil formulations for each additive type were significantly lower in elastic modulus than the formulations with less amount of silicone oil additive. A visible trend on this graph was that as the amount of silicone oil added was increased, the elastic modulus decreased. This trend is followed except for the two formulations just previously discussed, 5wt% 500 cSt and 5000 cSt. Another trend, noticeable in the 10 and 20wt% linear chain silicone oil formulations, was that as the viscosity, in other words molecular weight, of the silicone oil increased, the elastic modulus increased. An important point to remember was that none of these additives

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31 Figure 3.5: Effect of nonfunctional additives at different amounts on the elastic modulus (calculated between 20-50% strain) for a silicone elastomer. Strain measurements were made from crosshead displacement. Figure 3.6: Stress-strain curve for unmodified silicone elastomer with strain measurements made from crosshead displacement. Notice the 2 different linear segments, which correspond to different moduli values at low strain versus high strain. Performed at room temperature with a 1in. gage length and 2 in/min crosshead displacement. 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 50 cSt500 cSt5000 cSttetrakisSilicone Oil AdditiveElastic Modulus (MPa) 0 wt% 5 wt% 10 wt% 20 wt% 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0.00.51.01.52.02.53.03.5 Strain (mm/mm)Stress (MPa) High Strain Modulus Low Strain Modulus 0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 0.00.51.01.52.02.53.03.5 Strain (mm/mm)Stress (MPa) High Strain Modulus Low Strain Modulus

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32 were chemically bound to the elastomer, thus the cross-link density was roughly the same in each elastomer formulation. The effect of the bulky molecule tetrakis(trimethylsiloxy)silane (abbrev. tetrakis), as compared to the linear chains on the modulus was only slight. In fact, the elastic modulus of the bulky oil was not significantly different from the small linear chain silicone oil, 50cSt. The physical interactions behind these trends are discussed in detail with the laser extensometer measurements where the standard deviations are smaller due to an increased accuracy in technique. However, many of the same trends were identified with the laser extensometer measurements. It was also noted that perhaps the elastomer films were not completely cured after 24 hours so the time of day that the samples were run could have slightly changed the time allowed to cure and thus had a small influence on the mechanical properties. Laser extensometer measurements While performing these tests with the crosshead displacement, however, it was evident that there was a considerable amount of slippage out of the pneumatic grips while under high elongations. This led to a lower value for strain in the modulus calculations and thus a higher reported modulus. As a result, new batches of all the elastomer Table 3.4: Effect of cure time at 80C for 5wt% 50 cSt silicone oil modified silicone elastomer. (n = 10) 0.14 0.92 5 hours 0.07 0.85 2 hours Std.dev. Modulus (MPa) Cure Time 0.14 0.92 5 hours 0.07 0.85 2 hours Std.dev. Modulus (MPa) Cure Time

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33 formulations were examined using a high elongation laser extensometer for strain measurements. This technique minimizes the influences of grip slippage. These samples were cured at 80C for 2 hours to make sure cure reaction was complete and then allowed to rest for 24 hours at room temperature before testing. To prove that curing was complete after 2 hours, a sample of 5wt% 50cSt was also cured at 80C for 5 hours. After comparison of the two different cure times, values shown in Table 3.4, it was evident that there was no significant difference in elastic moduli between the two samples. As a result, it was concluded that 2 hours at 80C was sufficient time for cure completion. When the stress-strain curves of the elastomers were examined with the laser extensometer, again two distinct linear portions of the curve were present, as seen in Figure 3.7. The elastic modulus calculated from the low strain region (30-50% strain) and high strain region (120-150% strain) is given in Figure 3.8 for all the elastomer formulations, including a 1kSt linear silicone oil additive. This graph again showed the significant difference in modulus between the two strain areas. After examination, the high strain modulus was on average 3.1 0.5 times as much as the respective low strain modulus for all the formulations. For example, the 5wt% 50cSt silicone elastomer formulation had a low strain modulus of 0.9 (0.1) and a high strain modulus of 2.41 (0.2), which was 2.7 times as high. This upturn change in modulus, around 80% strain, occurred in every sample in the same regime. As a result, the factor must lie within the elastomer, rather than the oil additives. The base elastomer resin was known to have a broad range of molecular weight vinyl-terminated PDMS chains. Thus, it was theorized that this change in modulus at high strains was due to the limited extension of the smaller

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34 Figure 3.7: Stress-strain curve for an unmodified silicone elastomer with strain measurements made from a laser extensometer. Two different slope regions are evident between high strain and low strain corresponding to 2 different moduli values. molecular weight chains in the elastomer network. Mark et.al. noticed the same upturn in modulus at high elongations and on overall elongations when he examined bimodal trifunctional and tetrafunctional PDMS networks.77 They were convinced of this theory when the upturns were reversible and their monodisperse PDMS networks showed no visible upturn. In other types of rubber elasto mers, an increase in modulus at high strains has been usually attributed to a strain-induced crystallization, where the entropy of the elastomer was decreased enough upon elongation to promote crystallization, which then acted as physical crosslinks.45 However, since PDMS elastomers have such a low glass transition temperature, they do not crystallize even under a high strain.47 The introduction of silicone oils into the elastomer network brought a few more interactions when the mechanical properties were examined. At both the high and low strain, the modulus decreased as the percent weight of oil additive increased. There were 0.0 1.0 2.0 3.0 4.0 5.0 6.0 050100150200250300Strain %Stress (MPa) Low Strain Modulus High Strain Modulus 0.0 1.0 2.0 3.0 4.0 5.0 6.0 050100150200250300Strain %Stress (MPa) Low Strain Modulus High Strain Modulus

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35 a few samples that were not significantly differe nt because of larger standard deviations, but the overall general trend was present. However, as the viscosity (or MW) of the silicone oil additive increased, there was no significant change detectable in the high or low strain elastic moduli. One critical interaction, which could contribute to differences in moduli values, was the presence of silica agglomerates in the network system. At normal conditions, the silica agglomerates are unreactive due to the trimethylsilylation of the particle’s surface. However, at a critical stress, induced in the network from tensile elongation, the silica agglomerates can break apart and then introduce reactive hydroxyl groups from the freshly exposed particle surfaces. These hydroxyl groups can then either react with the close polydimethylsiloxane chains’s silicon-oxygen backbone linkages and increase the cross-links in the region or produce hydrogen bonding between the particle and the PDMS chains.48 These interactions should increase the elastic modulus of the elastomer. Once the reactive sites are inhibited, silicone chains are able to slip over the silica surface with ease.48, 78 The addition of nonfunctional silicone oils should introduce even higher chain mobility and thus a higher ease of gliding over the surface. As a result, as the molecular weight of the PDMS chains decreased or the amount of additive increased, there was an increase in chain mobility and thus a lower elastic modulus. This was evident in the values provided in Figure 3.8 where the percent weight additive was a detectable factor. Polmanteer and Lentz have shown that this same generation of reactive sites on the silica filler particles can also occur under high shear mixing conditions of the silicone elastomer resin.49 As the filler aggregates are broken in this instance, the silanol reactive sites can again introduce hydrogen bonding or chemically react with the polydimethylsiloxane chains, as previously mentioned. Even though the elastomer mixed

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36 in this experiment was not under high shear, the relatively low strain that was necessary to introduce this occurrence during mixing, leads to the conclusion that the effect of the silica particles occurred at both points of modulus measurement. Boonstra et.al introduced a stress-strain graph, which displayed reactive versus unreactive silica particles in vinyl-cured silicone elastomer.48 The two curves both had upturns around 100% elongation, but the reactive particle filled elastomer had a higher modulus at both low and high strain. This graph proved that the silica particle reactivity had little influence on the upturns seen on all the elastomer formulations’ stress-strain graphs, but did influence the relative elastic moduli values. The elongation at failure should be influenced by the same factors as just previously discussed, but also strongly direct ed by the number of chain ends present in the network. As the number of chain ends increase, the elongation should decrease as the untied ends introduce more defects, which ultimately leads to failure. The chain ends also are unable to carry any of the stress loads so the surrounding chains undergo a larger stress then being applied to compensate. The low molecular weight additives, introduce more chain ends from a set volume into the system. However, comparisons and trends were difficult to declare with the elongation at failure values obtained because of large standard deviations. This lack of reproducibility of the ultimate mechanical properties of silicone elastomers were observable in these experiments and also stated by other researchers.47 As a result, the elastic modulus was calculated and compared in these experiments as an observance of mechanical properties, along with its role in adhesion mechanics and rubber elasticity.

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37 Figure 3.8: Comparison of elastic moduli values between the different freshly cured silicone elastomer formulations from both the low strain and high strain regions. All of the strain measurements were collected from a laser extensometer. When the low strain elastic moduli data from the laser extensometer and crosshead displacement are compared, similar trends, which were previously discussed, are evident. Examination of a graph of the comparison, shown in Figure 3.9, shows that again an increase in percent weight of silicone oil, significantly decreased the elastic modulus, except for the 10wt% 5000 cSt formulation which was not detectible as a result of the larger standard deviation on the 5wt% 5000 cSt formulation. This could be attributed to the higher molecular weight fo rmulations having a more visibly lubricious (oily) surface and thus more grip slippage and higher standard deviation. As predicted earlier concerning the effect of grip slippage on modulus, the crosshead displacement modulus data, where slippage was an issue, was on average 1.6 ( 0.1) times higher than the extensometer modulus data. As a result, the extensometer data was closer to the true 0.0 0.5 1.0 1.5 2.0 2.5 3.0unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% 1000000 20% 1000000 5% Tetrakis 20% TetrakisSilicone Formulation (wt%; cSt)Elastic Modulus (MPa) Low Strain% Modulus High Strain% Modulus

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38 Figure 3.9: Comparison of elastic moduli values between the different freshly cured silicone elastomer formulations from the two sources of strain values, the laser extensometer and crosshead displacement. All modulus values are from the low strain region. mechanical properties of these formulations. However, on a large-scale view, the changes seen in modulus between the formulations were miniscule and differences should have little influence on the biological adhesion. Aged silicone measurements Since many of the applications for these silicone elastomers could be over a few years lifespan, the mechanical properties of aged silicone formulations were examined. All the formulations were cured at 80C for 2 hours, and then aged in air approximately 128 days at room temperature. The samples were then mechanically tested at room temperature with the laser extensometer. Again the stress-strain curves exhibited two distinct linear portions and so both were examined. The moduli calculated for these aged samples are shown in Figure 3.10. The high strain moduli were 2.7 (0.3) times higher 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% tetrakis 20% tetrakisSilicone Formulation (wt%; cSt)Elastic Modulus (MPa) extensometer crosshead

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39 Figure 3.10: Comparison of elastic moduli values between the different 128-day aged silicone elastomer formulations from both the low strain and high strain regions. All of the strain measurements were collected from a laser extensometer. than the moduli at low strain. Just as in 1-day-old samples, a noticeable trend was as the amount of oil additive was increased, the elastic modulus decreased. However, when the change in molecular weight (or viscosity) was examined, most formulations were not significantly different, except for 10, 20wt% 5000 cSt and 5, 20wt% 1kSt, which had lower moduli and happened to be the visibly oily surfaced samples. The decrease in elastic modulus for these four samples was obviously due to the large migration of the large chains. It was theorized that as the silicone oils chains totally migrated to the surface, there was an increase in free volume left in the network elastomer since the crosslink density should not have changed. Thus the network chains had an increase in chain mobility, which, as discussed earlier, would lower the modulus. 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% 1000000 20% 1000000 5% tetrakis 20% tetrakisSilicone Elastomer Formulation (wt%; cSt)Elastic Modulus (MPa) Low Strain High Strain

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40 Figure 3.11: Comparison of the low strain region elastic moduli values between the different silicone elastomer formulations at two different resting times after cure, 1 day before testing (fresh) and 128 days before testing (aged). When compared to the extensometer moduli values of the 1 day old (fresh) samples, some of the aged samples showed a markedly higher elastic moduli over time at both low and high strains, as shown in Figure 3.11 and 3.12. The same trends that were noticed and discussed with the 1-day-old samples were observable with both of the strain regions. The complete initial curing of the silicone elastomer was previously verified in Table 3.4 where there was minimal difference in moduli between curing at 80C for 2 hours versus 5 hours. The lower molecular weight silicone oil additives increased the modulus in the aged samples as compared to the freshly prepared elastomers. This was attributed to excess curing agent in the network. The slow decomposition of the residual hydride groups caused a postcuring hydrolysis reaction with water in the system where 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% 1000000 20% 1000000 5% tetrakis 20% tetrakisSilicone Elastomer Formulation (wt%; cSt)Elastic Modulus (MPa) Fresh Aged

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41 Figure 3.12: Comparison of the high strain region elastic moduli values between the different silicone elastomer formulations at two different resting times after cure, 1 day before testing (fresh) and 128 days before testing (aged). the hydride groups were converted to silanol groups.79 The increase in silanol groups increased the modulus by either reacting with the silicone backbone or the production of more hydrogen bonding, just as in the case of the silica particles. This effect was more noticeable with the low molecular weight additives than the unmodified elastomer because of the increase in mobility of the water and curing agent molecules, which thus decreased the reaction time. For the case of high molecular weight additives, this postcure reaction was less of an effect than the oil migration, which was just previously discussed. Soaked silicone measurements Since the application for the marine industry would ultimately be in a seawater environment and many laboratory tests are performed in distilled water, these environments were examined for their effect on mechanical properties on the silicone 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% 1000000 20% 1000000 5% Tetrakis 20% TetrakisSilicone Elastomer Formulation (wt%; cSt)Elastic Modulus (MPa) Fresh Aged

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42 elastomer formulations. For each formulation, 2 approximately 14-day-old dogbone specimens were soaked for 3 hours at 80C in an environment, DI wate r or seawater. The calculated elastic moduli values at high strain are provided in Figure 3.13. Overall, there was little difference in either environment as compared to the 1-day-old elastomer samples, previously presented, which was just exposed to air. This led to the conclusion that there was little uptake of water and the elastomer was hydrolytically stable. The low strain modulus values also had little water uptake influence observable. J.J. Kennan et al also observed little significant change in mechanical properties and advancing contact angle when a hydrosilylation silicone elastomer was exposed to saline water at elevated temperatures.80 However, if there was water uptake, it would probably have been more obvious on the effect of elongation at break. The water molecules would have acted as defects and decreased the percent elongation at break, just as previously discussed with the effect of chain ends. However, ultimate tensile properties are not easily reproducible, as seen in these experiments and noted by other researchers, so little physical mechanics could be determined from the elongation at break.47 Surface Energetics Goniometer Another factor affecting adhesion is the surface energetics at an adhesion interface. As a result, the surface energy of the silicone elastomer formulations must be characterized to be able to later compare the adhesion to different formulations. Initially many of the silicone formulations were examined through contact angle measurements from a goniometer. Typically a Zisman plot (cos versus lv) is used to calculate the critical surface tension ( c) of the solid substrate from contact angle measurements.

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43 Figure 3.13: Comparison of the effect of e nvironment (air, DI water, and seawater) on elastic moduli at high strain region for the various freshly cured silicone elastomer formulations. This relationship comes from Young’s interfacial equation of equilibrium, sv – sl = lv cos (2) where sv, sl, and lv are the surface tensions for the surface-vapor, surface-liquid, and liquid-vapor interfaces, respectively, and is the contact angle. Theoretically, as cos approaches 1 ( = 0), the liquid completely wets the surface and then sv = lv. This critical surface tension point is determined by extrapolating a linear regression line from the data of multiple liquids on a Zisman plot. However, since the solid substrate being examined was poly(dimethylsiloxane) elastomer, a low surface energy material, a problem was encountered. There was an induced hydrogen bonding, thus nonlinearity, when polar liquids (usually with a surface 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5unmodified 5% 50 10% 50 20% 50 5% 500 10% 500 20% 500 5% 5000 10% 5000 20% 5000 5% 1000000 20% 1000000 5% Tetrakis 20% TetrakisSilicone Elastomer Formulations (%; cSt)Elastic Modulus (MPa) Fresh (Air) DI Water Soaked Seawater Soaked

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44 tension greater than 50 mN/m) was intr oduced to the surface, such as water.81, 82 Young’s equation does not take intermolecular forces into factor. Thus in order to counteract this nonlinearity, Good proposed a modified Zisman plot of cos versus 1/ lv 1/2 for calculation of critical surface tension of low surface energy materials.53. This relationship comes from the introduction of a geometric mean combining rule (equation 3) for an i-j interface, which allows sl to be eliminated from the equation when combined with Young’s equation and thus results in equation 4.53, 83 ij = i + j – 2 ( ij)1/2 (3) cos = 2 ( sv / lv)1/2 – 1 (4) In equation 4, the cos is directly proportional to 1/ lv 1/2, which thus promotes the modified Zisman plot. An example of th is plot is provided in Figure 3.14 for an unmodified silicone elastomer. From this plot, the surface energy of the silicone elastomer substrate was extracted. There were many assumptions with this type of surface analysis, which were discussed in Chapter 2. Some of the natural variability in data points was attributed in part to some of the assumptions not being completely met. The static contact angles measured with the goniometer for the 5 different liquids are provided in Table 3.5. Some of these angles were previously reported by Wade Wilkerson in his University of Florida master’s thesis.3 This table also provides the calculated surface energies of the various elastomer formulations from a modified Zisman plots. As desired, the surface energies of the formulations were all relatively the same. All the formulations that were not included in this table were also assumed to have relatively similar surface energy values. This should be the case, since the elastomer and the silicone oils all had the same chemical composition. Any small variation of the

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45 Figure 3.14: A modified Zisman plot for an unmodified silicone elastomer from digital capture contact angle measurements. The equation for the linear regression and respective regression correlation are provided on the graph. surface energies was attributed to either the packing difference of the methyl groups on the surface, discussed more with the digital capture contact angles, or standard technique error. Usually, the silicone oils have a tighter packing of the methyl groups on the surface than the silicone elastomer.84 However, this could not be detected with the standard deviations from the goniometer technique. Since all of the elastomer samples were cured against glass, the effect of this particular cure surface was examined. The other option that was presented during preparation was curing the silicone elastomer with no glass cover plate, which left the silicone to cure with an air interface. This technique was rarely performed because the control over film thickness and uniformity was lost. However, when the surface energies were compared, given in Table 3.6, there was a slight, noticeable difference. The elastomer cured facing air had a slightly lowe r surface energy than the glass surface cured elastomer. This small difference was again contributed to the packing (density) of the y = 13.52x 1.916 R2 = 0.954-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000.050.100.150.200.251 / (Surface Energy)^1/2Cos Theta H20 EthGly DMF ACN Prop y = 13.52x 1.916 R2 = 0.954-0.6 -0.4 -0.2 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.000.050.100.150.200.251 / (Surface Energy)^1/2Cos Theta H20 EthGly DMF ACN Prop

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46 Table 3.5: Contact angle measurements and calculated critical surface tension values (modified Zisman plot) from a goniometer of a few silicone elastomer formulations. surface methyl groups due to varying interfacial environments. The closer packing of the methyl groups on the surface, contributed to a lower contact angle.53 Thus the high energy of glass enhanced the motility and packing factor of the methyl groups at the surface during cure.50 Digital capture Another technique useful for measuring contact angle was taking a digital image of the liquid droplet on the surface. With ImageTool 2.0 software, the contact angle could be determined from the digital image. Images could also later be examined and compared. This technique gave a permanent documentation of the surface energetics of the substrate, as seen in Figure 3.15. When compared to digital image measurements, provided in Table 3.7, for an unmodified elastomer substrate, the goniometer surface energy was slightly higher at 23.0 0.4 mN/m, than the digital capture calculated surface energy, 21.5 0.3 mN/m. This difference was attributed to a larger standard deviation on the contact angle measurements for all liquids with the goniometer technique. The goniometer did not have an enlarged view of the surface-liquid interface as the digital images, so there was decreased accuracy with the goniometer technique. This digital 23.7 0.425 46 54 64 109 20% 50 cSt23.3 0.331 44 56 66 110 5% 50 cSt23.0 0.432 48 55 67 109 Unmodified23.1 0.3 23.3 0.2 (mN/m) c26 261-Prop48 48ACN52 55DMF64 65MeI103 20% 5000 cSt 108 5% 5000 cStWater Samples Contact Angles (Degrees) 23.7 0.425 46 54 64 109 20% 50 cSt23.3 0.331 44 56 66 110 5% 50 cSt23.0 0.432 48 55 67 109 Unmodified23.1 0.3 23.3 0.2 (mN/m) c26 261-Prop48 48ACN52 55DMF64 65MeI103 20% 5000 cSt 108 5% 5000 cStWater Samples Contact Angles (Degrees)

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47 Table 3.6: The effect of cure surface on the contact angles and calculated critical surface (from modified Zisman plot) on an unmodified silicone elastomer. technique had the advantage of also being capable of capturing videos in order to examine the effect of time, which will be important in later experiments. Similar sources of error with the goniometer were present with the digital capture method, such as dust, swelling, solvent evaporation, and unsymmetrical droplet, even though they were minimized. For permanent documentation, some of the silicone elastomer formulations were examined with the digital capture system. The contact angles and calculated surface energies are provided in Table 3.7. There was a clear trend that the linear oil additives, which had partially migrated to the surface, gave the substrate a slightly higher surface energy, of approximately 22.6 mN/m, than the unmodified silicone elastomer, which was measured as 21.5 mN/m. This trend can be attributed to the packing density of the methyl groups at the surface interface. Silicone oil has a significantly higher density of methyl groups than the crosslinked silicone elastomer.84 According to Hoernschemeyer, the increase of surface packing resulted in a higher concentration of attractive centers.50,80 This caused more interactions between the so lid and liquid and resulted in a lower contact angle, which was evident with the contact angles provided in Table 3.7. The lowering of contact angles resulted in an increase in calculated surface energy. However, this theory (mN/m)1-Prop ACN DMF MeI Water Cure Surface22.0 0.3 35 48 58 72 105 Air 22.9 0.4 30 45 54 67 103 GlasscContact Angles (Degrees) (mN/m)1-Prop ACN DMF MeI Water Cure Surface22.0 0.3 35 48 58 72 105 Air 22.9 0.4 30 45 54 67 103 GlasscContact Angles (Degrees)

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48 Figure 3.15: Digital images of each of the 5 liquids used to measure contact angles and subsequently calculate surface energy (modified Zisman plot) on an unmodified silicone elastomer. Table 3.7: Average contact angle measurements and calculated critical surface tension (modified Zisman plot) for various silicone elastomer formulations from the digital capture system. Water Propanol ACN DMF EthGly Water Propanol ACN DMF EthGly 21.6 0.235 55 64 88 110 20% Tetrakis22.6 0.227 52 55 80 104 20% 500022.7 0.229 48 57 86 105 20% 50(mN/m)1-Prop ACN DMF Ethgly Water Samples22.5 0.230 52 57 89 107 5% 5021.5 0.338 56 61 88 114 unmodifiedcContact Angles (Degrees) 21.6 0.235 55 64 88 110 20% Tetrakis22.6 0.227 52 55 80 104 20% 500022.7 0.229 48 57 86 105 20% 50(mN/m)1-Prop ACN DMF Ethgly Water Samples22.5 0.230 52 57 89 107 5% 5021.5 0.338 56 61 88 114 unmodifiedcContact Angles (Degrees)

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49 contradicts Zisman’s original theory that an increase in surface packing results in a larger force-field around the surface groups, which should repel the liquids and increase the contact angles (decrease the surface energy).55 All of the data presented here follows Hoernschemeyers’ concepts. In the case of the bulky molecule additive, tetrakis(trimethylsiloxy)silane, a similar surface energy and contact angles as the unmodified was observed, evident in Table 3.7. This was attributed to it having a similar methyl group packing density as the unmodified silicone elastomer. A source of error that was noticeable in the digital images fr om the highly oily surface of 20wt% 5000 cSt, was the deformation of the substrate surface (the silicone oil) by a DMF droplet, as shown in Figure 3.16. The interface deformation from the DMF droplet looks comparable to the drawing of Rusanov’s model for contact angle-induced deformation in Figure 3.16.50. This occurrence goes against the assumption that the solid substrate is rigid thus this was a source of error for this formulation and other oily surfaced substrates. Another issue was that the excessive oil on the surface meant that the contact angles were a liquid-liquid interaction as compared to a solid-liquid interaction. Overall, the surface energies of all of these silicone elastomer formulations only varied minutely and this factor should have little effect on adhesion between comparisons of the various formulations with biological organisms.

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50 Figure 3.16: Digital image of a DMF droplet on a visibly oily surface of a 5wt% 5000 cSt silicone elastomer sample (left). The deformation of the surface caused by the liquid droplet was observable. This relates to Rusanov’s model for contact angle induced surface deformation (right). (adapted from Andrade)50

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51 CHAPTER 4 ENTEROMORPHA SPORE SETTLEMENT/RELEASE ASSAY Introduction Key roles in adhesion to a substrate that must be examined are lubricity and roughness of the substrate surface. In order to evaluate the influence of roughness on adhesion, engineered topographies, of various shapes and dimensions, were produced on silicone elastomer substrates. High fidelity of these patterns was verified during replication processes with White Light Interference Profilometer (WLIP) or Scanning Electron Microscope (SEM). The lubricity of the surface was controlled by the incorporation of various nonfunctional silicone oils into a silicone elastomer substrate. These chemical modifications were characterized and discussed in the previous chapter. These additives allowed for similar surface chemistry and bulk properties between every sample. To evaluate these engineered material factors, Enteromorpha spores were chosen as the target biological organism to examine settlement and adhesion to the engineered substrates. Materials Silicone Elastomer Substrate All samples used in this Enteromorpha spores assay were silicone elastomer substrates prepared from Dow Corning’s Silastic T2 formulation. This is a 2-part, hydrosilylation polymerization reaction with cured properties as discussed in Chapter 3. This particular elastomer was chosen for its transparency (important for the

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52 hydrodynamic flow cell), low-shrinkage (im portant for topography reproduction), and few undisclosed additives (important for chemical modifications). Two linear, trimethylterminated polydimethylsiloxane oils (Gelest, Inc.), of 50 cSt and 5000 cSt viscosity, were separately added to the elastomer resin during mixing of the base resin and curing agent. These chemical modifications provided lubricious surfaces, while generally holding all other factors constant, such as elastic modulus and surface energy. This was all proven in Chapter 3 with characterization of many different nonbonding silicone oil additives in the Silastic T2 silicone elastomer. Epoxy As part of the replication process, a low shrinkage epoxy was necessary for reproduction of engineered topographies The chosen epoxy was EPON 828 Resin, which is a difuntional bisphenol A/epic hlorohydrin derivative. A Jeffamine D-230 hardener (Huntsman), which is a polyoxypropylenediamine with an approximate molecular weight of 230 g/mol (n = 2-3), was used at a ratio of 9.7 g of the resin to 2.7 g of the hardener. The two parts were weighed and mixed thoroughly in order to undergo the reaction shown in Figure 4.1. After completely degassing in vacuum for 30 minutes, the mixture was cured at 60C for 5 hours in order to increase the reaction rate. According to Huntsman, the manufacturer, at room temperature the reaction takes 7 days. The chosen cure temperature was also slightly lower than the deflection temperature of 68C for the cure system (Huntsman product information sheets). Glass Slide Preparation All silicone elastomer samples were coupled to borosilicate glass slides (75mm X 25mm X 1mm) for rigidity. The coupling agent chosen was allyltriethoxysilane (ATS)

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53 Figure 4.1: Epoxy cure reaction. Reaction at each of the NH’s is possible, which leads to a crosslinked matrix. from Gelest, Inc. For the coupling agent solution, a small amount of Aldrich’s Glacial Acetic Acid was needed. In order to prevent silicone elastomer adhesion to the glass molds and silicon wafers, hexamethyldisilazane (HMDS) from Gelest, Inc. was applied to the surfaces. Biological Algae Material Enteromorpha linza fertile algae plants were collected from Wembury beach, UK. The zoospores from these plants were then gathered and prepared for experiments as described by Dr. Maureen Callow in the literature.10 H2NCHCH2CH3OCH2CHNH2n CH3H2C O CH OC CH3CH3O CH O CH2HC O CH2 N H R' H R R CH OH N H R' N N O OH C CH3CH3ONN CHCH2CH3OCH2CH n CH3n=2-3 CHCH2CH3OCH2CH n CH3Epon 828 EpoxyJeffamine D230 Hardener Reaction Mechanism Crosslinked Epoxy H2NCHCH2CH3OCH2CHNH2n CH3H2C O CH OC CH3CH3O CH O CH2HC O CH2 N H R' H R R CH OH N H R' N N O OH C CH3CH3ONN CHCH2CH3OCH2CH n CH3n=2-3 CHCH2CH3OCH2CH n CH3Epon 828 EpoxyJeffamine D230 Hardener Reaction Mechanism Crosslinked Epoxy

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54 Methods Unpatterned Elastomer Samples All of the unpatterned silicone elastomer coated glass slides were prepared as previously described in Chapter 3. These samples were used for examination of the effect of silicone oil additives on Enteromorpha spore settlement and allowed for controls in the topographical studies. Patterned Elastomer Samples All of the engineered topographies were prepared through a few replication steps, beginning with an etched silicon wafer. St andard photolithographic techniques were used to prepare 5m wide ridges separated by 5m, 10m, and 20m wide valleys or 5m wide square pillars separated by 5m, 10m, or 20m wide flat areas on 3in. diameter silicon wafers in the arrangement shown in Figure 4.2. The length of ridge topographies was also varied from 60m, 800m, or 10,000m. Each silicon wafer was first coated with a Clariant AZ1529 positive photoresist and then exposed to UV light source through a patterned mask. Two different pattern depths, 1.5m and 5m, were prepared. Chuck Seegert etched all of the 1.5m deep pattern ed wafers by a reactive ion etching (RIE) process at the University of Florida, while Unaxis (Tampa, Fl) etched the 5m deep patterns through a standard Bosch process. In order to achieve a positive copy of the topographies from the silicon wafer, 3 intermediate replication steps had to be performed, as shown in Figure 4.3, before the patterns could be cast into the final silicone elas tomer slides. The first step was to make a direct copy off of the silicon wafer with solvat ed polystyrene (PS). In a glass flask, 5g of PS was mixed with 30 mL of chloroform. The mixture was allowed to stir and dissolve

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55 Figure 4.2: Placement of topography on the etched silicon wafers. Each large quadrant of patterns had 3 smaller rectangular areas, which corresponded to the 3 different valley/flat-area widths, 5m, 10m, 20m. for at least 2 hours. Meanwhile, the silicon wafer was cleaned with an ethanol wash, dried, and treated with hexamethyldisilazane. A small amount of dissolved PS was then placed on the clean silicon wafer, which was completely leveled for even thickness and allowed to dry for 24 hours. In order to remove the dried PS, the wafer was placed on a block of dry ice. This allowed for removal of the PS film due to the difference in thermal expansion coefficients. The PS was brittle and thin, so attachment to a glass plate for mechanical support was necessary. However, PS was chemically stable and so the backside was argon plasma treated in order to increase adhesion to glass with a quick cure epoxy. The patterned PS film was a negative of the topographies on the wafer, thus the pillars were now divets. 10,000mlong ridges 25mm 5mm 10mm60m long ridges 800m long ridges 5m wide Pillars 10,000mlong ridges 25mm 5mm 10mm60m long ridges 800m long ridges 5m wide Pillars

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56 Figure 4.3: Replication process from patterned silicon wafer to final patterned silicone elastomer coated glass slides. The second replication step involved curing Dow Corning’s Silastic T2 silicone elastomer on the patterned polystyrene in order to obtain a positive of the original topographies. Since the PS was so brittle, conti nuous replication off of this mold was not feasible. As a result, additional replication steps were necessary to obtain a durable mold. Approximately 20g of the silicone base was mixed with 2g of the silicone curing agent. After degassing, a small amount was poured on top of the secured PS replicate. This was allowed to cure in air environment at room temperature. After 24 hours, the silicone elastomer could be demolded. At this point, the patterns could be cut and rearranged to the desired arrangement, as shown in Figure 4.4. This particular PillarsSilicon wafer Indentations Polystyrene Silicone Elastomer Epoxy Silicone SlidePillars Positive Copy Negative Copy Glass Slide PillarsSilicon wafer Indentations Polystyrene Silicone Elastomer Epoxy Silicone SlidePillars Positive Copy Negative Copy Glass Slide

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57 Figure 4.4: Topography layout on the silicone elastomer coated glass slides as compared to the direction of hydrodynamic flow. topography arrangement was chosen for a flow cell apparatus so that the impact of the hydrodynamic flow as compared to the direction of the ridges could be examined. The desired patterns in both spore settlement and release assays were the 10,000m length ridges and the pillars of the 3 different valley widths. Since 2 ridge pattern sets were needed, one for each flow direction, two silicone elastomer replications off of the PS were needed to make one appropriately patterned elastomer mold. The cut patterns were then placed pattern side down on a clean 4”x 4”x 1/12” glass plate. Cured silicone elastomer makes a natural seal with the glass so that when additional silicone mixture was poured on top the patterns were not lost. This glass plate was then set on a larger (8”x 8”x 3/8”) glass plate, which had 4 4mm. thick spacers. Then approximately 120g of silicone base was mixed with 12 g of silicone curing agent and degassed. This mixture was poured on top of the inverted elastomer patterns and pressed to the spacer’s thickness with another clean 8”x 8”x 3/8” glass plate. This mold was allowed to cure at 80C for 1 hour. Upon removal of the cured silicone elastomer from the glass plate, a “well” (or recess) was formed from the thickness of the 4”x 4”x 1/12”glass plate with the patterns facing upward in the middle. FLOWValley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 Valley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 10,000 m length ridges orthogonal to flow 10,000 m length ridges parallel to flow 5 mwide pillers FLOWValley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 Valley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 Valley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 Valley Width [um] Feature Width [um] Key 5 5 5 5 5 5 20 10 5 20 10 5 10,000 m length ridges orthogonal to flow 10,000 m length ridges parallel to flow 5 mwide pillers

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58 The next replication step involved the creation of an epoxy mold from the silicone well just described. The epoxy was first mixed in a weight ratio of 9.7g Epon 828 resin to 2.7g of Jeffamine D230. The mixture was allowed to degas completely under vacuum. The silicone well was placed on a large glass plate so that there were minimal air pockets underneath the silicone elastomer. The silicone elastomer was then placed in a vacuum oven at 80C for 30 minutes to ensure complete cure and to remove residual moisture. The epoxy mixture was then slowly poured into the silicone well up to the brim. After pouring of the epoxy, the mold could not be moved much or ripples would result in the cured epoxy. A 5”x 5”x 1/8” glass plate, which had a spray thermoset mold release dry coating (Stoner, Inc), was slowly lowered on top of the silicone well encapsulating the epoxy. The epoxy does not self-release air bubbles, so care was taken to not introduce any bubbles in this step. The mold was then placed into an oven at 60C for 3 hours to cure. The epoxy was allowed to cool to room temperature before removal from the silicone in order to prevent epoxy deformation. This epoxy had negative patterns, as compared to the silicon wafer, and was used as the repeatable master mold for patterned silicone slide production. In order to reproduce the positive topographies onto silicone elastomer bound to a glass slide, the general mold setup shown in Figure 4.5 was used. The epoxy mold was placed on the bottom glass plate with the patterns facing upwards while an ATS treated glass slide was taped to the upper plate, treated side exposed. The preparation and application of ATS coupling agent was described in detail in Chapter 3. Appropriate metal spacers were attached to the bottom plate that would make an approximately 700m thick elastomer film. The thickness of the epoxy mold, the glass slide, and the

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59 Figure 4.5: Final mold setup for reproduction of a patterned silicone elastomer coated glass slide from an epoxy master mold. double-sided tape had to be considered. The silicone elastomer was mixed in a 10:1 weight ratio of base resin to curing agent. After degassing for 25 minutes, the silicone mixture was poured on top of the epoxy patterns. The top glass plate was then slowly lowered onto the silicone mixture with the glass slide towards the elastomer and centered over the patterns. A 5 lb weight was placed on top of the molds to ensure accurate thickness. This setup was then placed in a 60C oven for 5 hours to increase the crosslinking reaction rate. After cooling and removing the patterned silicone coated slides from the epoxy, the excess silicone elastomer around the glass slides was trimmed off. The film thickness was then measured with a micrometer. The samples that were of appropriate thickness (600-800m) and of good pattern fidelity were then individually placed in polyethylene bags and shipped to Dr. Maureen Callow’s group at the University of Birmingham, UK for Enteromorpha studies. Adhesion Fidelity In order to physically endure the hydrodynamic current in a flow cell as a coating, the silicone elastomer had to be completely adhered to the glass slides. Every sample was tested for adhesion according to ASTM D3806, which was a quick, qualitative hand Metal Spacers Epoxy Mold (Patterns up) Glass slide (ATS treated side up) Metal Spacers Epoxy Mold (Patterns up) Glass slide (ATS treated side up)

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60 pull-test to measure relative adhesion to the glass slides. If the sample coating failed adhesively, the slides were not shipped and discarded. Topography Fidelity In order to quantify the biological growth or release response to surface topography, maintaining the fidelity of the patterns were necessary throughout the replication processes. The first step was to visually examine the smoothness and clarity of every patterned sample. Images of the engineered topographies were then taken periodically with either a JEOL 6400 Scanning Electon Microscope (SEM) or a Wyko NT 1000 (Veeco Metrology software) White Light Interference Profilometer (WLIP). A Au/Pd coating was applied to the SEM samples and examined under 100x to 10,000x magnification in order to detect any significan t defects. WLIP technique also gave a nondestructive, noncontact 3-dimensional image of the patterned surfaces. Spore Settlement Assay Once the silicone elastomer coated glass slide samples arrived at Dr. Callow’s laboratory, they were incubated in filtered, sterilized artificial seawater (Instant Ocean) for 24 hours at 20C. The slides were then placed in individual polystyrene petri dishes and 10 mL of a 2 x 106 spores/mL Enteromorpha zoospore concentration were added to each dish. The zoospores were then incubated on the substrates for 1 hour at 20C in the dark. The nonadhered spores were then washed off of the substrates with artificial seawater and fixed with a 2% glutaraldehyde in seawater solution for 10 minutes. Settlement counts were then taken from images examining the autoflorescence of the chlorophyll.61 On the unpatterned elastomer slides, spores were examined and counted at 1mm intervals down the center of the slide’s l ong-axis for a total of 30 measurements for each slide. Three replicate slides were run for each formulation. For the flat areas in

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61 between the patterns and on the actual topographies, the spores were counted at 0.5mm intervals along the 10mm length of the pattern squares. Exact counting methods and calculations have been previously described by Dr. Callow.61 Spore Removal Assay For examining the adhesion of the Enteromorpha zoospores, the same experimental methods for settlement were performed, as just described, but the elastomer substrates were additionally placed into a flow cell after spore settlement counts were taken. One difference in methods from before was that the spores were only allowed to settle for 45 minutes as compared to the hour-long immersion time. This change was due to the inability of the flow cell to detach the spores if they were allowed to settle more than an hour on any substrate. After settlement, the elastomer coated slides were placed into a flow cell and run at a 55Pa maximum wall velocity for 15 minutes.86 There was obviously no spore fixation step in this experiment. The number of spores that remained attached after exposure to the hydrodynamic flow was then counted as described for the spore settlement assay. The spore densities from before and after the flow cell exposure on the different topographical areas were then compared and presented as percent spore release. Results and Discussion Silicone Elastomer Fidelity Before any topographically modified silicone elastomer samples were sent to Dr. Callow for Enteromorpha spore assays, the fidelity of the patterns through the many replication steps from the original silicon wafer was verified. All samples where the topographies and flat areas were not visua lly acceptable were immediately discarded.

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62 Figure 4.6: SEM and WLIP images of an epoxy mold and silicone elastomer coated glass slide. A) SEM at 800X magnification of patterned epoxy with the negative topographical features (10,000m length ridges at the trans ition point from 5m wide ridges to 10m wide ridges) replicated from a silicone well. B) SEM (350X magnification) of silicone elastomer with 10,000m length ridges at the transition from 5m wide valleys to 10m wide valleys that was replicated off of an epoxy mold from the last step in the replication process. C) WLIP image of the 10,000m length ridges with 5m valleys from the silicon wafer. D) WLIP image of the 60m length ridges with 5m wide ridges from an epoxy replicate. Then random samples were periodically examined with either SEM or WLIP. A few of these images are shown in Figure 4.6. These images showed that there were minimal defects in the samples. It was also noticeable, both visually and microscopically, that some of the silicone oils were partially filling in the patterns as they migrated to the surface. The 20wt% 5000cSt elastomer formulation filled in the patterns to the extent that the patterns disappeared. As a result, an examination of the spores on the patterns C D A50m B100m C D A50m B100m

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63 was not feasible and so this formulation was not included in the topographical experiments. Each sample was also examined for suitable adhesion between the silicone elastomer and the glass slide. A hand pull-test was performed on every sample to ensure that it would withstand the shear stress exerted by the flow cell. Only samples that passed were sent to Dr. Callow’s laboratory for testing. The rest were discarded as unusable. Another factor of the process that was examined before samples were sent was that the polyethylene (PE) bags, which every sample was individually stored and shipped in, had little effect on the surface properties of the elastomer. This factor was examined by measuring contact angles and calculating the surface energy of unpatterned, unmodified silicone elastomer samples that were both stored and not stored in the PE water for an hour to see if their surfaces reoriented differently after exposure to the PE bags. The results of this study are shown in Table 4.1. The contact angles were measured from 5 different liquids by the goni ometer technique discussed in Chapter 3. The surface energy was then calculated from a modified Zisman plot (cos versus1/ lv 1/2) as also previously described in Chapter 3. From comparison of the measurements, there was no significant alteration of the silicone elastomer surface from storage in the PE bags.

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64 Table 4.1: Measured contact angles and calculated surface energies examining the effect of storage and shipment of unmodified silicone elastomer samples in polyethylene bags. Spore Settlement Assay The unpatterned samples of the various elastomer formulations were examined to distinguish whether the oil additives influenced spore settlement. A trend that was slightly observed, from the collected data shown in Figure 4.7, was that as the wt% or molecular weight (or viscosity) increased, the amount of spores that settled was increased. The 5wt% 5000 cSt formulation was the most significantly different from the unmodified elastomer sample. An uncoated glass slide was included in the experiment as a reference point. The glass had noticeably higher settlement than any of the elastomer formulations. Dr. Maureen Callow noted that the spores that settled on the 5000 cSt elastomer samples were visually “embedded” into the oily surface film. This introduced an additional adhesion mechanism than on the unmodified elastomer samples. Even if the spores were not cued to adhere, they got trapped to the surface. This was an influence (mN/m)1-Prop ACN DMF MeI Water Samples 23.0 (0.4) 25 49 51 63 102 No bag (1hr soak) 23.2 (0.2) 30 48 52 66 110 Bag (1hr soak) 23.0 (0.1) 31 50 53 67 112 No bag 23.0 (0.2) 30 48 54 69 108 PE BagcStatic Contact Angle (degrees) (mN/m)1-Prop ACN DMF MeI Water Samples 23.0 (0.4) 25 49 51 63 102 No bag (1hr soak) 23.2 (0.2) 30 48 52 66 110 Bag (1hr soak) 23.0 (0.1) 31 50 53 67 112 No bag 23.0 (0.2) 30 48 54 69 108 PE BagcStatic Contact Angle (degrees)

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65 Figure 4.7: The settlement of Enteromorpha spores on the different unpatterned silicone elastomer formulations. and contributed to the small differences in settlement seen between the different formulations rather than chemical settlement cue variations. As a result, the trend observed correlated with the amount of excess silicone oil on the surface. The topographical and silicone oil additive influences were examined with the spores in order to determine if there was an influence on their settlement cues. The spore settlement densities observed on the different samples are shown in the 3 graphs in Figures 4.8 – 4.10 according to the dimension of the spacings between patterns. The settlement data for the flat areas between both the 1.5 and 5m deep patterns are shown on each graph for comparison. The 5m deep valley patterns had noticeably higher spore settlement for the unmodified, 20% 50cSt, and 5% 5000cSt elastomer formulations at all three valley widths (5, 10, and 20m). However, the 5% 50 cSt formulation at 5m depth valley pattern was not significantly diffe rent from the flat, 1.5m deep, or pillar 0 200 400 600 800 1000 1200 1400glassUM5% 5020% 505% 500020% 5000Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement)2 0 200 400 600 800 1000 1200 1400glassUM5% 5020% 505% 500020% 5000Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement)2

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66 Figure 4.8: The settlement of Enteromorpha spores on 5m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features. Figure 4.9: The settlement of Enteromorpha spores on 10m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features. 0 200 400 600 800 1000 1200 1400 1600 1800 2000012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 5 m wide spacing2 0 200 400 600 800 1000 1200 1400 1600 1800 2000012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 5 m wide spacing2 0 200 400 600 800 1000 1200012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 10 m wide spacing2 0 200 400 600 800 1000 1200012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 10 m wide spacing 0 200 400 600 800 1000 1200012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 10 m wide spacing2

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67 Figure 4.10: The settlement of Enteromorpha spores on 20m wide spaced features on both the topographies of the 2 depths and 2 types of features and on the flat areas between the features. samples at all three valley widths. When the unmodified sample was specifically examined, the 5m deep valleys had approximately 3 times as much spore settlement as the flat area in between the patterns for the 5m wide valleys. The two trends observed previously with the increased settlement on the unpatterned silicone oil modified samples were not as evident in the data from the flat areas in between the patterns in this experiment. Perhaps the topographies slightly influenced the settlement on the flat areas or the topographies highly dominated the settlement cues of the spores. However, this aspect could not be extracted from the data collected as a result of different spore batches being incomparable between experiments because of large statistical differences between each batch from different seasons and tidal cycles.10, 57 Another experiment would need to be designed in order to look at this particular factor. 0 100 200 300 400 500 600 700 800 900012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 20 m wide spacing2 0 100 200 300 400 500 600 700 800 900012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 20 m wide spacing 0 100 200 300 400 500 600 700 800 900012345Silicone Elastomer Formulation (wt%; cSt)Spores / mm (Settlement) 5 micron flat 1.5 micron flat 5 micron deep valleys 5 micron deep pillars 1.5 micron deep valleys 1.5 micron deep pillars Unmod.5% 5020% 505% 5000 20 m wide spacing2

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68 Figure 4.11: Fluorescent microscope images of Enteromorpha spores settled on 3 different 5m deep patterned areas on unmodified silicone elastomer. (A) 5m wide ridges with 5m wide valleys. (B) 5m wide ridges with 10m wide valleys. (C) 5m wide pillars with 5m spacing. (Images courtesy of Dr. Maureen Callow) The spores did, however, choose to settle in the most energetically favorable areas on the samples. The spores settled preferentially in valleys, rather than on top of the 5m ridges, and against the walls pillars, rather than on top of the pillars, as evident in Figure 4.11. Furthermore, the spores settled in the bottom corners of the valleys, between the valley floor and ridge sidewall, rather than on the flat space of th e valleys between the ridges like on the unpatterned samples. Thus there was an active topographical settlement cue for the spores. Originally the 5m topography dimensions were established to be smaller than the size of the spores, which were thought to be approximately 10m, so that AB C 50m 50m 50m AB C 50m 50m 50m

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69 they would be unable to securely attach to the bottom of the valleys. However, from the images in Figure 4.11, the spores were obviously still able to migrate completely into the valleys. Thus studies must progress to smaller dimensions in order to get the unstable and incomplete adhesion underneath the spores. Spore Removal Assay The significance of the silicone oil additives was to increase the lubricity of the surface and thus decrease the force required for removal of biological organisms. This factor was examined by placing the samples in a flow cell with a hydrodynamic wall shear stress of 55MPa for 15 minutes and obtai ning the percent spore release as compared to settlement counts on the same samples. The more densely populated patterns (5m wide pillars/ridges with 5m wide spacing/valleys) were the patterns examined in this experiment because they seemed to influence settlement the most in the previous study. When the flat areas between the 5m deep patterns were examined, most of the elastomer formulations had the same relative spore settlement and removal, as shown in Figure 4.12. However, the 5wt% 5000cSt formulation did show a slightly higher settlement than the other formulations, which followed the trend seen in the previous spore settlement assay. It also had a slightly lower spore density after flow, which demonstrated a slightly higher percent spore removal of 79% than the other formulations that had approximately 67% as compared to the flat areas and pillars for all elastomer formulations. The 5m deep and wide valley pattern spore density data both before and after flow in the parallel direction is shown in Figure 4.13. In this graph, all the silicone oil modified samples had lower initial spore settlement and lower spore densities than the unmodified elastomer formulation of this pattern type. However, the spore removal for all of the formulations was roughly the same. As a result, the percent spore removal was mainly a factor

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70 Figure 4.12: Spore density measurements before and after immersion in a flow cell for the flat regions between the 5m deep patterns of the different elastomer formulations. Figure 4.13: Spore density measurements before and after immersion in a flow cell for the 5m deep and wide valley pattern set parallel to flow direction of the different elastomer formulations 0 100 200 300 400 500 600 700 800 900 1000 Unmod5% 5020% 505% 5000Silicone Elastomer Formulation (wt%; cSt)Spores / mm2 Before Flow After Flow 0 200 400 600 800 1000 1200 1400 1600 1800 2000 Unmod5% 5020% 505% 5000Silicone Elastomer Formulations (wt%; cSt)Spores / mm2 Before Flow After Flow

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71 of the pattern type rather than the oil additives. The increased settlement trend with oil additive seen previously was not observed in this data set. The flat area between the 5m pattern sets definitely had a lower spore density of approximately 200 spores/mm2 on the surface than the pillars and valleys after hydrodynamic flow conditions. The pillars had a much lower spore density after flow than the valleys though. When the percent spore removal measurements were compared for all of the different pattern types and silicone elastomer formulations, additional trends were observed, as seen in Figure 4.14. The flat areas between the patterns had on average a higher spore removal than the other patterns. However, the pillars did have similar removal as the flat areas for the unmodified elastomer and the 20wt% 50cSt formulation. There was also a slightly higher percent removal for the 5wt% 5000cSt formulation with the different pattern types except for the pillars, which had erratic measurements. When the direction of flow with respect to the valleys was examined, the valleys parallel to the direction of flow had a slightly higher percent spore removal than the orthogonal flow direction for all the elastomer formulations. This was expected due to the laminar hydrodynamic flow through the parallel valleys. However, both valley direction sets were significantly lower than the flat and pillar regions for all the formulations. In the same experiment, patterned silicone elastomer coated slides with 1.5m depth features were also examined for spore settlement and release. As shown in Figure 4.15, there was no significant difference between the two depths on pillar patterns or on the flat area between patterns for most of the silicone elastomer formulations. However, there was an increase in settlement for the 5wt% 5000cSt as compared to the other

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72 Figure 4.14: Comparison of the percent spore removal for the different 5m deep pattern sets and the different silicone elastomer formulations. Figure 4.15: Enteromorpha spore settlement densities on the flat areas between the patterns and on the pillars for the different elastomer formulation samples with either 1.5m or 5m deep pattern features. 0 10 20 30 40 50 60 70 80 90 100012345 Silicone Elastomer Formulation (wt%; cSt)% Spore Removal flat parallel valleys orthoganol valleys pillars Unmod. 5% 5020% 505% 5000 0 10 20 30 40 50 60 70 80 90 100012345 Silicone Elastomer Formulation (wt%; cSt)% Spore Removal flat parallel valleys orthoganol valleys pillars Unmod. 5% 5020% 505% 5000 0 100 200 300 400 500 600 700 800 900 1000Unmod.5% 50 20% 505% 5000Unmod.5% 5020% 505% 5000Silicone Elastomer Formulation (wt%; cSt)Spores / mm2 (Settlement) 1.5 microns deep 5 microns deep Flat AreaPillars Area 0 100 200 300 400 500 600 700 800 900 1000Unmod.5% 50 20% 505% 5000Unmod.5% 5020% 505% 5000Silicone Elastomer Formulation (wt%; cSt)Spores / mm2 (Settlement) 1.5 microns deep 5 microns deep Flat AreaPillars Area

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73 formulations on both flat and pillar regions when the pattern depth was increased from 1.5m to 5m. This increase was contributed to the infill of the patterns by the viscous silicone oil. On the 1.5m patterns, the oil provided a relatively flat surface by almost completely filling the patterns, where as the oil did not completely fill in the 5m deep patterns. Thus the impact of the patterns on settlement was not significantly noticeable on the 1.5m deep patterns. When the samples were then exposed to a hydrodynamic flow, additional trends were noticed, as seen in Figure 4.16. The flat area between the 5m deep patterns had consistently more spore release as compared to the flat area between the 1.5m deep patterns for all formulations. It was also evident that the elastomer samples with the oil additives had more removal from the 1.5m deep pillars than the flat areas between the 1.5m deep patterns. The percent spore removal from the flat areas between the 5m deep patterns was always equal to or greater than the 5m pillars and always greater than the 1.5m deep patterned elastomer slides for all formulations. Overall, the 5m deep areas had more spore removal with flow except that some of the data from the pillars was somewhat disordered. The spore removal for the 5m pillars was significantly lower than all the other pattern areas and depths for the 5wt% 50cSt and 5wt% 5000cSt elastomer formulations. Perhaps there was another mechanism interacting with these formulations. These trends could not be compared to the settlement assay on the previous study, unfortunately, due to uncontrollable variations in Enteromorpha spore aggression and motility. Overall, even though some trends were seen in each experiment, no overall statistically significant trends could be claimed possibly due to low spore densities on the samples.

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74 Figure 4.16: Percent Enteromorpha spore removal on the flat areas between the patterns and on the pillars for the different elastomer formulation samples with either 1.5m or 5m deep pattern features after exposure to hydrodynamic flow. 0 10 20 30 40 50 60 70 80 90Unmod.5% 50 20% 505% 5000Unmod.5% 5020% 505% 5000Silicone Elastomer Formulation (wt%; cSt)% Spore Removal 1.5 micron deep 5 micron deep Flat AreaPillars Area 0 10 20 30 40 50 60 70 80 90Unmod.5% 50 20% 505% 5000Unmod.5% 5020% 505% 5000Silicone Elastomer Formulation (wt%; cSt)% Spore Removal 1.5 micron deep 5 micron deep Flat AreaPillars Area

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75 CHAPTER 5 QUATERNARY AMMONIUM SALT CHEMICAL MODIFICATIONS Introduction The chemical species on the surface of a substrate is crucial to the surface energetics and biological response. The ability to control the biological response, depending on the application, would be crucial to many fields. In the case of marine structures, which need a designed coating to prevent biological growth, an antimicrobial chemical species on the surface could greatly influence biological settlement. Biofilm formation is the first step to adhesion of biological organism whether in the body or marine environment. Thus in order to prevent larger organisms from adhering to a surface, the initial stage needs to be blocked. However, it is also necessary for only the targeted organisms to be influenced by the antimicrobial action. Thus the biocide cannot be incorporated freely into a release coating. This release mechanism also provides a short life for the activity and productivity of the coating. As a result, binding the antimicrobial molecule to the surface both prevents toxic release and provides a longer productivity time and is a reasonable solution. Thus, the chemistry behind coupling a quaternary ammonium salt to various substrates, glass, silicone elastomer, and polysulfone, is presented and analyzed as a feasible solution to preventing microbial fouling on a surface.

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76 Materials Biocide The chosen quaternary ammonium salt biocide compound was Dow Corning’s 5700 Antimicrobial Agent (now sold by Aegis Environments). This compound is octadecylaminodimethyltrimethoxysilylpropyl ammonium chloride, structure shown in Figure 5.1, and comes in a 50wt% methanol solution. Some experimental protocols required that this biocidal silane be coupled to a borosilicate glass slide (75mm x 25mm x1mm). For comparison, a long alkyl silane molecule, octadecyltrimethoxysilane, was also coupled to a glass slide. This silane has the chemical structure shown also in Figure 5.1 and was obtained from Gelest, Inc. Silicone Elastomer Substrate As described in Chapter 3, the silicone elastomer chosen was Dow Corning’s Silastic T2 resin. This elastomer is a hydrosilylation cure reaction with platinum Figure 5.1: Chemical structures of Dow Co rning’s 5700 Antimicrobial Agent (top) and a trifunctional alkoxy silane (bottom) with same length unreactive alkyl chain. Si OCH3H3CO OCH3CH2CH2CH2N CH3CH3C17H34CH3ClOctadecylaminodimethyltrimethoxysilylpropyl ammonium chloride n-Octadecyltrimethoxysilane Si OCH3H3CO OCH3C17H34CH3 Si OCH3H3CO OCH3CH2CH2CH2N CH3CH3C17H34CH3ClOctadecylaminodimethyltrimethoxysilylpropyl ammonium chloride n-Octadecyltrimethoxysilane Si OCH3H3CO OCH3C17H34CH3

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77 coordination catalyst. The elastomer was mixed as 10 parts of the base resin with 1 part of the curing agent by weight and then degassed. The mixture was cured on glass slides and adhered with allyltriethoxysilane coupling agent by the methods described in Chapter 3 for the preparation of unpatterned elastomer samples. The elastomer was allowed to cure for 24 hours at room temperature. For coupling of the biocide to the silicone elastomer surface, tetraethoxysilane (TEOS) from Gelest, Inc. was used. Polysulfone Substrate For the coupling of the biocide to a more rigid polymer substrate, UdelTM polysulfone from Union Carbide was used, structure shown in Figure 5.2. The typical properties designated by the manufacturer are shown in Table 5.1. The addition of sulfonate groups to the polysulfone used Fi sher’s purified grade chlorosulfonic acid, 1,2dichloroethane (DCE), and methanol. In order to redissolve the sulfonated polysulfone, HPLC grade N,N, dimethylformamide (DMF) from Aldrich was used as the solvent. A 3-glycidoxypropyltrimethoxysilane (GPS) coupling agent (from Aldrich) was used to adhere the quaternary ammonium salt to the polymer backbone. All densities and molecular weights necessary for molar ratio calculations are provided in Table 5.2. Figure 5.2: Chemical structure of UdelTM poly(ether sulfone), which was used as a more durable substrate as compared to silicone elastomer for chemical modifications. OC CH3CH3O S O On OC CH3CH3O S O On

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78 Table 5.1: Typical material properties for poly(ether sulfone) as provided by the distributor, Amoco Products. Table 5.2: Density and molecular weight values for various chemicals and polymers necessary for the molar ratio calculations in the binding of the quaternary ammonium salt to the polysulfone backbone. 190C Glass Transition Temp. 69 Hardness (Rockwell) (23C) 40-100% Elongation at Break (23C) 0.5-0.7% Linear Shrinkage 171C Deflection Temperature 79 MPa Tensile Strength (23C) 2.7 GPa Elastic Modulus (23C) Result Test 190C Glass Transition Temp. 69 Hardness (Rockwell) (23C) 40-100% Elongation at Break (23C) 0.5-0.7% Linear Shrinkage 171C Deflection Temperature 79 MPa Tensile Strength (23C) 2.7 GPa Elastic Modulus (23C) Result Test 236.34 1.07 (3-Glycidoxypropyl) trimethoxysilane 73.09 0.95 N,N, Dimethylformamide 116.52 1.75 Chlorosulfonic acid 98.96 1.25 1,2 Dichloroethane 442.53 1.24 Polysulfone (repeat unit) Molecular weight (g/mol) Density (g/mL) Chemical 236.34 1.07 (3-Glycidoxypropyl) trimethoxysilane 73.09 0.95 N,N, Dimethylformamide 116.52 1.75 Chlorosulfonic acid 98.96 1.25 1,2 Dichloroethane 442.53 1.24 Polysulfone (repeat unit) Molecular weight (g/mol) Density (g/mL) Chemical

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79 Methods Glass Slide Grafting In order to examine the effect of the two different silanes on interfacial properties, they were adhered to a glass slide. The glass surface was first ultra-cleaned to clear any residue or contaminates from the surface by burning the slide through a clean flame on the Bunsen burner and then soaking them in a 10Normal acetic acid solution for 5 minutes. The slides were allowed to drip dry and then immediately placed in the silane solution. A 1vol% quaternary ammonium silane solution was prepared in DI water and stirred vigorously for 5 minutes. A 1vol% octadecyltrimethoxysilane was prepared in a 95% ethanol aqueous solution, which had been adjusted to a pH of 4-5 with acetic acid. This solution was allowed to mix for 10 minutes to undergo hydrolysis. After the glass slide soaked in the appropriate silane solution for 10 minutes, they were dried at 60C for 1 hour. This method results in a chemical reaction between the silanol groups on the glass surface and the hydrolyzed methoxy groups on the silane molecule for both solutions. Silicone Grafting Since many of the current research for foul-release coatings are with a silicone elastomer substrate, the quaternary ammonium antimicrobial was grafted to the surface of the elastomer. The surfaces of silicone elastomers are known to have a higher concentration of silica particles than the bul k so the biocide was grafted to the surface particles with tetraethoxysilane (TEOS) coupling agent. The TEOS was prepared in a 10vol% solution in 200-proof ethanol. The pH of this solution was reduced to 4-5 with acetic acid in order to accelerate the hydrolysis reaction. The solution was then allowed to mix for 10 minutes. Meanwhile, the cured silicone elastomer (adhered on a glass

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80 slide) was wiped intently with toluene in orde r to clear the surface of residual silicone oil. The elastomer was then immersed in the TEOS solution for 10 minutes. A 1vol % quaternary ammonium solution was prepared in ethanol and allowed to mix for 20 minutes. The silicone elastomer was then removed from the TEOS solution and immersed in the antimicrobial silane solution for 10 minutes. Afterwards, the slide was rinsed with ethanol to remove any excess agents and heated at 60C for 1 hour. The modified elastomer was then immediately cooled to room temperature and tested with digital contact angle images. Polysulfone grafting Two methods were performed for binding the quaternary ammonium compound to the polysulfone, solution and surface reactions. By performing the chemical reactions in solution, the quaternary ammonium biocide was introduced into the bulk polymer, rather than just on the surface. This treatment method could affect the mechanical and morphological properties of the polysulfone. The initial step in this chemical modification was to sulfonate the polysulfone in order to increase its hydrophilicity and directionalize the silanating reaction. First 10wt% of the unmodified polysulfone was dissolv ed in 1,2-dichloroethane. Argon gas was bubbled though the mixture for 2 minutes before sealing the tri-neck flask in order to force any oxygen and moisture out. The polysulfone was allowed to dissolve while stirring for 24 hours. The argon gas line was then replaced back onto the flask creating a continuous gas flow. Chlorosulfonic acid was quickly added in a 1:1 mol ratio to the polysulfone repeat unit. After stirring for 30 minutes, the rest of the polymer was crashed out with methanol and then rinsed 3 more times with a methanol wash. The resulting polymer was then quickly sliced into small pieces, in order to increase the surface area

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81 for solvent to diffuse from, and placed in a vacuum oven at 75C for 24 hours. The obtained polymer is shown in Figure 5.4. The next step was to use a GPS coupling agent to adhere the biocide to the sulfonated polysulfone (SPSF). The SPSF was redissolved (5wt%) in DMF and allowed to stir for 24 hours. Since the sulfonation reaction would have left theoretically one sulfonate group on every polysulfone repeat un it, proven by previous researchers, where the silane was also added in a 1:1 mol ratio to the polysulfone repeat unit.87 The silane was allowed to evenly distribute in the solution by stirring for 5 minutes. Then to accelerate the hydrolysis reaction, water adjusted to a pH of 1.5 with nitric acid was added in a 3:1 molar ratio to the silane. The solution was again allowed to stir for 5 minutes. The quaternary ammonium salt was then added in a 1:1 mol ratio with the glycidoxypropyltrimethoxysilane. This final solution was allowed to stir for 1 hour at which point the polymer was dried on a glass slide or polyimide film at 60C for 24 hours. Before characterization, the films were dried again at 150C for 1 hour in order to prove that all of the DMF solvent was completely evaporated from the polymer. The biocide was also surface grafted onto a dried sulfonated polysulfone (SPSF) film. First the dried sulfonated polysulfone, prepared as previously described, was resolvated into DMF. This solution was then dried at 60C for 4 hours and then 120C under vacuum for 1 hour onto either a section of polyimide (Kapton) film or glass coverslip. Meanwhile, a 1vol% aqueous solution of glycidoxypropyltrimethoxysilane was prepared. The DI water was dropped to a pH of 4-5 by the addition of acetic acid and thoroughly mixed before the silane was added. This silane solution was allowed to mix for 10 minutes before the SPSF film was introduced. Subsequently, the film was

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82 soaked for 5 minutes in the GPS silane solution and then dried for 20 minutes at 60C. A 1vol% aqueous solution of the quaternary ammonium salt was then prepared as previously described with the glass slide gr afting. The GPS silanated SPSF film was then allowed to soak in the biocide solution for 10 minutes. The film was then dried at 60C for 20 minutes and stored under dry conditions until analysis. SEM/EDS In order to examine the morphological properties of the polysulfone chemical modifications, the dry films were examined with a JEOL 6400 Scanning Electron Microscope (SEM). These images were obtained with a 10kV accelerating voltage at magnifications ranging from 100x to 10000x of Au/Pd coated specimens. In order to examine the chemical species exposed on the surface, an Electron Density Spectrum (EDS) analysis was also performed on the modified polysulfone films. FTIR In an attempt to examine the chemical bonds present after each modification step with the solution grafting of the polysulf one, Fourier-transform infra-red (FTIR) spectroscopy analysis was performed. Dry films were prepared on NaCl crystals by drying each solution at 150C for 30 minutes. Each film was analyzed with a Nicolet 20SX spectrometer in transmission mode using 64 scans at a 4 cm-1 resolution. Peaks were analyzed using ACDLabs Freeware 5.0 SpecViewer software. Contact Angle In order to examine the molecular compounds on the surface of the various modified substrates, glass, silicone elastomer, and polysulfone, the technique of contact angle was used. Both static contact angle techniques, sessile drop and captive air bubble were used with Ultrapure water as the liquid interface. These techniques have been

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83 described in detail in previous chapters. An approximate 2L bubble or droplet was used depending on the technique. From the digital capture goniometer setup, the surface response to a water/air/solid interface was obtained by either still pictures or video through the Viviewer software. The contact angles were measured from the still images with UTHSCSA ImageTool 2.0 software. Still frames from the captured digital videos were seized with Adobe Premiere software. Results and Discussion Glass Slide Grafting The interfacial properties of a substrate have shown to have a large impact on biological adhesion. As a result, the surface energetics of the quaternary ammonium salt on a glass surface was examined. First, an untreated glass slide’s effect on contact angle measurements was examined for a comparison. From the captive air bubble technique with ultrapure water, it was evident, as shown from the captured video frames in Figure 5.3, that the air bubble immediately attached to the untreated glass surface. However, when an air bubble was introduced to the water-quaternized glass substrate, by the same method, an interesting phenomenon occurred as compared to an unmodified glass substrate. Trying to press the air pocket to the surface did not induce the bubble to settle, as was the case for the untreated glass surface. As seen in Figure 5.4, the air pocket underwent large deformation when pressed upon the quaternized glass substrate and would not leave the micropipet tip to settle on the surface. After gentle tapping of the micropipet, the air droplet jumped to the surface, but did not immediately settle to the glass surface. Instead the bubble glided across the surface until a favorable location for settlement was found. This observance was captured by a digital video. Some time

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84 progressive still frames of this movement and settlement behavior are shown in Figure 5.5. Sometimes the air bubble would move only a short distance and settle to the surface, as shown in Figure 5.5, and other times it would progress all the way to the edge of the sample where another interface was introduced, as is observed in Figure 5.6. This observation was theorized to be a factor of the cationic ammonium atom strong attraction to the water molecules to the extent that the air droplet did not have enough energy to displace the water molecules. This meant that there was always a minuscule water layer between the air bubble and the substrate so that the air bubble could not settle on the surface. However, due to inconsistencies of the surface silane monolayer, the air pocket was able to eventually find a weakness in the water adhesion strength and settle on the glass surface. While unstable, the air bubble was pulled along the surface by the extremely hydrophobic long alkyl chains, which were attracted to the air rather than the water. The alkyl chains were so long and mobile that they would grab for the air pocket and cause it to move. The interfacial effects of the hydrophilic quaternary ammonium and hydrophobic alkyl chains were noted by Vaidya et al ., but they only reported the lowering of water’s surface tension.88 In order to verify that the lack of settlement of the air bubble was due to the cationic nitrogen, a silane with the same long alkyl chain, octadecyltrimethoxysilane (ODS), was also coupled to a glass slide and the interface examined. Harder et al have previously shown that a self-assembled monolayer of long polyethylene glycol terminated chains on a surface can prevent th e absorption of proteins to the surface. 89 They contributed this to a steric repulsion effect that was due to the mobility, otherwords compressibility, of the monolayer chains and the strong bonding of water to the surface,

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85 Figure 5.3: Single frames from a digital vide o of the placement of an air droplet at the untreated glass (at the top of each image) and water interface with a microliter pipet. This setup is the captive air bubble contact angle technique. Images from A) to D) are in sequential time order. A B C D A A B B C C D D

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86 Figure 5.4: Single frames from a digital video of the attempt to place of an air droplet at the quaternary ammonium surface grafted glass (at the top of each image) and water interface with a microliter pipet. A) Deformation of the air bubble is observed as it is pressed against the modified glass surface. B) The same air bubble transforms completely back to a sphere after deformation observed at modified glass surface. A B A B

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87 Figure 5.5: Single frames from a digital vide o of the placement of an air droplet at the quaternary ammonium surface grafted glass (at the top of each image)and water interface with a microliter pipet. This air bubble only moved a short distance until attaching to surface. Images from A) to D) are in sequential time order. A B C D A B C D

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88 Figure 5.6: Single frames from a digital video of the placement of two air droplets (which split upon release from the micropipet from a single bubble) at the quaternary ammonium surface grafted glass (at the top of each image) and water interface with a microliter pipet. These air bubbles glided completely across the camera view on the modified glass surface until they reached the side of the water holder. Images from A) to D) are in sequential time order. A B C D A B C D

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89 which prevented the protein chains to reach the substrate. They also noted that the thickness of the monolayer and density of coverage were major factors in obtaining the protein resistance, while the chemistry of the termination groups was only slightly influential. However, in the case of ODS bound to the surface, no repulsion of the air droplet from the surface was observed. Figure 5.7 shows a few sequential frames from the digital video capture of the interaction. The air droplet quickly settled onto the ODS modified glass, but the contact angle was definitely larger as compared to the angle made with untreated, observable between Figures 5.7 and 5.3. This was attributed to the hydrophobicity of the alkyl chains making the surface attract the air bubble more than the untreated hydrophilic glass surface. However, the contact angle of the quaternary ammonium modified glass was not significan tly different from the untreated glass surface, also evident in Figures 5.5 and 5.3. This was theorized to be a result of the air bubble still having contact with a similar hydrophilic surface from the cationic amine after displacing the thin water layer. As a result, the repulsion and mobility of the air bubble was definitely concluded to be mainly a factor of the quaternary ammonium species on the surface. Silicone Elastomer Grafting Silicone elastomers are currently studied for their foul-release properties in the marine and biomedical fields. Thus chemical modification of the silicone elastomer into an active, antimicrobial surface is crucial. However, there is one characteristic, which increases these elastomers’ foul-release properties which also makes them hard to surface modify. This property is the high chain mobility, surface rearrangement potential, and low surface energy of the silicone elastomers. As a result, when the quaternary

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90 Figure 5.7: Single frames from a digital vide o of the placement of an air droplet at the octadecyltrimethoxysilane treated glass–water interface with a microliter pipet. Images from A) to D) are in sequential time order. No movement of the air droplet is observed. A B C D A B C D

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91 ammonium salt was coupled onto the silicone elastomer surface, there was no change evident in the water sessile drop contact angles. As shown in Figure 5.8, the measured contact angle for the modified silicone elastomer in air was approximately 111. However, when the silicone was immediately placed and stored in water after the chemical modifications, a captive air bubble in water had an approximately 50 contact angle. This is also evident in Figure 5.8. Thus it was concluded that when the silicone was exposed to air, the surface chains were mobile enough to tuck the quaternary ammonium salt back into the bulk elastomer so that its lowest energy state with the methyl groups on the surface was accomplished. As a result, the method for binding a quaternary ammonium salt to the surface of a silicone elastomer with a tetraethoxysilane coupling agent presented here was successful, but was only surface active when immersed in water. Polysulfone Grafting One large problem with the use of silicone elastomers as a foul-release coating for marine applications is its lack of durability. As a result, polysulfone (PSF) was examined Figure 5.8: Digital image of the water-air-silicone elastomer intersection by two contact angle methods: A) sessile drop and B) captive air bubble. AB AB

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92 as a potential substitute coating. Thus, the binding of the antimicrobial agent onto this polymer was also attempted. The chemical structures for each of the intermediate and the final reaction steps are shown in Figure 5.9. The first step was sulfonating the polysulfone to directionally guide the furt her chemical modifications. The introduction of the sulfonic group onto the polysulfone backbone should increase the polymer’s hydrophilicity and change its solubility. A change in solubility was detected when the SPSF was easily dissolved in ethanol and DMF, whereas PSF was insoluble with these solvents. Evidence for the change in solubility was even detectable during the chemical Figure 5.9: Chemical structures of the modifi ed polysulfone after each chemical reaction. The final result (bottom structure) is of the quaternary ammonium salt bound to the polysulfone. SO3H OC CH3CH3O S O On SPSF-GPS silanated-Quaternized SPSF-GPS silanated Sulfonated PSF (SPSF) O C CH3CH3O S O OnSO2CH2CH OH OSi OCH3OCH3OCH3O O C CH3CH3O S O OnSO2CH2CH OH OSi OCH3OCH3O Si OCH3OCH3CH2CH2CH2N CH3CH3C17H34CH3Cl O SO3H OC CH3CH3O S O On SPSF-GPS silanated-Quaternized SPSF-GPS silanated Sulfonated PSF (SPSF) O C CH3CH3O S O OnSO2CH2CH OH OSi OCH3OCH3OCH3O O C CH3CH3O S O OnSO2CH2CH OH OSi OCH3OCH3O Si OCH3OCH3CH2CH2CH2N CH3CH3C17H34CH3Cl O

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93 reaction when the SPSF crashed out of the 1,2 dichloroethane, unlike the PSF. The change in hydrophilicity was also observed with the SPSF, which visibly wetted a glass surface when cast into a film and the detection of water absorption from humidity. This method for sulfonation of polysulfone was previously demonstrated and verified. 87 For each of the 3 chemical modification steps, FTIR was used to examine whether the appropriate chemical bonds were present. These graphs are shown in Figure 5.11. All of the dried films were neat on a NaCl crystal, but a small peak at ~1700 cm-1 was due to minute amounts of residual solvent. The sulfonation of polysulfone was evident Figure 5.10: FTIR spectras of the different steps in the binding of quaternary ammonium salt to polysulfone. All films were neat on NaCl crystals. A) polysulfone (PSF). B) sulfonated polysulfone (SPSF). C) GPS sila nated SPSF. D) Quaternary ammonium salt grafted to the silanated SPSF. 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.1 0.2 0.3 0.4 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Absorbance A B C D 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.1 0.2 0.3 0.4 Absorbance 4000 3000 2000 1000 Wavenumber (cm-1) 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Absorbance A B C D

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94 by the introduction of the symmetric O=S=O stretching peak of the sulfonate group at ~1028 cm-1, which was not present in the unmodified polysulfone. This peak was also observed in the subsequent two reactions. Then the silanation of GPS onto the sulfonate group was evident by the addition of a peak at 1230 cm-1, which corresponded to the stretching vibration of the C-O bond. This peak was also observed in the subsequent final reaction, but not the previous modifi cations. With the quaternary ammonium bound film, there was a significant increase in the intensity of the CH2 stretch peak at 2925cm-1 relative to the surrounding peaks as compared to the previous reactions. Baudrion et al also observed a large intensity increase of the CH2 stretch peak when the quaternary ammonium salt was bound to poly(vinyl alcohol).90 This increase was a result of the addition of the long alkyl chain on the quaternary ammonium salt to the polymer. However, many of the additional characteristic peaks are not seen on these graphs due to the limitation of the technique to detect bonds at low concentrations, which was probably the case in these films. The lack of large distinguishable characteristic peaks by the quaternary ammonium salt was also observed and declared by Gan et al and Wang et al .66, 91 Observance of the morphology as a result of modifying the polysulfone in bulk solution was inspected with SEM. Visible in the images in Figure 5.11, are rectanglular crystal formations from the glycidoxytrimet hoxysilane which phase segregated from the bulk SPSF. These are large enough to be seen under the natural eye. The same crystals were noticeable in SEM images of the quaternary ammonium treated samples. As a result, the crystal formation was contributed to the GPS silanating of the SPSF chemical reaction step. Probably the concentration of GPS (1vol%) was too high in solution and

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95 Figure 5.11: SEM images of glycidoxypropyltrimethoxysilane silanated SPSF by solution reaction. A) 400X magni fication. B) 3300X magnification. the silane hydrolyzed with other silane molecules to form polymer chains, which then agglomerated with other GPS chains. These crystal formations were also noticeable in the SEM images of the surface-treated quaternary ammonium salt, which further enhances the concept of too high of GPS concentration. Another theory was that the above a critical concentration of ions, the ionic species aggregated to form separate domains.92 An EDS analysis, shown in Figure 5.12, was also performed on the surface of the quaternary ammonium grafted polysulfone sample in order to detect the surface composition. This was to answer the question whether the biocide groups were active on the surface when the chemical modifications were performed in solution. However, no definitive conclusions came from this analysis since the characteristic peak, chlorine, for the biocide composition was obscured behind the gold coating peak. The presence of nitrogen was detected, but could not be defin itely attributed to the quaternary ammonium salt since there was nitrogen also present in atmosphere of the SEM chamber. AB100m10m AB100m10m

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96 Figure 5.12: EDS graph and identifications of chemical atoms present on the surface. In order to study the surface chemistry present after the solution chemical reactions for binding the quaternary ammonium salt to the surface from another direction, contact angles were examined. Observing the surface interfacial properties by this technique are sensitive to even miniscule changes in the surface chemistry, thus the addition of the quaternary ammonium salt should drastically change the observed wettability. As evident in the water droplet images of Figure 5.13, the contact angle observed with sulfonated polysulfone (45) was drastically more hydrophilic than the energetics observed on a quaternary ammonium modified polysulfone surface (contact angle of 73 ). Due to this change in interfacial properties, some quaternary ammonium salt was assumed to be present on the surface. However, the quantity and density of the biocide on the surface has not been studied, which are factors that could affect the biocidal properties of the material. Thus the chemical modifications were achieved, but many properties need to be adjusted to enhance the coatings effectiveness such as a better morphology, surface concentration, and less imperfections in the monolayer coverage of biocide. S S

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97 Figure 5.13: Contact angle images of 2L dr oplets of water on A) sulfonated polysulfone (SPSF) and B) quaternary ammonium salt grafted onto silanated SPSF AB AB

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98 CHAPTER 6 CONCLUSIONS AND FUTURE WORK Conclusions The capability of engineering a material’s surface to either improve biocompatibility or to prevent biofouling is of great importance. There are many material properties that can be manipulated in order to affect settlement and adhesion of biological organisms to a surface: elastic modulus, surface chemistry, surface roughness, and surface lubricity. Silicone elastomers have excellent foul-release properties, but their biofouling properties have the potential to be enhanced. As a result, the lubricity of a hydrosilylation cured silicone elastomer was controlled as an independent variable by the incorporation of nonfunctional poly(dimethylsiloxane) chains (silicone oil) of varying viscosity and quantity to ultimately reduce the force required for organism removal. In order to verify that the surface lubricity of the elastomer was the singl e variable factor in the design, mechanical properties and surface energetics were examined for all of the different elastomer formulations. When the tensile mechanical properties were examined, a relatively constant elastic modulus (as compared to the unmodified elastomer where E = 2.4 MPa at high strain from laser extensometer) was obtained. However, there were many interactions with the silica particles and the elastomer network that could be identified as the cause for slight variations and trends between the different lubricity formulations. There were also two characteristic linear regi ons on the stress-strain curves observed with

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99 all of the formulations, at low and high strain, which were contributed to critical extensions of the molecular weight distribution of PDMS chains in the base elastomer resin. Initially crosshead displacement was used as the strain measurements, but extensive slippage from the grips resulted in more accurate modulus values from a laser extensometer. One clear trend observed was as the quantity of PDMS oil incorporated into the bulk elastomer increased, the elastic modulus slightly decreased. After aging the samples for approximately 128 days, there were significant differences in the observed modulus values. For the low molecular weight PDMS chain additives, there was an increase in elastic modulus after aging. This event was contributed to the excess curing agent present in the elastomer network, which introduced a post curing hydrolysis reactions and thus increase hydrogen bonding or crosslink density. Soaking the different formulations in DI water and simulated s eawater environments did not significantly change the elastic modulus values. Examination of the surface energetics by contact angle measurements also proved that there were minimal differences between the different lubricious elastomer formulations (unmodified elastomer’s c = 21.5 mN/m with digital capture method). A unique method for calculating surface energetics from contact angles for low surface energy materials, a modified Zisman plot (cos versus 1/ 1/2), was revealed and discussed. The necessity for this special method was a result of the induced hydrogen bonding from polar liquids and thus non linearity in the standard Zisman plot. Two methods were used to obtain contact angle measurements, goniometer and digital capture, and then compared. The new digital capture system with computer web camera and software proved to be easier, provide lasting documentation of surface energetics, and give slightly more reproducible angle measurements than the standard goniometer.

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100 Any small deviations from the unmodified silicone elastomer with the PDMS oil additives were contributed to either surface packing density of methyl groups or error produced from induced deformations in the surface created by having a liquid surface. In order to examine the effect that surface lubricity and topography introduce on bioadhesion and settlement, Enteromorpha algae spores were chosen as the biological organism. Their settlement cues and ability to release under hydrodynamic flow was quantitatively observed. A method for reproduction of high quality samples was introduced and then verified with Scanning Electron Microscopy and White Light Interference Microscopy. There were four replications steps from the initially patterned silica wafer to obtain a durable epoxy mold in which to make large numbers of elastomer sample reproductions from. The topographical features that were examined were 10,000m long ridges of 5m width separated by 5, 10 or 20m wide valleys, positioned in both parallel and orthogonal directions to flow, and 5m wide pillars surrounded by either 5,10,or 20m spacing. These pattern sets were examined at both 5m and 1.5m feature depths. Some of the different lubric ious elastomer formulations (0, 5 and 20 wt% with 50 and 5000cSt) with the specific patterns were coupled to a glass slide and sent to Dr. Maureen Callow’s laboratory at the Univ ersity of Birmingham, UK for settlement and removal testing of the Enteromorpha spores. In the spore settlement assay, the elastomer formulations with 5000cSt and 20wt% 50cSt silicone oil additive showed an increase in the quantity of spores that settled as compared to the unmodified elastomer due to the spores becoming embedded and stuck onto the oily surface. This meant that there was an additional factor with those sample sets than just chemical settlement cues by the spores. When topography was introduced into the experiment, the spores

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101 preferential settled around the pillars and in the valleys rather than on top of the features. The 5m deep valley patterns influenced the settlement of the spores the most by significantly increasing the quantity with almost every formulation and almost every valley width. This experiment showed that there was a definite surface topographical settlement cue with the spores. To examine the ease of removal from these different topography and lubricious formulations, samples were first exposed to spore settlement, as in the previous assay, and then introduced to a hydrodynamics flow cell with a 55Pa wall shear stress. Valley patterns parallel to flow only had a slightly higher spore release as compared to valleys positioned orthogonal to flow, but overall the valleys had less removal than any other patterns. The patterns with 5m spacing were examined for spore settlement and removal from directly on the patterns and on the flat areas between pattern sets. The flat areas between pattern sets (5m depth) only saw a significant change between formulations with an increase in percent spore removal with the 5wt% 5000cSt samples. The 20wt% 5000cSt samples could not be quantified due to the excess PDMS oil infilling and completely hiding the patterns. Removal from the flat areas between the patterns (5m depth) was either better than or equal to the removal from the pillars (depending on oil formulation) and was significantly better than the valley patterns. When the 5m deep patterns were compared to the 1.5m deep, the deeper patterns influenced the flat areas to have the most spore removal. No significant trends were observed for spore removal on the pillar patterns between the different depths. As a result, there was definitely an affect on the removal of algae spores from the flat areas between the patterns that was dependent on the pattern dimensions and shape. Thus

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102 topographical features and lubricious surf aces do influence the removal of biological organisms. Binding a biological biocide to a surface could influence the settlement of organisms and when coupled with the topographical cues could ultimately significantly reduce the biofouling properties of a surface. Three chemical methods for binding a quaternary ammonium salt biocide, octadecylaminodimethyltrimethoxysilylpropyl ammonium chloride, to a glass, silicone elastomer, and polysulfone surface has been proposed. When coupled to a glass surface and examined with captive air bubble contact angle technique, the air bubble moved across the surface until it found an inconsistent monolayer coating where it then settled onto the surface. The bubble was unable to settle initially due to the tightly held water molecules to the ionic species and was moved along the surface as a result of the strong hydrophilic alkyl chains grabbing for the air bubble. As a result, the binding of the quaternary ammonium salt to a glass surface through hydrolysis of the alkoxy groups on the biocide was deemed successful. When compared to octadecyltrimethoxysilane coupled onto a glass surface in the same manner, which lacks the cationic ammonium species, an air bubble did not move like with the quaternary ammonium salt, but a decrease the contact angle was observed as a result of the long alkyl chains present. When the quaternary ammonium salt was bound to a hydrosilylation-cured silicone elastomer with tetraethoxysilane, a water droplet on the surface was unable to detect any chemical change. However, when examined in water with an air bubble, the contact angle observed was much lower. Thus it was concluded that the binding reaction was successful, but when exposed to air the silicone elastomer’s highly mobile surface chains were able to reorient and place the methyl groups back on

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103 the surface and pull the biocide into the bulk el astomer. In order to graft the quaternary ammonium salt to polysulfone, there were three chemical reactions to obtain an active biocidal surface: Sulfonation, glycidoxypropyltrimethoxy silanation, and quaternary ammonium salt silanation. The different stages of the reactions were examined and verified with solubility changes, Fourier-transform infrared spectroscopy (FTIR) and contact angle measurements. Thus all of the proposed methods for binding a quaternary ammonium salt biocide to the surface were successful for three different surfaces. Future Work There are many steps that can be taken to further enhance the knowledge of controlling biological interactions on a surface. These developments include improving technique and further experimentation of bi ological cues, some which are listed below. Surface energetics This study has enhanced the visualization and calculation of low surface energy materials, but increase in accuracy could be achieved if the contact angles were calculated from the dimensions of the droplet rather than just by direct observation. Johnson and Dettre describe this technique, which could then be easily applied to the digital images of the droplets on the surface.55 This allows an averaging of all the angles along the perimeter of droplet as compared to single points being examined. Examination of the dynamic contact angles and hysteresis could also be performed with the digital capture system by tilting the surface and observing the movement of the liquid-solid interface.

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104 Topography In order to have the critical topographical dimensions for preventing adhesion of many different type and size biological organi sms, a hierarchical patterning has to be achieved. However, as the topographies become on the nano-scale, the silicone elastomer will have the problem of pattern deformation due to stresses invoked by the biological organisms, as described by Schmidt and von Recum with macrophage cells.6, 93 This occurrence will counteract the instability that the patterns are meant to induce on the biological adhesion. As a result, a more rigi d polymer coating, such as polysulfone, that also has biofouling properties naturally or chemically activated should be used to study the effects of hierarchical patterns on biological adhesion. Spores After samples have been tested for the settlement and adhesion characteristics of Enteromorpha spores, the surfaces should be examined with elemental analysis to observe if any adhesion residue was left. This would provide the chemical composition of the biological adhesive and a chemical mapping of the surface to examine where the adhesive is being placed. It would also provide insight on the adhesion failure mechanism: cohesive or adhesive failure. A cross-sectioned TEM image of the patterns would also provide useful information on whet her the liquid adhesive is flowing all the way down into the bottom of the patterns or if energetics prevents it from wetting. Quaternary Ammonium Salt Grafting There are many advances for enhancing the chemical binding presented here for optimization of its biocidal properties. Further verifications of surface composition could be made with C13 NMR. The surface concentration of biocide can be characterized and optimized by adjusting the ratios during chemical modification. A perfect monolayer

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105 would be desired to tightly hold in the water boundary layer that was discussed here. However the long alkyl chains makes molecular packing on the surface difficult, so the ideal concentration would be need to be unc overed. Changing the counter ion to iodide has also been shown to increase biocidal properties.72 A unique dye, fluoroscein, has been shown to bind to quaternary amino groups only and would be useful to examine the binding concentrations of the biocide on the surfaces.94 A step that correlates with varying the surface concentrations of biocid e would be to assess the effectiveness of these different concentrations by measur ing growth and inhibition of biological organisms, especially bacteria and diatoms in the initial biofilm settlement. Also interesting would be the detection of the repulsion forces from the bound water layer between the quaternary ammonium salt and the air bubble by using the atomic force microscope.95 However, the most critical assay that needs to be performed is a protein absorption experiment on the quaternary ammonium grafted surfaces. Measurement of bovine serum albumin (BSA) on the surface could be simply and accurately performed by a method proposed by Hsiue et al .96

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106 LIST OF REFERENCES 1. Cella J. Advanced Non-Toxic Silicone Fouling-Release Coatings: Environmental Security Technology Certification Program US Department of Defense, 1999:22. 2. Liu D, Maguire RJ, Lau YL, Pacepavicius GJ, Okamura H, Aoyama I. Transformation of the New Antifouling Compound Irgarol 1051 by Phanerochaete Chrysosporium Water Research 1997; 31:2363-2369. 3. Wilkerson WR. Contribution of Modulus to the Contact Guidance of Endothelial Cells on Microtextured Silicone Elastomers. Biomedical Engineering Deparment. Gainesville: University of Florida, 2001:153. 4. Braber ETd, Ruijter JEd, Smits HTJ, Ginsel LA, Recum AFv, Jansen JA. Effect of Parallel Surface Microgrooves and Surface Energy on Cell Growth. Journal of Biomedical Materials Research 1995; 29:511-518. 5. Tan J, Saltzman WM. Topographical Control of Human Neutrophil Motility on Micropatterned Materials with Various Surface Chemistry. Biomaterials 2002; 23:3215-3225. 6. Schmidt JA, Recum AFv. Macrophage Response to Microtextured Silicone. Biomaterials 1992; 13:1059-1069. 7. Dalton BA, Walboomers XF, Dziegielewski M, Evans MDM, Taylor S, Jansen JA, Steele JG. Modulation of Epithelial Tissue and Cell Migration by Microgrooves. Journal of Biomedical Research 2001; 56:195-207. 8. Rovensky YA, Slavnaja IL, Vasiliev JM. Behaviour of Fibroblast-Like Cells on Grooved Surfaces. Experimental Cell Research 1971; 65:193-201. 9. Neff JA, Caldwell KD, Tresco PA. A Novel Method for Surface Modification to Promote Cell Attachment to Hydrophobic Substrates. Journal of Biomedical Materials Research 1998; 40:511-519. 10. Callow ME, Callow JA, Pickett-Heaps JD, Wetherbee R. Primary Adhesion of Enteromorpha (Chlorophyta, Ulvales) Propagules: Quantitative Settlement Studies and Video Microscopy. J ournal of Phycology 1997; 33:938-947.

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109 40. Bennett DR, Statt WH. Primate Absorption and Elimination Balance Studies Including Pulmonary, Urinary, Biliary and Fecel Excretion of t-Butanol, Trimethylsilanol, Dimethylsilanediol, a nd Hexamethyldisiloxane. Toxicology and Applied Pharmacology 1977; 21:445. 41. Company GS. Environmental Fate and Effects of PDMS (In Relation to Release Coatings): GE Answer Center. 42. Garrido L, PFeiderer B, Papisov M, Ackerman JL. In Vivo Degradation of Silicones. Magnetic Resonance in Medicine 1993; 29:839-843. 43. Baney RH, Voight CE, Mentele JW. St ructure-Solubility Relationships in Polymers. In: Harris FW, Seymour RB, eds. New York: Academic Press, 1977:225-232. 44. Hillborg H, Gedde UW. Hydrophobicity Changes in Silicone Rubbers. IEEE Transactions on Dielectrics and Electrical Insulation 1999; 6:703-717. 45. Mark JE. Interpretation of Polymer Properties in Terms of Chain Conformations and Spatial Configurations. Accounts of Chemical Research 1979; 12:49-55. 46. Cochrane H, Lin CS. The Influence of Fumed Silica Properties on the Processing, Curing, and the Reinforcement Properties of Silicone Rubber. Rubber Chemistry and Technology 1993; 66:48-60. 47. Baker D, Charlesby A, Morris J. Reinforcement of Silicone Elastomer by Fine Particles. Polymer 1968; 9:437-448. 48. Boonstra BB, Cochrane H, Dannenberg EM. Reinforcement of Silicone Rubber by Particulate Silica. Rubber Chemistry and Technology 1975; 48:558-576. 49. Polmanteer KE, Lentz CW. Reinforcement Studies--Effect of Silica Structure on Properties and Crosslink Density. Rubber Chemistry and Technology 1975; 48:795-809. 50. Andrade JD. Surface and Interfacial Aspects of Biomedical Polymers. Vol. 1. New York: Plenium Press, 1985. 51. Johnson KL, Kendall K, Roberts AD. Surface Energy and the Contact of Elastic Solids. Proc. B. Soc. Lond. A. 1971; 324:301-313. 52. Fox HW, Zisman WA. The Spreading of Liquids on Low-Energy Surfaces. III. Hydrocarbon Surfaces. Journal of Colloid and Interface Science 1952; 7:428-442. 53. Good RJ. Contact Angle, Wetting, and Adhe sion: A Critical Review. Journal of Adhesion Science Technology 1992; 6:1269-1302.

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110 54. Johnson RE. Conflicts Between Gibbsian Thermodynamics and Recent Treatments of Interfacial Energies in So lid-Liquid-Vapor Systems. The Journal of Physical Chemistry 1959; 63:1655-1658. 55. Johnson RE, Dettre RH. Wettability and Contact Angles. Surface and Colloid Science 1969; 2:85-153. 56. Lobban CS, Harrison PJ. Seaweed Ecology and Physiology. New York: Cambridge University Press, 1994. 57. Evans LV, Hoagland KD. Algal Biofouling. Studies in Environmental Science. Vol. 28. New York: Elsevier, 1986. 58. Callow ME, Callow JA. Substratum Location and Zoospore Behaviour in the Fouling Alga Enteromorpha Biofouling 2000; 15:49-56. 59. Bold HC, Wynne MJ. Introduction to the Algae: Structure and Reproduction. Englewood Cliffs, New Jersey: Prentice-Hall, Inc., 1978. 60. Callow ME, Callow JA, Ista LK, Coleman SE, Nolasco AC, Lopez GP. Use of Self-Assembled Monolayers of Different Wettabilities to Study Surface Selection and Primary Adhesion Processes of Green Algal ( Enteromorpha ) Zoospores. Applied and Envionmental Microbiology 2000; 66:3249-3254. 61. Callow ME, Jennings AR, Brennan AB, Seegert CA, Gibson A, Wilson LH, Baney R, et al. Microtopographic Cues fo r Settlement of Zoospores of the Green Fouling Alga Enteromorpha Biofouling 2002; 18:229-236. 62. Fletcher RL, Callow ME. The Settlement, Attachment and Establishment of Marine Algal Spores. British Phycology Journal. 1992; 27:303-329. 63. Nurdin N, Helary G, Sauvet G. Biocidal Polymers Active by Contact. III. Aging of Biocidal Polyurethane Coatings in Water. Journal of Applied Polymer Science 1993; 50:671-678. 64. Hazziza-Laskar J, Helary G, Sauvet G. Biocidal Polymers Active by Contact. IV. Polyurethanes Based on Polysiloxanes with Pendent Primary Alcohols and Quaternary Ammonium Groups. Journal of Applied Polymer Science 1995; 58:77-84. 65. Bukovsky M, Mlynarcik D, Ondrackova V. Immunomodulatory Activity of Amphiphilic Antimicrobials on Mouse Macrophages. International Journal of Immunopharmacology 1996; 18:423-6. 66. Wang HH, Lin MS. Biocidal Polyurethane and its Antibacterial Properties. Journal of Polymer Research Tawain 1998; 5:177-186.

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111 67. Clarkson N, Evans IV. Raft Trial Experiments to Investigate the Antifouling Potential of Silicone Elastomer Polymers with Added Biocide. Biofouling 1995; 9:129-143. 68. Grapski JA, Cooper SL. Synthesis and Characterization of Non-Leaching Biocidal Polyurethanes. Biomaterials 2001; 22:2239-2246. 69. Kenawy E-R. Biologically Active Polymers. IV. Synthesis and Antimicrobial Activity of Polymers Containing 8-Hydroxyquinoline Moiety. Journal of Applied Polymer Science 2001; 82:1364-1374. 70. Augusta S, Gruber HF, Streichsbier F. Synthesis and Antibacterial Activity of Immobilized Quaternary Ammonium Salts. Journal of Applied Polymer Science 1994; 53:1149-1163. 71. Denyer SP. Mechanisms of Action of Antibacterial Biocides. International Biodeterioration & Biodegradation 1995; 36:227-245. 72. Baudrion F, Perichaud A, Vacelet E. Infl uence of Concentration and Structure of Quaternary Ammonium Salts on their Antifouling Efficiency Tested against a Community of Marine Bacteria. Biofouling 2000; 14:317-331. 73. Denyer SP, Maillard JY. Cellular Impermeability and Uptake of Biocides and Antibiotics in Gram-Negative Bacteria. Journal of Applied Microbiology Symposium Supplement 2002; 92:35S-45S. 74. Russell AD. Mechanism of Bacterial Resistance to Biocides. International Biodeterioration & Biodegradation 1995; 36:247-265. 75. Wynne KJ, Swain GW, Fox RB, Bullock S, Uilk J. Two Silicone Nontoxic Fouling Release Coatings: Hydrosilation Cured PDMS and CaCo3 Filled, Ethoxysiloxane Cured RTV11. Biofouling 2000; 16:277-288. 76. Homma H, Kuroyagi T, Izumi K, Mirley CL, Ronzello J, Boggs SA. Diffusion of Low Molecular Weight Siloxane From Bulk to Surface. IEEE Transactions on Dielectrics and Electrical Insulation 1999; 6:370-375. 77. Andrady AL, Llorente MA, Mark JE. Model Networks of End-linked Polydimethylsiloxane Chains. IX. Gaussian, Non-Gaussian, and Ultimate Properties of Trifuntional Networks. Journal of Physical Chemistry. 1980; 73:1439-1445. 78. Kwan KS, Harrington DA, Moore PA, Hahn JR, Degroot JV, Burns GT. Synthesis and Use of Colloidal Silica for Reinforcement in Silicone Elastomers. Rubber Chemistry and Technology 2001; 74:630-644.

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112 79. Karlsson A, Singh SK, Albertsson A-C. Controlled Destruction of Residual Crosslinker in a Silicone Elastomer for Drug Eelivery. Journal of Applied Polymer Science 2002; 84:2254-2264. 80. Kennan JJ, Peters YA, Swarthout DE, Owen MJ, Namkanisorn A, Chaudhury MK. Effect of Saline Exposure on the Su rface and Bulk Properties of Medical Grade Silicone Elastomers. Journal of Biomedical Materials Research 1997; 36:487-497. 81. Schmidt JA, Recum AFv. Surface Characterization of Microtextured Silicone. Biomaterials 1992; 13:675-681. 82. Shafrin EG, Zisman WA. Constitutive Relations in the Wetting of Low Surface Energy Surfaces and the Theory of the Retraction Method of Preparing Monolayers. The Journal of Physical Chemistry 1960; 64:519-524. 83. Girifalco LA, Good RJ. A Theory for the Estimation of Surface and Interfacial Energies. I. Derivation and Application to Interfacial Tension. The Journal of Physical Chemistry 1957; 61:904-909. 84. Gedde UW, Hallebuych A, Hedenqvist M. Sorption of Low Molar Mass Silicones in Silicone Elastomers. Polymer Engineering and Science 1996; 36:2077-2082. 85. Hoernschemeyer D. The Relationship of Contact Angles to the Composition and Morphology of the Surface. The Journal of Physical Chemistry 1966; 70:26282633. 86. Shultz MP, Finlay JA, Callow ME, Callow JA. A Turbulent Channel Flow Apparatus for the Determination of the Adhesion Strength of Microfouling Organisms. Biofouling 2000; 15:243-251. 87. Macdonald J. Interfacial Design for a Bioactive Composite. Materials Science and Engineering. Gainesville, Fl: University of Florida, 2001. 88. Vaidya A, Chaudhury M. Synthesis of Surface Active Quaternary Amino Polyfluorosiloxanes. Journal of Applied Polymer Science 2000; 77:1700-1708. 89. Harder P, Grunze M, Dahint R, Whitesides GM, Laibinis PE. Molecular Conformation in Oligo(ethylene glycol)-Terminated Self-assembled Monolayers on Gold and Silver Surfaces Determines their Ability to Resist Protein Adsorption. Journal of Physical Chemistry B 1998; 102:426-436. 90. Baudrion F, Perichaud A, Coen S. Chem ical Modification of Hydroxyl Functions: Introduction of Hydrolyzable Ester Function and Bactericidal Quaternary Ammonium Groups. Journal of Applied Polymer Science 1998; 70:2657-2666.

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113 91. Gan LH, Deen GR, Gan YY, Chew CH. S ynthesis and Properties of Piperazine Derivatives and their Quaternary Ammonium Amphiphilic Salts. Journal of Colloid and Interface Science 1996; 183:329-338. 92. Huang Z, Yu Y, Huang Y. Ion Aggregation in the Polysiloxane Ionomers Bearing Pendent Quaternary Ammonium Groups. Journal of Applied Polymer Science 2002; 83:3099-3104. 93. Balaban NQ, Schwarz US, Riveline D, Goichberg P, Tzur G, Sabanay L, Mahalu D, et al. Force and Focal Adhesion A ssembly: A Close Relationship Studied Using Elastic Micropatterned Substrates. Nature Cell Biology 2001; 3:466-472. 94. Tiller JC, Liao C-J, Lewis K, Klibanov AM. Designing Surface that Kill Bacteria on Contact. PNAS 2001; 98:5981-5985. 95. Morra M. On the Molecular Basis of Fou ling Resistance. Journal of. Biomaterials. Science. Polymer Edn. 2000; 11:547-569. 96. Hsiue G-H, Lee S-D, Chang PC-T, Kao C-Y. Surface Characterization and Biological Properties Study of Silicone Rubber Membrane Grafted with Phospholipid as Biomaterial via Plasma Induced Graft Polymerization. Journal of Biomedical Materials Research 1998; 42:134-147.

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114 BIOGRAPHICAL SKETCH Amy Louise Gibson was born on March 14, 1978, in Gainesville, FL, to David and Betty Gibson. She lived her life in the small, serene town of Evinston, FL, with her two younger brothers, Evin and Will. Some of her early fond memories were of attending engineering seminars and meetings with her father to spend quality time. This led to early interest in the math and science fields. From an early age she was introduced to the wonderful hobby of music from her parents. Starting with piano, vocal, and guitar lessons throughout elementary school, her love for music grew even more as she started playing the trumpet in middle school. These ambitions led her to enthrall herself in marching, symphonic, and jazz bands, along with group and solo ensembles. These hobbies taught her how to be a leader and to strive for excellence. After graduating from Eastside High School in 1996, she attended the University of Florida where she studied materials science and engineering. Again, she continued her musical diversion through participating in the University of Florida’s Pride of the Sunshine Marching Band for her first 2 years. As an undergraduate, she became an active participant and leader in the student chapter of The Metals, Minerals, and Materials professional society. During this time, she became especially interested in polymer materials, which later became her specialty. While doing her senior research with Bob Hadba, under Dr. Eugene Goldberg, on drug-delivery microspheres, she became attracted to the field of biomaterials. She graduated with honors with her bachelor degree in May 2000.

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115 With peaked interest in biomedical applied polymer materials, she decided to continue her education at the University of Florida, under the supervision of Dr. Anthony Brennan, in the Materials Science and Engineering Department. This experience gave her the confidence and knowledge to be successful in industry. She not only made valuable friends, but also learned a lot about her own interactions and people-skills with working in a large group. She found that her strong organizational skills gave her a distinct place in the group early on. After receiving her Master of Science degree in fall 2002, she will take another career leap into working as a product design engineer in the biomedical industry field in Miami Lakes, FL There she hopes to again make a valuable, distinct place for herself and eventually use her organizational and people skills to work towards a managerial position. As a child, she dreamed of being an astronaut all the way to becoming a radiologist (a medical field which she thought at the time did not require much blood). Somehow this degree has turned out to be a combination of both her engineering and medical field interests (w ithout the blood contact) that she always desired.


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Title: Chemical modification of silicone elastomer for controlled biological interactions on microtextured surfaces
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Creator: Gibson, Amy Louise ( Author, Primary )
Publication Date: 2002
Copyright Date: 2002

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Material Information

Title: Chemical modification of silicone elastomer for controlled biological interactions on microtextured surfaces
Physical Description: Mixed Material
Creator: Gibson, Amy Louise ( Author, Primary )
Publication Date: 2002
Copyright Date: 2002

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Source Institution: University of Florida
Holding Location: University of Florida
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CHEMICAL MODIFICATION OF SILICONE ELASTOMER FOR CONTROLLED
BIOLOGICAL INTERACTIONS ON MICROTEXTURED SURFACES


















By

AMY LOUISE GIBSON


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2002




























Copyright 2002

by

AMY LOUISE GIBSON



























This thesis is dedicated to my loving parents David and Betty Gibson for their continuous
love and support throughout my educational career.















ACKNOWLEDGMENTS


I would, first of all, like to acknowledge my advisor and committee chairman, Dr.

Anthony Brennan, for the confidence, motivation, and valuable advice that he has

provided me through my graduate years at the University of Florida. My gratitude also

goes to my other committee members, Dr. Ronald Baney and Dr. Christopher Batich,

who have provided me with insightful direction and inspiration, especially with their

silicone chemistry and interfacial behavior expertise.

Of course, the help and advice from my fellow group members through this

strenuous process cannot go unnoted. I would like to thank the past group members,

Jeanne Macdonald, Jeremy Mehlem, Wade Wilkerson, and Chuck Seegert, who provided

me with the initial direction and enthusiasm for polymer research. My deepest gratitude

and appreciation go to the group members who have seen me through this entire process:

Clay Bohn, Leslie Wilson, Adam Feinberg, Brian Hatcher, and Nikhil Kothurkar.

Without their support, expertise, and friendship this whole process would not have been

possible. The recent addition of Rob Sparkman, Thomas Estes, Michele Carmen, and

Kiran Karve to the research laboratories has been instrumental to my research and I give

them my thanks for all of their hard work and dedication. Special thanks also go to the

members of Dr.Eugene Goldberg's group, past and present, who have given me

knowledgeable advice and assistance in my research and life through the years.









Assistance was provided by Dr. Maureen Callow, Dr. Jim Callow, and Dr. John

Finley at the University of Birmingham, UK, by performing all of the Enteromorpha

spore settlement and release assays. All of their hard work and flexibility were much

appreciated and provided me with the opportunity for a significant collaboration.

My biggest thanks go to my family and friends whose emotional support and love

have driven me through my academic years. My close friends, Athena West and Ginny

Miliusis, deserve much thanks and appreciation for keeping my life exciting and for

pushing me through the hard times. My gratitude and love also go to Brett Almond, who

gave me the confidence and motivation to overcome any hurdle that I reached. Lastly, I

am completely and forever indebted to my family who has given me nothing but love and

support. They should be credited with the success that I have had through my life and

educational path.
















TABLE OF CONTENTS

page

A C K N O W L E D G M E N T S ................................................................................................. iv

L IST O F T A B L E S ...................................................................... .. ... .. .......... ix

LIST OF FIGURES ......................... .......... ... ....................x

A BSTR A C T ......... .............. ................................................ ... .. ....... .. xv

CHAPTER

1 IN TR OD U CTION ....................... ........................ ........... ..... .. 1

2 BACK GROUND ..................... .......................... .. .. ... ................ 5

B io fo u lin g .................................................................................................... . 5
A dh esion ...................................................................................... . 6
M ech an ics .......................................................... ........ ...... 6
Coupling Agents ........................................ 8
R oughness Factor ......................................... 9
Silicones .................................................. 9
Introdu action .................................................................................. . 9
M echanical Properties............................. .............. 10
Surface Energy/Chem istry .............................................................. ............. 13
Enteromorpha Algae Spores ............... ......................... 16
Quaternary Ammonium Salts .......................... ........ ................... 18
Introduction ................ ... ......... .................. 18
Biocidal Mechanism .. ... ........ .................. .............. 18


3 SILICONE ELASTOMER CHEMICAL MODIFICATION AND
CHARACTERIZATION ..... ......... .. ........ ........ ........20

Introduction ............................ ............. ...... 20
M a te ria ls ................................................................... 2 1
Silicone Elastomer ...... ......... ......... .......... ......... 21
N onfunctional A dditives ...................... ....... ......... .. .............. .............. 22
Surface Energy Liquids............................. .............. 22
M e th o d s ....................................................................................................................... 2 4









Sam ple P reparation .... .... ........................................................ ..... ............. 24
M echanical Properties..................................................... .......................... 26
Surface Energetics ..................................................... .............. 27
Results and D discussion ................ ............... .. ........ .............. 29
M echanical Properties.................................................... ........................... 29
Crosshead displacement measurements.............................. .............. 29
Laser extensometer measurements .................................................. ........ 32
A ged silicone m easurem ents................................................. .... ................ 38
Soaked silicone measurements .................... ........................ ........... 41
Surface Energetics ..................................................... .............. 42
Goniom eter ......................................... 42
D digital capture............................. .............. 46


4 ENTEROMORPHA SPORE SETTLEMENT/RELEASE ASSAY................................51

Intro du action ..................................................... 5 1
M ateria ls ..................................................................... 5 1
Silicone Elastom er Substrate .......................................... .......................... 51
E p o x y ................................................................................................... 5 2
G lass Slide Preparation ............................................ .. .. ..... ............. 52
Biological Algae M material ...................................... ........... ..... .............. 53
M methods .................................... ......... ..... 54
Unpatterned Elastomer Samples ....................................... .............. 54
P atterned E lastom er Sam ples...................................................... .... .. .............. 54
A dhesion Fidelity ............. .. ... ..... ...... ............ ...... ........ .. ............ .. 59
T opography Fidelity............................... ............ ........................ ................ .. 60
Sp ore S ettlem ent A ssay ........................................................................................ 60
Spore R em oval A ssay ...................... .. .. ......... .. ........................ .............. 61
Results and Discussion .......... ............................................ .... ............. 61
Silicone E lastom er Fidelity ...................... .. .. ............................... .............. 61
Sp ore S ettlem ent A ssay ........................................................................................ 64
Spore R em oval A ssay ...................... .. .. ......... .. ........................ .............. 69


5 QUATERNARY AMMONIUM SALT CHEMICAL MODIFICATIONS ................75

In tro du ctio n ................................................. ...... ......... ...... 7 5
M materials .................... ............................... ........ ......... ...... 76
Biocide ....................... ....... ........................... .... ...... ......... 76
Silicone Elastom er Substrate ........................................ .......................... 76
P oly sulfone Sub strate...................................... .......................... ................ .. 77
Methods .................................................. .................... 79
G lass Slide G rafting ................................................. ........ .. ......... 79
Silicone G rafting .................. ............................................. ............ .. 79
Polysulfone grafting................................ .......... ........................ ................ .. 80
SEM/EDS .......................... ............... 82









F T IR ...................................................................................................................... 8 2
Contact Angle ......................................... 82
Results and Discussion .......... ............................................ .... ............. 83
G lass Slide G rafting ............................................... .. ....... .. ......... .. 83
Silicone E lastom er G rafting ........................................................ ... ................. 89
Polysulfone Grafting ........... .. ............... ............... .. ...... .. .......... 91


6 CONCLUSIONS AND FUTURE WORK ........................................... ...............98

C onclu sions............................. .................... 98
Future Work ......................................... 103
Surface energetic .................. ............. .... ........ ......... ........ 103
T o p o g ra p h y ................................................................ ................................. 1 0 4
S p o re s ............................. .... ............................................................... ...... 1 0 4
Quaternary A m m onium Salt Grafting ...................................... .................... 104


LIST OF REFEREN CES .................................................................. ............... 106

B IO G R A PH ICA L SK ETCH ........... ..................................................... .....................114
































viii















LIST OF TABLES


Table Page

3.1 Typical properties of Dow Coming's Silastic T2 addition cured silicone elastomer.
(From Dow Coming's product information sheets) .............................................. 22

3.2 Chemical structures and properties of the nonfunctional silicone oil additives from
Gelest, Inc. (Information obtained from Gelest, Inc. catalog)............................. 23

3.3 List of chemicals used for contact angle measurements with their corresponding
liquid surface tensions. ...................... .. .......................... .. ........ ................23

3.4 Effect of cure time at 800C for 5wt% 50 cSt silicone oil modified silicone
elastom er. (n = 10) .................................................... ................ 32

3.5 Contact angle measurements and calculated critical surface tension values
(modified Zisman plot) from a goniometer of a few silicone elastomer formulations. 46

3.6 The effect of cure surface on the contact angles and calculated critical surface (from
modified Zisman plot) on an unmodified silicone elastomer. .....................................47

3.7 Average contact angle measurements and calculated critical surface tension
(modified Zisman plot) for various silicone elastomer formulations from the digital
capture sy stem ..................................................... ........ ...... 48

4.1 Measured contact angles and calculated surface energies examining the effect of
storage and shipment of unmodified silicone elastomer samples in polyethylene
b ag s ......................................................... ..................................64

5.1 Typical material properties for poly(ether sulfone) as provided by the distributor,
A m oco P rodu cts........................................................................................... 7 8

5.2 Density and molecular weight values for various chemicals and polymers necessary
for the molar ratio calculations in the binding of the quaternary ammonium salt to
the polysulfone backbone. ................................................ ................................ 78















LIST OF FIGURES


Figure Page

2.1 Chemical species on the surface of fumed silica and trimethylsilylated silica
particles. .......... ............... ... ............. ....................... 12

2.2 The chemical reaction for the trimethylsilylation of a fumed silica particle by
addition of hexam ethyldisilazane. .............................................................. ... ..... 12

2.3 Setup and labeled interfaces/contact angle for both the (A) sessile drop technique
and (B) captive air bubble technique. ........................................ ....................... 13

2.4 Energy balance approach at the point of triple phase (liquid-solid-vapor)
intersection for the derivation of Young's equation. The forces are labeled
previously in Figure 2.3 .......................................... .. .... .. ............... 14

3.1 Dow Coming's Silastic T2 silicone elastomer crosslinking reaction.......................21

3.2 Mold setup for preparation of silicone elastomer films (left) and silicone elastomer
coated glass slides (right)...................................................................... .................. 2 5

3.3 Digital picture of the microscope goniometer setup (left) and the digital capture
setup (right) for contact angle measurements. .................................... ...............28

3.4 Computer screen image capture of the ImageTool software for measuring contact
angles from digital images of the liquid droplets. The image is of a 21tL droplet of
water on an unmodified silicone elastomer substrate. .............................................29

3.5 Effect of nonfunctional additives at different amounts on the elastic modulus
(calculated between 20-50% strain) for a silicone elastomer. Strain measurements
were made from crosshead displacement. ........................................ ............... 30

3.6 Stress-strain curve for unmodified silicone elastomer with strain measurements
made from crosshead displacement. Notice the 2 different linear segments, which
correspond to different moduli values at low strain versus high strain. Performed at
room temperature with a lin. gage length and 2 in/min crosshead displacement........31









3.7 Stress-strain curve for an unmodified silicone elastomer with strain measurements
made from a laser extensometer. Two different slope regions are evident between
high strain and low strain corresponding to 2 different moduli values. .....................34

3.8 Comparison of elastic moduli values between the different freshly cured silicone
elastomer formulations from both the low strain and high strain regions. All of the
strain measurements were collected from a laser extensometer.................................37

3.9 Comparison of elastic moduli values between the different freshly cured silicone
elastomer formulations from the two sources of strain values, the laser extensometer
and crosshead displacement. All modulus values are from the low strain region.......38

3.10 Comparison of elastic moduli values between the different 128-day aged silicone
elastomer formulations from both the low strain and high strain regions. All of the
strain measurements were collected from a laser extensometer.................................39

3.11 Comparison of the low strain region elastic moduli values between the different
silicone elastomer formulations at two different resting times after cure, 1 day
before testing (fresh) and 128 days before testing (aged) ...........................................40

3.12 Comparison of the high strain region elastic moduli values between the different
silicone elastomer formulations at two different resting times after cure, 1 day
before testing (fresh) and 128 days before testing (aged) ...........................................41

3.13 Comparison of the effect of environment (air, DI water, and seawater) on elastic
moduli at high strain region for the various freshly cured silicone elastomer
form ulations. .............................................................................43

3.14 A modified Zisman plot for an unmodified silicone elastomer from digital capture
contact angle measurements. The equation for the linear regression and respective
regression correlation are provided on the graph ........................................... ............. 45

3.15 Digital images of each of the 5 liquids used to measure contact angles and
subsequently calculate surface energy (modified Zisman plot) on an unmodified
silicone elastom er. .................................................... ................. 48

3.16 Digital image of a DMF droplet on a visibly oily surface of a 5wt% 5000 cSt
silicone elastomer sample (left). The deformation of the surface caused by the
liquid droplet was observable. This relates to Rusanov's model for contact angle
induced surface deformation (right). (adapted from Andrade)50...............................50

4.1 Epoxy cure reaction. Reaction at each of the NH's is possible, which leads to a
crosslinked m atrix.................................. ............... ......... ..... ............. 53

4.2 Placement of topography on the etched silicon wafers. Each large quadrant of
patterns had 3 smaller rectangular areas, which corresponded to the 3 different
valley/flat-area widths, 5pm 10pm 20 m ...................................... ............... 55









4.3 Replication process from patterned silicon wafer to final patterned silicone
elastom er coated glass slides. ............................................. .............................. 56

4.4 Topography layout on the silicone elastomer coated glass slides as compared to the
direction of hydrodynam ic flow ...................... ..... ......................... ............... 57

4.5 Final mold setup for reproduction of a patterned silicone elastomer coated glass
slide from an epoxy m aster m old ....................................................... ..................59

4.6 SEM and WLIP images of an epoxy mold and silicone elastomer coated glass slide.
A) SEM at 800X magnification of patterned epoxy with the negative topographical
features (10,000tm length ridges at the transition point from 5[tm wide ridges to
10tm wide ridges) replicated from a silicone well. B) SEM (350X magnification)
of silicone elastomer with 10,000atm length ridges at the transition from 5atm wide
valleys to 10atm wide valleys that was replicated off of an epoxy mold from the last
step in the replication process. C) WLIP image of the 10,000atm length ridges with
5atm valleys from the silicon wafer. D) WLIP image of the 60atm length ridges
with 5atm wide ridges from an epoxy replicate. ................................... .................62

4.7 The settlement of Enteromorpha spores on the different unpatterned silicone
elastom er form ulations....... ................................................................. .. .... ... .... 65

4.8 The settlement of Enteromorpha spores on 5pm wide spaced features on both the
topographies of the 2 depths and 2 types of features and on the flat areas between
the features. ..............................................................................66

4.9 The settlement of Enteromorpha spores on 10pm wide spaced features on both the
topographies of the 2 depths and 2 types of features and on the flat areas between
the features. ..............................................................................66

4.10 The settlement of Enteromorpha spores on 20pm wide spaced features on both the
topographies of the 2 depths and 2 types of features and on the flat areas between
the features. ..............................................................................67

4.11 Fluorescent microscope images of Enteromorpha spores settled on 3 different 5atm
deep patterned areas on unmodified silicone elastomer. (A) 5atm wide ridges with
5atm wide valleys. (B) 5atm wide ridges with 10atm wide valleys. (C) 5atm wide
pillars with 5atm spacing. (Images courtesy of Dr. Maureen Callow)......................68

4.12 Spore density measurements before and after immersion in a flow cell for the flat
regions between the 5 tm deep patterns of the different elastomer formulations.........70

4.13 Spore density measurements before and after immersion in a flow cell for the 5atm
deep and wide valley pattern set parallel to flow direction of the different elastomer
form u nation s ............................................................................. 70

4.14 Comparison of the percent spore removal for the different 5atm deep pattern sets
and the different silicone elastomer formulations .................................... ............... 72









4.15 Enteromorpha spore settlement densities on the flat areas between the patterns and
on the pillars for the different elastomer formulation samples with either 1.5 am or
5 m deep pattern features .................................................. ............................... 72

4.16 Percent Enteromorpha spore removal on the flat areas between the patterns and on
the pillars for the different elastomer formulation samples with either 1.5tam or 5tam
deep pattern features after exposure to hydrodynamic flow......................................74

5.1 Chemical structures of Dow Coming's 5700 Antimicrobial Agent (top) and a
trifunctional alkoxy silane (bottom) with same length unreactive alkyl chain.............76

5.2 Chemical structure of UdelTM poly(ether sulfone), which was used as a more
durable substrate as compared to silicone elastomer for chemical modifications........77

5.3 Single frames from a digital video of the placement of an air droplet at the untreated
glass (at the top of each image) and water interface with a microliter pipet. This
setup is the captive air bubble contact angle technique. Images from A) to D) are in
sequential tim e order ......................................... ............... .. ........ .... 85

5.4 Single frames from a digital video of the attempt to place of an air droplet at the
quaternary ammonium surface grafted glass (at the top of each image) and water
interface with a microliter pipet. A) Deformation of the air bubble is observed as it
is pressed against the modified glass surface. B) The same air bubble transforms
completely back to a sphere after deformation observed at modified glass surface.....86

5.5 Single frames from a digital video of the placement of an air droplet at the
quaternary ammonium surface grafted glass (at the top of each image)and water
interface with a microliter pipet. This air bubble only moved a short distance until
attaching to surface. Images from A) to D) are in sequential time order. .................87

5.6 Single frames from a digital video of the placement of two air droplets (which split
upon release from the micropipet from a single bubble) at the quaternary
ammonium surface grafted glass (at the top of each image) and water interface with
a microliter pipet. These air bubbles glided completely across the camera view on
the modified glass surface until they reached the side of the water holder. Images
from A) to D) are in sequential time order. ...................................... ............... 88

5.7 Single frames from a digital video of the placement of an air droplet at the
octadecyltrimethoxysilane treated glass-water interface with a microliter pipet.
Images from A) to D) are in sequential time order. No movement of the air droplet
is ob serve ed .......................................................... ................ 9 0

5.8 Digital image of the water-air-silicone elastomer intersection by two contact angle
methods: A) sessile drop and B) captive air bubble. ............. ..................................... 91

5.9 Chemical structures of the modified polysulfone after each chemical reaction. The
final result (bottom structure) is of the quaternary ammonium salt bound to the
p o ly su lfo n e ......................................... .. .. ..............................................9 2









5.10 FTIR spectras of the different steps in the binding of quaternary ammonium salt to
polysulfone. All films were neat on NaCl crystals. A) polysulfone (PSF). B)
sulfonated polysulfone (SPSF). C) GPS silanated SPSF. D) Quaternary ammonium
salt grafted to the silanated SP SF ..................................................... .....................93

5.11 SEM images of glycidoxypropyltrimethoxysilane silanated SPSF by solution
reaction. A) 400X magnification. B) 3300X magnification ....................... ..............95

5.12 EDS graph and identifications of chemical atoms present on the surface....................96

5.13 Contact angle images of 24L droplets of water on A) sulfonated polysulfone (SPSF)
and B) quaternary ammonium salt grafted onto silanated SPSF ................................97















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

CHEMICAL MODIFICATION OF SILICONE ELASTOMER FOR CONTROLLED
BIOLOGICAL INTERACTIONS ON MICROTEXTURED SURFACES

By

Amy Louise Gibson

December 2002


Chairman: Dr. Anthony Brennan
Major Department: Materials Science and Engineering

The ability to control a biointerface in terms of biological growth and adhesion to

a material's surface is beneficial to many fields, including marine coatings and

biomedical devices. By examining some of the individual factors that influence these

biointerfaces, a step towards developing a systematic model for biological interactions

with materials can be established. As a result, a material could be engineered to elicit a

specific biological response. In the case of anti-fouling coatings for marine applications,

an engineered surface to prevent biofouling is desired. In particular, silicone elastomers

have been heavily studied recently for their foul-release properties, which means that

only a small force is required to remove adhered biological organisms. In order to

increase effectiveness of these elastomer release coatings, the factors of surface lubricity

and topography are examined in this study as a function of biological growth and

adhesion. The lubricity of the silicone elastomer surface was controlled by the









incorporation of nonfunctional poly(dimethylsiloxane) linear oils. This chemical

modification allowed the surface chemistry and bulk mechanical properties to stay

relatively constant with only the lubricity as a variable. These design criteria for the

surface lubricity factor were proven to be effective through meticulous testing. An

increase in surface lubricity was expected to increase the removal of organisms under a

constant force. When Enteromorpha algae spores were examined for settlement/removal

characteristics with a hydrodynamic flow cell, however, the predicted trend for surface

lubricity was only observed with the excessively oily surfaces. The factor of surface

topography was examined through engineered microtextured silicone elastomer surfaces

of 5[tm wide pillars or ridges separated by 5,10 or 20tm wide valleys/spaces. The factor

of topographical depth was also inspected with both 5 and 1.5am deep features. With the

Enteromorpha spores, the release from the microtextured, unmodified silicone elastomer

surfaces was significant for the pillar features and the larger depth. The interaction

between the coupling of these two factors on settlement and adhesion was also in the

experimental design. The ridges had less release of spores for all elastomer formulations.

There were definite topographical settlement cues as the spores preferentially adhered in

the valleys, as opposed to on top of the ridges, and around the pillars. Through

chemically functionalizing the surface with a quaternary ammonium antimicrobial salt,

the potential for preventing the growth of biological organisms onto the surface is a

prospect. Chemical modifications for binding a quaternary ammonium salt to glass,

silicone elastomer, and polysulfone are presented and analyzed for their fidelity and

interfacial properties. This entire study presented significant aspects and directions for

designing biointerfaces for the purpose of controlling biological interactions.














CHAPTER 1
INTRODUCTION



The ability to engineer a surface in order to control or prevent biological adhesion

is of great importance. Whenever a man-made structure or device is placed into a

biological environment, there is an immediate settlement cascade that initiates on the

surface. In the field of anti-fouling coatings for ship hulls, biological adhesion to the

surface is not desired because an increase of biofouling on the hull causes an increase in

the hydrodynamic drag and thus an increase in fuel consumption and a decrease in

optimum speeds.1 Even a 100tm thick biological growth can cause a 6% increase in fuel

consumption.2 It is also costly to dry dock the large ships in order to clean off the

biofouling or reapply the anti-fouling coating, so increasing the time in between dry

docking by increasing coating efficiency would be cost beneficial. In the biomedical

field, the contact guidance of cells, cellular response in a directional orientation, can

allow the desired biological response to be achieved on the surface of a medical device or

implant. One particular area of need is with tissue grafts where the mechanical stability

in particular directions is desired. The exact cues which allow for control of biological

interactions with synthetic materials is the key for engineering surfaces.

The contact guidance of cells has been shown to be directly influenced by the

chemistry and topography of a material's surface.35 Cellular alignment and growth can

either be enhanced or inhibited depending on topographical dimensions. The mechanical

stability of cells has been shown to increase when settled within valley patterns due to the









increase in adhesion contact area.6-8 However, if the biological organisms are unable to

settle within the valley topographies, then the adhesion contact area should decrease and

thus the mechanical stability of the organism should also decrease. Cells and other

organism have also shown specificity to various surface chemistries.9' 10 Hydrophobic

surface monolayers have shown larger cell adherence then with hydrophilic surfaces.11

The friction at an interface also has been observed to have a major impact on the

adhesion between two substrates.12, 13 As a result, the adhesion of a biological organism

should decrease as a surface becomes more lubricious.

The purpose of this study was to examine the role of lubricity and topographical

surface modifications on biological settlement cues and adhesion forces. The surface

grafting of a chemical biocide, quaternary ammonium salt, was also introduced as a

chemical modification to prevent biological adhesion. In particular, the settlement and

adhesion of marine algae Enteromorpha zoospores were examined. This biological

organism is a major nuisance in the biofouling of ship hulls and thus its cues are an

important role to examine. The substrate chosen for these modifications was a silicone

elastomer because of its foul-release properties.

One specific aim of this study was to characterize the chemical modifications of

the silicone elastomer in order to verify that only the surface lubricity was variable. In

particular, the elastic modulus and surface energy was characterized for the different

formulations since these two variables have been shown to influence adhesion.14 With

the addition of nonfunctional silicone oils, the lubricity of the surface was predicted to

change, but the elastic modulus and surface energy values should have stayed relatively









constant. This was expected due to similar molecular chemistries between the elastomer

and additives and the nonfunctionality of the silicone oil additives.

Another specific aim was to examine the effects of the lubricity and topographical

surface modifications on settlement and relative adhesion with Enteromorpha spore

assays. An increase in surface lubricity was predicted to decrease adhesion, thus increase

the number of spores removed after subjected to a hydrodynamic flow, which had a 55Pa

wall shear stress. The amount of removal was quantified as the difference of the spore

densities on the surface from before and after exposure to a hydrodynamic flow. The

spore adhesion to the surface should decrease as the force required to remove the

organism decreases or the amount removed under a constant force increases. The

addition of pillar topographies on the surface was expected to disrupt the standard

settlement by providing an increased adhesion surface area. However, having a high

density of pillars over the surface should ultimately disrupt the settlement so that the

spores never obtain stability and could thus be more easily removed. The micropatterned

valleys were predicted to enhance settlement in a directional manner as the spore should

settle in the valleys where they have the highest adhesion contact area. However, again

when the width of the valleys was smaller than the spore, there should be a decrease in

the stability of the spore's adhesion and thus easier removal from the surface. There was

also predicted to be an increase in removal when the valleys were placed in the parallel

direction with flow, as a laminar flow would then be localized down the valleys and

spores would be subjected to a slightly higher shear force. Coupling both topographical

and lubricity factors together should enhance the control of biological adhesion to

engineered surfaces.









The third specific aim of this study was to chemically bind a biocide to the

surface in order to prevent biological adhesion. A quaternary ammonium salt, which has

antibacterial properties, was grafted to the surface to prevent biofilm formation. The

initial step in any biological-material interaction cascade is the formation of a biofilm.15-

17 Reducing this initial step should decrease the biological settlement and adhesion of

other organisms. The polymer substrates chosen for chemical modification were silicone

elastomer and polysulfone. The polysulfone was investigated due to its higher durability

as compared to the elastomer and its history of naturally reducing biofouling as a dialysis

membrane.18,19 This chemical surface modification allows for an anti-fouling coating for

either marine structures or medical devices without the leaching of toxic chemicals into

the biological environment.














CHAPTER 2
BACKGROUND


Biofouling

In the recent past, tributyltin (TBT) based paint coatings have been the standard

for use on the hulls of ships and other immersed man-made structures to prevent

biofouling. However, this anti-fouling (AF) coating releases an environmentally toxic

biocide chemical into the water. Some toxicity readings around the water layer of a

ship's hull were classified as safe, but one thing was not considered. When the ship is

docked in a bay or other semi-closed body of water for a long period of time, the time

released toxic material continuously rises in quantity and concentration in the

surrounding waters. In the 1970s, the effects of TBT in the waters were observed in

abnormal shell and reproduction of oysters.20 Since then abnormalities have been

reported in other non-target organisms such as marine vertebrates and invertebrates. As a

result, a ban was placed restricting the application of a TBT coating on small vessels in

1982.20, 21 The goals of the US Navy, following the passing of the US Organotin

Antifouling Paint Control Act in 1988 and the ban set by the International Maritime

Organization, are to have the application of all TBT coatings universally banned by 2003

and the use of them banned by 2008.22 Thus a replacement coating is necessary. Many

of the coatings have been switched to copper based paints, which perform under the same

mechanism, but these are claimed to be less toxic to non-target organisms.23 However,

these copper based coatings are also showing signs of accumulation and toxicity and their

long-term effects are currently unknown.21 Currently, significant research has focused on

5









silicone elastomers as foul-release coatings (FR), from which either movement of the

ship or effortless cleaning can easily remove organisms. However, the elastomer lacks

durability as compared to the other coatings, which is a critical component for ship hulls.

Thus there are also steady attempts to alter this mechanical property, while keeping the

elastomer's release properties.


Adhesion

Mechanics

In order to control adhesion of biological organisms to a substrate, some

fundamental fracture mechanics have to be considered. The term adhesion is defined as

the force required to separate two surfaces, whereas, interfacial surface energy is the

work that is required to separate the surfaces. Currently, there are four methods classified

for obtaining adhesion: chemical bonding, electrostatic interactions, mechanical

interlocking, and diffusion.14 Adhesion of biological organisms is complicated not just

because one mode is followed, but a combination of modes. Thus a surface must be

engineered to be favorable in preventing adhesion by multiple mechanisms. Currently,

silicone elastomers are being studied for their combination of reducing adhesion by the

smooth residual silicone oil layer on the surface preventing adsorption, the formation of

only weak chemical bonds, and the high compliance aiding in removal. 14

For prediction of the force required to break an adhesive from a silicone elastomer

substrate, basic fracture mechanics should be examined. Griffith formulated an equation

(eqn. 2.1) for the critical stress (o5) required to propagate a crack in a plate

c5 = EGo / 7a(1 2) (2.1)

from an uniaxial direction, where E, Go, a, u are the elastic modulus, Griffith's critical

fracture energy per area, half the crack length, and Poisson's ratio, respectively.24 He









then applied this equation to the stress over a set crack area (A=7ta2), known as the critical

pull-off force (Pc) (eqn 2.2).

Pc = rEGEa3/ (1 2) (2.2)

Quite a few years later, Kendall followed Griffith's fracture analysis to model adhesion to

elastomer substrates.25'26 He used a circular disc to design the force for removal from the

elastomer. He then derived the critical pull off force, assuming a thin elastomer film and

the radius of the disc being smaller than the size of the elastomer film, as

Pc = 7a2 (2GoK/t)1/2 (2.3)

where Go, a, t, and K are the critical fracture energy, radius of the contact area, elastomer

film thickness, and bulk modulus (K= E /3(1 2u)). These equations show that there is a

proportional relationship between the critical pull-off force and (EG)1 2, which are

material properties.14 In this case, fracture energy is directly related to the work of

adhesion, which is then equal to double the critical surface tension (y ) of the

elastomer.27 28 As a result, the adhesion correlates with (2EY~)1/2. The elastic modulus

and surface energy are all parameters that can be engineered with a material. Another

term that is used considerably in the literature is the compliance of a material, defined as

the inverse of the elastic modulus, which measures its softness.

These fracture mechanic equations assume that the applied force is in the normal

direction to the elastomer film. However, when the force is applied at an angle, as would

be the case when a ship is moving or in a laboratory hydrodynamic flow cell, there is the

additional component of interfacial slippage that has to be considered. According to

Newby et al., there is evidence that the adhesion of a viscoelastic adhesive on silicone

elastomers is controlled heavily by interfacial slippage rather than thermodynamics.29

When peeling a viscoelastic adhesive, an extension deformation occurs behind and









contraction occurs in front of the moving crack tip.30 If slippage is allowed to occur by

the substrate, then the work required to move the crack tip is lowered, which results in a

lower adhesion strength. The more mobile chains are on the substrate surface, the lower

the friction observed. When force is applied in the normal direction the release mode is

peeling from nucleated voids within the contact area, but when the force is directed at an

angle, failure occurs by a fingering process (like webbed fingers) as described by Newby

et al. which starts at the edges and moves inward.29' 31 These finger adhesion release

deformations were also shown to increase in length (i.e., amplitude) slightly as the

modulus or the thickness of the elastomer coating increased and significantly as the

rigidity of the adhesive increased.32

Coupling Agents

Sometimes coupling agents of double or more functionality are used to promote

adhesion through a chemical bond between two molecules or materials. If the coupling

agent only has a single functionality, then it can be used as an end-capping agent. The

most common types of coupling agents between polymers are organofunctional silane

based molecules.33 Typically the four end groups are 1 vinyl or other organic functional

group (R) and 3 alkoxy groups (OR1) so that the general formula is RSi(OR1)3. The

alkoxy groups first hydrolize into silanols relatively quickly and then slowly undergo a

condensation reaction to make a siloxane chain when in water. The reaction rates are

dependent on the pH of the solution. The longer the alkoxy side chains are, the slower

they hydrolyze. Solubility of the silane molecules decreases as the condensation reaction

progresses and thus as the solution becomes hazy, the coupling agent becomes

unusable.33 When the silane is added to an aqueous alcohol solution, the coupling agent









has a longer work time as an equilibrium reaction is reached between the hydrolysis of

the alkoxy groups to silanol and the reesterification of the silanols by the alcohols.

Roughness Factor

With the fracture mechanisms discussed previously, the surfaces were assumed to

be completely smooth. However, even the smoothest substrate has molecular roughness

on the surface. As a result, the factor of surface roughness, random or engineered, on

adhesion strength needs to be examined. The biological adhesive usually starts as a

liquid and then either stays liquid or cures. Depending on the viscosity of the liquid, the

adhesive might not fill in all of the small crevices because of its inability to displace the

air pockets.34 When solidification of the adhesive occurs, there are stress concentrations

that occur at the focal points of the roughness. The stress at these focal points is much

higher than the applied force, so less applied force is needed to fracture the adhesion. If

the voids are relatively close together then the fracture crack can propagate even easier.34

The unfilled crevices can be on the molecular level or micron scale depending on the size

of the biological organism.


Silicones

Introduction

Poly(dimethylsiloxane) (abrev. PDMS) elastomers are widely used in coatings

and medical devices because of its relative biocompatibility, high resistance to chemical

attack, and elastomeric properties. This material also has a high oxygen permeability,

which promotes its use in contact lenses and membrane oxygenators.35 An additional

benefit with PDMS is its low toxicity to the environment. The polysiloxane chains can

hydrolyze to dimethylsilanediol oligomers over time. When these silanediols are in a

microbial or UV light environment, degradation into CO2 and SiO2 (silica) occurs, which









are all naturally occurring materials.36-38 In the literature, there have been discrete

references to the possible side product Me3SiOH, which is known to be a nervous system

depressant, introduced upon degradation, but there has been little published follow-

up.39'40 There are a few mineral catalysts that seem to increase the degradation rate

slightly.36 However, this degradation process is very slow for PDMS chains and even

slower in silicone elastomers. Many different marine and soil environments have been

examined for silicone toxicity and little adverse effects on growth or reproduction in

aquatic animals have been discovered.41' 42

Polydimethylsiloxane (PDMS) chains have a silicon-oxygen backbone, which has

a longer bond length of 1.63 A than carbon bonds in many of the standard organic

polymers.43 This property presents large bond rotation and thus large chain mobility and

restructuring ability. As a result, the nonpolar and polar groups can reorient on the

surface to their most favorable position depending on its environment. 44 PDMS at its

lowest energy state is in the all-trans conformation due to this arrangement having the

most favorable van der Waals interactions where the methyl groups are separated by four

bonds.45 This ability to recovery to its lowest energy state is beneficial when trying to

predict the material properties when exposed to diverse environments.

Mechanical Properties

Elastomers have extremely low tensile values without reinforcement (-0.3 5MPa)

which limits their applications.39 46 In order to increase the mechanical properties and

tear strengths of elastomers, reinforcement particles are incorporated into the bulk. In

particular, silica particles are most widely used for reinforcement in silicone elastomers

because they have shown to provide the highest change in mechanical properties.47 Three

factors of the silica particles influence the resulting mechanical properties from









reinforcement: size, structure (pore volume), and surface interaction.48'49 There are 3

interactions that have to be considered when introducing silica particles: chemical filler-

elastomer bonds, physical interference in the elastomer, and filler-filler interactions.47

With fumed silica, there are hydrophobic and hydrophilic portions on the surface, as

shown in Figure 2.1. The isolated hydroxyl groups introduce the hydrophilic portions

and the hydroxyl groups that have reacted again with the silica surface introduce a

nonpolar, hydrophobic portion.48 In some cases, the silica particles are made unreactive

by the introduction of hexamethyldisilazane, which reacts to produce trimethylsilylated

silica surfaces, as shown in the reaction in Figure 2.2. The fumed silica increases the

crosslink density of the elastomer network and thus has been shown by Boonstra et al. to

increase elastic modulus at all strain ranges, but decrease elongation as compared to

trimethylated particles.48 During synthesis of the particles, agglomeration also occurs.

The packing factor of these agglomerates changes the sizes of pores within the particle

clusters. Agglomeration also introduces polymer chains that can be occluded within the

irregularities of the clusters and thus be secluded from distribution of stresses within the

elastomer network. Baker et al. have shown that the smallest fillers produce the highest

reinforcement.47 The elastic modulus and tensile strength have also been shown by

Polmanteer and Lentz to increase as the filler structure, measured by pore size, increases

for silicone elastomers.49 With the multiple factors involved in particle reinforcement,

declaration of trends of just one single factor alone is difficult.












Hydrophilic

i
W^


Fumed Silica


S 'Si-(CH3)3
Si
(CH3)3

Trimethylsilylated Silica


Figure 2.1: Chemical species on the surface of fumed silica and trimethylsilylated silica
particles.


CH3 H CH3
H3C-Si-N-Si CH3
CH3 CH3

Hexamethyldisilazane


2 H20 O- 2 H3C-Si-OH
I


NH3


Trimethylsilanol


OH
Si

Si OH

Si
OH

Fumed Silica


CH3
HO-Si-CH3 1-

CH3



Trimethylsilanol


OH
Si CH3

Si O-Si-CH3

Si CH3
OH

Trimethylsilylated Silica


Figure 2.2: The chemical reaction for the trimethylsilylation of a fumed silica particle by
addition of hexamethyldisilazane.


H20









Surface Energy/Chemistry

The surface energetic of silicone elastomer is very complicated since it is such a

low surface energy material with highly mobile methyl groups at the surface being its

lowest energy state. A basic method for examining the surface energetic of a solid

substrate is with static contact angles, either through sessile drop technique or captive air

bubble technique. The diagrams in Figure 2.3 show the general setup and variables for

the two contact angle measurement techniques. Measurement by contact angle provides

information about the outer angstroms of the surface and is a result of adhesive forces

between the two phases and cohesive forces within the liquid.50' 51 This is one of the

most sensitive techniques for examining the actual surface and it is relatively simple to

perform.50 However, analysis of the contact angles can be confusing because of the many

assumptions and factors that have to be considered. Both static techniques follow

Young's interfacial equilibrium equation,

7sv Tsl 71v cos 0 (2.1)

where ysv, Tsl, and y1v are the surface tensions for the surface-vapor, surface-liquid, and

liquid-vapor interfaces, respectively, and 0 is the contact angle. This is derived from an

A B
A YLV
Vapor


YSL YSV




Figure 2.3: Setup and labeled interfaces/contact angle for both the (A) sessile drop
technique and (B) captive air bubble technique.









energy balance approach at the sessile drop intersection point, shown in Figure 2.4, with

a Helmholtz free energy (F) equation (eqn. 2.2) over a small distance, dx.

(dF)T,V,n = ysvdx ysidx ylvdx cos 0 (2.2)

At equilibrium, dF = 0, and Young's equation is the resultant.50 The experimental

technique involves measuring the contact angles (0) of different liquids, of various

surface tensions (ylv), on the desired substrate. However, there are still two unknowns in

Young's equation, y7s and ysl. As a result, W.A. Zisman proposed a plot of cos 0 versus

1iv which he found to be empirically linear.52 Theoretically, then at 0 = 0, in other words

cos 0 = 1, the liquid completely wets the substrate surface. At this point, Zisman claims

that si = 0 and, as a result, the surface tension of the solid vapor interface can be

calculated from a linear regression on the Zisman plot. This is then termed the critical

surface tension (y ) of the solid. For a homogenous solid, the critical surface tension is

the same as the surface free energy assuming that there is no other forced elastic strains

on the solid and no solvent adsorption.53 54





Vapor




dx cos 0




Figure 2.4: Energy balance approach at the point of triple phase (liquid-solid-vapor)
intersection for the derivation of Young's equation. The forces are labeled previously in
Figure 2.3.









There are many assumptions that coincide with this technique for surface

analysis.50, 51 First, the droplet (or air bubble) is assumed to be small enough that there is

minimal gravitational effect and thus little distortion of the droplet shape.51 A major

assumption when examining a thermodynamic system is that everything is at

equilibrium.50 With the factors of solvent volume, due to evaporation, and surface

rearrangement, equilibrium is hard to obtain and hold for contact angle measurement.

This also implies that time and temperature are factors which can alter the contact angle.

Some other basic assumptions are that the surface is rigid (i.e., nondeformable), smooth,

homogeneous, does not reorient, and that the solvents have exact, known surface tensions

that do not swell the surface. These assumptions are obviously not all met in many

scenarios, but care can be taken in interpretation of measurements if the limitations are

defined. Many factors can not be controlled easily which led to R.E. Johnson and R.H.

Dettre's claim about contact angle measurements that "the limitation on accuracy is not

the measuring technique, but rather the reproducibility of the surface being studied".5

When one or more of these assumptions are not met, a contact angle hysteresis is

observed. This is defined as the difference of the contact angle advancing over a solid-air

interface and the contact angle receding back over the solid-liquid interface. Silicone

elastomers are known to have large contact angle hysteresis due to their highly mobile

backbone chain and thus surface rearrangement. When a static surface energy method is

performed, the measured contact angle is observed between the advancing and receding

angles from a dynamic method, but closest to the advancing angle.53

One of the large factors that influence contact angle measurements and introduces

a hysteresis is surface roughness. Wenzel first attempted to define a relationship between

the measured contact angle and the surface roughness with equation 2.3 where 0' is the









cos O' = r cos Oo (2.3)

observable contact angle on the rough surface and Oo is the intrinsic contact angle. The

roughness ratio, r, is defined as

r = A / A' (2.4)

where A' is the apparent surface area and A is the true surface area taking the roughness

into account.50' 55 This means that the ratio, r, is always greater than 1. According to

Johnson and Dettre, the trends observed from many studies were that as the roughness

increased, the advancing angle increased and the receding angle decreased. 5 This

means that when static conditions are examined, as the roughness increases, the contact

angle increases and thus the critical surface tension calculated increases. However, this

theory does not consider the size, the shape, and the exact location that the droplet edge

falls in reference to the rough features. These are all factors that are still undefined in

their relationship to measured contact angles. This roughness influence on the spreading

of liquids needs to be considered when examining the settlement of biological organisms,

which initially excrete a liquid cement to increase adhesion on engineered topographies.


Enteromorpha Algae Spores

There are many different types of seaweeds that are present in waters, including

brown, red, and green algae. All types are unicellular at some point in their life cycle in

the form of spores or zygotes.56 One type of green algae that is of particular interest is

the Enteromorpha species genus, which is the most common and taxing algae fouling on

ship hulls and other submerged marine structures.57' 58 This type of algae, from the

Ulvaceae family, is of high nuisance because it is tolerant to a wide variation of water

salinities and can thus travel and survive with ships to many freshwater and ocean

environments.59 The Enteromorpha reproduce by the release of quadriflagellate, pear-









shaped zoospores (7-10pm long), which attach to substrates.60 The cells then divide and

grow into a tubular type organization.59' 61 The mechanism behind the settlement and

adhesion of these spores to a substrate is of interest when trying to design an anti-fouling

coating. As motile spores, they lack a cell wall and search for a substrate on which to

colonize. Once a spore identifies an acceptable surface, a glycoprotein is secreted in

order to form an adhesion.60 The spore is then settled and a cell wall is developed and

eventually a new algae plant is produced. When performing algae laboratory studies,

there are always considerations that have to be recognized during interpretation of results,

mostly due to the culture having more ideal, uniform conditions than in the environment.

One important factor is that there is no longer competition or grazing from other

biological organisms in a laboratory environment.56 This is just one reason out of many

for large standard deviations that are encountered on biological organism studies.

There are many known cues that are involved in spore settlement, such as

negative phototaxis (light), thigmotaxis (roughness), and chemotaxis (chemical). 10, 62 All

of these factors have been shown to cause spores to not evenly distribute on a surface.

The adhesion of the spore to a substrate can also be influenced by these factors with the

spreading and bonding of the chemical secretion.61 All of the rough surfaces examined

previously were random and an increase in settlement was assumed to be due to an

increase in surface area contact. However, if a surface was engineered with regular

patterns at defined aspect ratios perhaps the contact area could be disrupted or decreased.

This is the basis for the topographical studies presented here.









Quaternary Ammonium Salts

Introduction

Recently, the antimicrobial action of surfaces has come into popularity with

disinfectants for textiles, medical devices, marine coatings, and food manufacturing.63-66

Quaternary ammonium salts are one of the most popular types of antimicrobial agents.

At first, this biocide was incorporated into the bulk resin and allowed to leach over time

in order to obtain a continually active surface. However, when attempted by researchers

with silicone elastomer, because of the favorable surface energetic with the methyl side

groups at the surface, the quaternary ammonium was unable to diffuse to the surface,

which was visible with simple contact angle measurements.67 This led to the motivation

of binding (or immobilizing) the quaternary ammonium to the surface so there was no

leaching or toxic effects to the environment and an increased productive time.68' 69 There

are other factors that influence the activity level of the quaternary ammonium salt, such

as the accessibility of the active centers (directly related to the length of the alkyl chains),

the concentration on a surface, the chemistry of the spacers along the surface (to change

concentration), and the counter ion.7

Biocidal Mechanism

Quaternary ammonium compounds interact with bacterial cells, but not by

chemically reacting. The extreme polarity and long hydrophobic alkyl chains interact

with the phospholipid bilayer of the cytoplasmic membranes to cause a phase separation

of the hydrophobic and polar regions.71 As a result, weakness and leakage of the

cytoplasmic membrane is caused by these compounds, which leads to a disruption of the

permeability and instability of the membrane. The absorption of this biocide is enhanced

by the cells negative charge.72 First the long alkyl groups face away from the cell in









order to introduce the cationic species. This causes the disruption of the cytoplasmic

membrane and results in displacement of potassium and magnesium ions, which

contribute to the membranes stability. The increase in permeability that results allows the

long alkyl chain to enter the cytoplasma and cause precipitation of the nucleic acids and

proteins.72 Many researchers have discovered that these cationic biocides are not as

effective for gram-negative bacteria as compared to gram-positive bacteria. This has

phenomena has been contributed to the presence of an outer membrane on only the gram-

negative bacteria. This outer membrane increases the impermeability of the antimicrobial

agents, even though some are still successful.73'74 As a result, binding the quaternary

ammonium salt to a surface should prevent or reduce the initial stage of biological

adhesion, biofilm formation from bacteria and diatoms, and ultimately reduce the later

stages of biological growth.














CHAPTER 3
SILICONE ELASTOMER CHEMICAL MODIFICATION AND
CHARACTERIZATION


Introduction

A factor of biological adhesion that should be examined is friction/lubricity of a

surface. However, the substrate materials must be fully characterized before analysis of

growth data can be performed. Currently, the substrate of silicone elastomer is being

studied for its foul-release properties in marine coatings and for medical devices." Thus

this substrate allows for application in many different fields. In order to engineer the

elastomer substrates with lubricity as the predominant variable, various nonfunctional

silicone oil additives were introduced into the bulk of the elastomer creating a lubricious

surface. Due to surface energetic, the lower surface energy silicone oils migrate and

coat the elastomer surface.76 As a result of similar chemical structures and

nonfunctionality between the silicone oils and elastomer, the elastic modulus and surface

energetic should stay relatively the same. However, this idea needed to be proven

through experimentation. For characterization of these chemical modifications, the

mechanical tensile properties, specifically Young's modulus, and the static surface

energetic were examined.









Materials

Silicone Elastomer

A silicone elastomer substrate was used as a basis for a chemically and

topographically engineered material in order to examine the biological response to these

modifications. In particular, the silicone elastomer substrate was Dow Corning's

Silastic T2, which is a hydrosilylation, platinum catalyzed poly(dimethylsiloxane)

elastomer. The two-part, vinyl-functionalized, addition cure reaction is shown in Figure

3.1. There are trimethylated silica particles also present in Dow Corning's formulation in

order to increase the material's mechanical stability. This specific silicone was chosen

for its transparency, low shrinkage, and few unknown/undisclosed additives. Thus it

allowed modification of this material to be controlled and engineered topographies be

reproduced with high fidelity. This resin was mixed in a 10:1 weight ratio of base resin

to curing agent, as directed by Dow Corning. The typical properties of this elastomer

prepared with this ratio are provided by the manufacturer and are shown in Table 3.1.

This material, in its original form, is referred to as "unmodified" elastomer.

CH3 CH3 CH3 H CH3 CH3
I I I I 3 1
H2C=C-Si-O Si-O Si-C=CH2+ H3C -Si-O Si-O Si-CH3
H I H
\Cy/ CH3 CH3
CH3 CH3 CH3 CH3 CH3 x CH3
Silastic T2 Base Resin Silastic T2 Curing Agent
Pt


I j PDMS
-Si-CH2CH--Si"J-NV


Silicone Elastomer
Figure 3.1: Dow Coming's Silastic T2 silicone elastomer crosslinking reaction.









Table 3.1: Typical properties of Dow Corning's Silastic T2 addition cured silicone
elastomer. (From Dow Corning's product information sheets)

Test Result
Base/Curing Agent Color Translucent/Transparent
Viscosity of Base Resin 50,000 cp
Viscosity of Curing Agent 550 cp
Mixed (10:1 weight) Specific Gravity 1.12
After Cure (24hrs at 250C):
Tensile Strength 800 psi (0.55 MPa)
Elongation 300 %
Tear Strength 120 ppi (Die B)
Linear Shrinkage < 0.1 %


Nonfunctional Additives

The role of nonfunctional silicone oils added to the bulk silicone elastomer was to

maintain similar chemistry and mechanical properties, but have a varying lubricious

surface. All the silicone oils used were either nonfunctional, trimethylsiloxy terminated

poly(dimethylsiloxane) (PDMS) linear chains, of varying chain length, or a bulky,

nonfunctional silane molecule. All the nonfunctional additives were purchased from

Gelest, Inc and their chemical structure, viscosity, and molecular weight are shown in

Table 3.2. All of the additives discussed were nonfunctional, thus none of them reacted

into the vinyl-functionalized bulk elastomer and the elastomer crosslink density was

maintained. Energetics drives these low surface energy silicone oils to migrate to the

surface, thus allowing a controlled release and complete surface coverage.

Surface Energy Liquids

In order to calculate surface energy from contact angles, a variety of liquids of

varying surface tensions must be used. In these experiments, 5 different liquids, all










Table 3.2: Chemical structures and properties of the nonfunctional
from Gelest, Inc. (Information obtained from Gelest, Inc. catalog)


silicone oil additives


Table 3.3: List of chemicals used for contact angle measurements with their
corresponding liquid surface tensions.


Surface Tension
Chemical Name
(mN/m)

HPLC grade Water 72.8

Diiodomethane
50.7
(methylene iodide)
N,N-Dimethylformamide
37.1
(DMF)
Acetonitrile
29.3
(ACN)

1-Propanol 23.8

Ethylene Glycol 47.7


Viscosity MW Sp.
Name Structure n
(cSt) (g/mol) Gravity


CH3 CH3 CH3 50 3,800 49 0.960
Poly(dimethyls iloxane), I I
trimethylsiloxy CH3-Si-O Si -O Si-CH3 500 17,300 231 0.971
terminated CH3 CH3 CH3 5000 49,400 664 0.973
1,000,000 308,000 4154 0.978



Tetrakis (CH3)3SiO OSi(CH3)3
Si ~3 384 -- 0.868
(trimethylsiloxy)silane (CH3)3SiO' OSi(CH3)3









obtained from Sigma Aldrich, were chosen with varying surface tensions, as shown in

Table 3.3. These liquids included, HPLC grade water, diiodomethane (methylene

iodide), N,N-dimethylformamide (DMF), acetonitrile (ACN), and 1-propanol. During

later experiments, the diiodomethane liquid was replaced with ethylene glycol as the

liquid with the surface tension in the range of 50 mN/m.


Methods

Sample Preparation

The unmodified silicone elastomer was always prepared according to Dow

Corning's recommendations. The base resin was weighed out as 100 parts to 10 parts of

the curing agent. Both were mixed thoroughly together in a polypropylene tri-cornered

beaker with a metal scoopula for approximately 5 minutes. The mixture was then

degassed in a vacuum desicator for approximately 30 minutes in order to get rid of the

many trapped air bubbles. Not all of the air bubbles disappear, but the elastomer self-

releases the residual small air bubbles within the first 30 minutes of cure. If the addition

of silicone oils was desired then they were added into the elastomer mixture at a 5, 10, or

20 weight % to the already mixed base and curing agent weight and subsequently

degassed in the same manner as the unmodified elastomer. The approximate work time

for the elastomer mixture was 1 hour. The elastomer was cured at either room

temperature for 24 hours or at 800C for 2 hours depending on experimental conditions.

For the preparation of free-standing films, the silicone elastomer was poured onto

a clean glass plate (7in x 7in x 3/8in) with the setup as shown in Figure 3.2. All glass

plates used in these experiments were cleaned with acetone, then ethanol, and lastly

wiped with hexamethyldisilazane (HMDS) (from Gelest, Inc) to cap any potential










Top Glass Plate


" Metal Spacers




attom Glass Plate


Glass Slides


Figure 3.2: Mold setup for preparation of silicone elastomer films (left) and silicone
elastomer coated glass slides (right).



silanol groups on the glass surface that would make the silicone elastomer adhere during

curing. On the bottom glass plate, there were 4 spacers of specific thickness, depending

on the thickness of the film desired, placed with double-sided tape. After pouring the

elastomer onto the bottom glass plate, the top glass plate, which was covered with a thin,

amorphous poly(ethylene terepthalate) (PET) sheet to ease film removal, was slowly laid

on top of the silicone elastomer, allowing it to rest on the spacers. A 5 lb weight was

needed on top of the mold to press the top glass plate completely down onto the spacers.

This mold setup provided smooth elastomer films of consistent thickness that would be

used for tensile mechanical testing.

For the preparation of silicone elastomer coated glass slides, the general mold

discussed above (Figure 3.2) was used. However, clean glass slides were placed on the

bottom glass plate with coupling agent treated side up just prior to pouring the elastomer

mixture. The slides were borosilicate glass of 75mm x 25mm dimensions and 1mm thick

(Fisher Brand). Before use, the slides were burned with a clean flame (blue color) from a

bunsen burner. Then they were wiped with a 1 Normal HCL solution. Lastly they were


PET sheet
\


I









treated with a 0.25 vol% allyltriethoxysilane (Gelest, Inc) coupling agent solution for 3

minutes on one side, rinsed with ethanol, and then dried in an oven at 1200C for 10

minutes. This coupling agent solution was prepared by first mixing 30 mL of ethanol

with 1.5 mL DI water. The pH of the solution was then dropped down to approximately

4.5 by adding Glacial Acetic Acid (Aldrich, Inc). The amount of acid added usually was

2 drops from a transfer pipet. After stirring for 3 minutes, 0.085 mL of

allyltriethoxysilane (ATS) was added and allowed to mix for an additional 5 minutes

before application to the glass slides. Another difference in the mold setup from the film

preparation was that the top glass plate was not covered with a PET sheet. This allows

the ultra smoothness of the glass to transfer to the elastomer surface on the glass slides.

These glass slides were used as a support mechanism for the elastomer film for surface

energy measurements and later flow cell experiments.

Mechanical Properties

Mechanical properties were examined of all the silicone elastomer formulations in

order to detect any significant changes in the bulk properties due to the silicone oil

additives. All the tensile mechanical testing experiments were performed on 1 mm thick

dogbones cut with an ASTM D1822-68 Type L die from silicone elastomer films. An

Instron model 1122 load tower with 90psi pneumatic grips, a 2001b calibrated load cell,

and Testworks 4.0 software was used for data collection and analysis of all experiments.

The tests were either performed with crosshead displacement or MTS high elongation

laser extensometer strain measurements, depending on the experimental protocol. The

age of the samples, either 1 day or 128 days old after cure, also depended on the

experimental design. Strain measurements were based on a 2 in/min crosshead

displacement and a 1 inch crosshead gauge length. All experiments conformed to ASTM









D412-97 at room temperature. For the extremely oily surfaced materials, a kimwipe was

wrapped around the gripped portion of the dogbones for better hold in the pneumatic

grips. For the crosshead displacement and the 1-day-old laser extensometer

measurements, 10 specimens were run for each formulation. For the other laser

extensometer measurements, only 2-3 specimens were available for each experiment.

Some of the mechanical properties experiments involved soaking the dogbones in

simulated seawater or DI water. The imitation seawater was mixed from InstantOcean

salts to a 1.0215 specific gravity, which was measured with a hydrometer. The specimens

were soaked in 15mL polypropylene centrifuge tubes of the desired liquid environment

for 3 hours at 800C. This time and temperature were chosen in accordance with coupling

agent adhesion test where 2 hours in boiling water was found to be equivalent to 30 days

immersion at room temperature.33 Placing the filled centrifuge tubes in a heated water

bath provided a constant heating environment. The liquid-filled tubes were allowed to

equilibrate to temperature before the elastomer dogbone specimens were added. After

the 3 hours, each silicone elastomer specimen was removed from the liquid and allowed

to equilibrate to room temperature for 5 minutes before being mechanically tested.

Surface Energetics

Various silicone elastomer formulations were examined with contact angle

measurements and calculated surface energies in order to detect any significant change in

the surface energetic due to the additives. A static sessile drop technique was performed

on silicone elastomer coated glass slides with both a goniometer and a digital capture

system. A variety of liquids, shown previously in Table 3.3, in 21L droplets were gently

placed on the solid substrate with a micropipet. With the goniometer, shown in Figure

3.3, the contact angles were read from the horizontal microscope lens piece. From the









digital capture setup, also shown in Figure 3.3, an image of a droplet on the surface was

taken from a web cam (3Com Homeconnect Web Cam) through the Viviewer software

on a computer. The lighting and contrast were adjusted with the software before image

was captured. The stages were aligned with a circular level and the sample was centered

and focused with a 3-dimensional micro-manipulator also before image capture. After

initial setup, the images were captured quickly after the liquids contacted the surface and

reached equilibrium in order to minimize interaction, evaporation, and contamination.

After all the images were collected, the UTHSCSA ImageTool 2.0 software was used to

measure the contact angles. The desired angle was drawn on the software, and then the

computer measured and reported the angle in a spreadsheet, as shown in Figure 3.4. From

all of these contact angles, the critical surface tension of the solid could be determined

through the modified Zisman plot (to be discussed later in the chapter). The angle on

both sides of the droplets was measured and each liquid was repeated 3-5 times

depending on the experiment.


Figure 3.3: Digital picture of the microscope goniometer setup (lett) and the digital
capture setup (right) for contact angle measurements.










NT iHA j i 1-A -A I I aL 3 a |Ba I IJHJ AM I


Figure 3.4: Computer screen image capture of the ImageTool software for measuring
contact angles from digital images of the liquid droplets. The image is of a 2pL droplet of
water on an unmodified silicone elastomer substrate.




Results and Discussion

Mechanical Properties

Crosshead displacement measurements

In order to examine the effect of additives on the bulk silicone elastomer, tensile

mechanical tests were performed. Initially, the nonfunctional additive formulations of

the silicone elastomer were examined with the crosshead displacement strain

measurements after curing at room temperature for 24 hours. The calculated elastic

modulus (E) results are shown in a graph in Figure 3.5. The software automatically

calculated the elastic modulus at the most linear portion of the graph as:

E = Ac / AS (1)









where Ac was the change in stress and As was the change in strain of two points in the

linear region of the stress-strain curve. However, upon examination of the stress-strain

curves for all of the silicone elastomer formulations, it was evident that there were two

linear regions on each graph, as shown in Figure 3.6. The kinetics for the increase in the

line slope at high elongations is examined more closely with the laser extensometer data

(the next section). All of the data in Figure 3.5 were recalculated so that the elastic

modulus was measured in the first linear portion at lower strain (20-50% strain). This

linear portion was chosen originally because of the low strain that a biological

environment would impose on a material. However, in later studies, the high strain

portion was also examined because of the high shear environment introduced to the

material as a foul-release coating. In Figure 3.5, the unmodified silicone (Owt% additive)

was only examined once, but compared next to each of the other sample sets. The

unmodified silicone had a noticeably higher elastic modulus, 1.4 MPa, than most of the

nonfunctional additive formulations. It was not statistically different, though, when

compared to the 5wt% 500cSt and 5000cSt formulations in this experiment because of

their large standard deviations. The 20wt% silicone oil formulations for each additive

type were significantly lower in elastic modulus than the formulations with less amount

of silicone oil additive. A visible trend on this graph was that as the amount of silicone

oil added was increased, the elastic modulus decreased. This trend is followed except for

the two formulations just previously discussed, 5wt% 500 cSt and 5000 cSt. Another

trend, noticeable in the 10 and 20wt% linear chain silicone oil formulations, was that as

the viscosity, in other words molecular weight, of the silicone oil increased, the elastic

modulus increased. An important point to remember was that none of these additives











0 wt% 5 wt%









Ih


10 wt% 20 wt%









llI


50 cSt


500 cSt 5000 cSt
Silicone Oil Additive


1.8
1.6
1.4
1. 1.2
~, 1.0

S0.6
o
. 0.4
0.2
0.2
0.0


Figure 3.5: Effect of nonfunctional additives at different amounts on the elastic modulus
(calculated between 20-50% strain) for a silicone elastomer. Strain measurements were
made from crosshead displacement.


SAJ
High Strain Modulus
5.0
2 4.0
3.0
3.0 Low Strain
S2.0 -Modulus
1.0
0.0
0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5
Strain (mm/mm)
Figure 3.6: Stress-strain curve for unmodified silicone elastomer with strain
measurements made from crosshead displacement. Notice the 2 different linear
segments, which correspond to different moduli values at low strain versus high strain.
Performed at room temperature with a lin. gage length and 2 in/min crosshead
displacement.


tetrakis









were chemically bound to the elastomer, thus the cross-link density was roughly the same

in each elastomer formulation. The effect of the bulky molecule

tetrakis(trimethylsiloxy)silane (abbrev. tetrakis), as compared to the linear chains on the

modulus was only slight. In fact, the elastic modulus of the bulky oil was not

significantly different from the small linear chain silicone oil, 50cSt. The physical

interactions behind these trends are discussed in detail with the laser extensometer

measurements where the standard deviations are smaller due to an increased accuracy in

technique. However, many of the same trends were identified with the laser

extensometer measurements. It was also noted that perhaps the elastomer films were not

completely cured after 24 hours so the time of day that the samples were run could have

slightly changed the time allowed to cure and thus had a small influence on the

mechanical properties.

Laser extensometer measurements

While performing these tests with the crosshead displacement, however, it was

evident that there was a considerable amount of slippage out of the pneumatic grips while

under high elongations. This led to a lower value for strain in the modulus calculations

and thus a higher reported modulus. As a result, new batches of all the elastomer




Table 3.4: Effect of cure time at 800C for 5wt% 50 cSt silicone oil modified silicone
elastomer. (n = 10)

Cure Time Modulus (MPa) Std.dev.

2 hours 0.85 0.07

5 hours 0.92 0.14









formulations were examined using a high elongation laser extensometer for strain

measurements. This technique minimizes the influences of grip slippage. These samples

were cured at 800C for 2 hours to make sure cure reaction was complete and then allowed

to rest for 24 hours at room temperature before testing. To prove that curing was

complete after 2 hours, a sample of 5wt% 50cSt was also cured at 800C for 5 hours.

After comparison of the two different cure times, values shown in Table 3.4, it was

evident that there was no significant difference in elastic moduli between the two

samples. As a result, it was concluded that 2 hours at 800C was sufficient time for cure

completion.

When the stress-strain curves of the elastomers were examined with the laser

extensometer, again two distinct linear portions of the curve were present, as seen in

Figure 3.7. The elastic modulus calculated from the low strain region (30-50% strain)

and high strain region (120-150% strain) is given in Figure 3.8 for all the elastomer

formulations, including a IkSt linear silicone oil additive. This graph again showed the

significant difference in modulus between the two strain areas. After examination, the

high strain modulus was on average 3.1 0.5 times as much as the respective low strain

modulus for all the formulations. For example, the 5wt% 50cSt silicone elastomer

formulation had a low strain modulus of 0.9 (0.1) and a high strain modulus of 2.41

(+0.2), which was 2.7 times as high. This upturn change in modulus, around 80% strain,

occurred in every sample in the same regime. As a result, the factor must lie within the

elastomer, rather than the oil additives. The base elastomer resin was known to have a

broad range of molecular weight vinyl-terminated PDMS chains. Thus, it was theorized

that this change in modulus at high strains was due to the limited extension of the smaller











6.0

5.0

r" 4.0 High Strain
Q- Modulus
U 3.0

,2.0
C 2.0 Low Strain
Modulus 0
1.0

0.0
0 50 100 150 200 250 300
Strain %

Figure 3.7: Stress-strain curve for an unmodified silicone elastomer with strain
measurements made from a laser extensometer. Two different slope regions are evident
between high strain and low strain corresponding to 2 different moduli values.



molecular weight chains in the elastomer network. Mark et.al. noticed the same upturn in

modulus at high elongations and on overall elongations when he examined bimodal

trifunctional and tetrafunctional PDMS networks." They were convinced of this theory

when the upturns were reversible and their monodisperse PDMS networks showed no

visible upturn. In other types of rubber elastomers, an increase in modulus at high strains

has been usually attributed to a strain-induced crystallization, where the entropy of the

elastomer was decreased enough upon elongation to promote crystallization, which then

acted as physical crosslinks.45 However, since PDMS elastomers have such a low glass

transition temperature, they do not crystallize even under a high strain.47

The introduction of silicone oils into the elastomer network brought a few more

interactions when the mechanical properties were examined. At both the high and low

strain, the modulus decreased as the percent weight of oil additive increased. There were









a few samples that were not significantly different because of larger standard deviations,

but the overall general trend was present. However, as the viscosity (or MW) of the

silicone oil additive increased, there was no significant change detectable in the high or

low strain elastic moduli. One critical interaction, which could contribute to differences

in moduli values, was the presence of silica agglomerates in the network system. At

normal conditions, the silica agglomerates are unreactive due to the trimethylsilylation of

the particle's surface. However, at a critical stress, induced in the network from tensile

elongation, the silica agglomerates can break apart and then introduce reactive hydroxyl

groups from the freshly exposed particle surfaces. These hydroxyl groups can then either

react with the close polydimethylsiloxane chains's silicon-oxygen backbone linkages and

increase the cross-links in the region or produce hydrogen bonding between the particle

and the PDMS chains.48 These interactions should increase the elastic modulus of the

elastomer. Once the reactive sites are inhibited, silicone chains are able to slip over the

silica surface with ease.48' 78 The addition of nonfunctional silicone oils should introduce

even higher chain mobility and thus a higher ease of gliding over the surface. As a result,

as the molecular weight of the PDMS chains decreased or the amount of additive

increased, there was an increase in chain mobility and thus a lower elastic modulus. This

was evident in the values provided in Figure 3.8 where the percent weight additive was a

detectable factor. Polmanteer and Lentz have shown that this same generation of reactive

sites on the silica filler particles can also occur under high shear mixing conditions of the

silicone elastomer resin.49 As the filler aggregates are broken in this instance, the silanol

reactive sites can again introduce hydrogen bonding or chemically react with the

polydimethylsiloxane chains, as previously mentioned. Even though the elastomer mixed









in this experiment was not under high shear, the relatively low strain that was necessary

to introduce this occurrence during mixing, leads to the conclusion that the effect of the

silica particles occurred at both points of modulus measurement. Boonstra et.al.

introduced a stress-strain graph, which displayed reactive versus unreactive silica

particles in vinyl-cured silicone elastomer.48 The two curves both had upturns around

100% elongation, but the reactive particle filled elastomer had a higher modulus at both

low and high strain. This graph proved that the silica particle reactivity had little

influence on the upturns seen on all the elastomer formulations' stress-strain graphs, but

did influence the relative elastic moduli values.

The elongation at failure should be influenced by the same factors as just

previously discussed, but also strongly directed by the number of chain ends present in

the network. As the number of chain ends increase, the elongation should decrease as the

untied ends introduce more defects, which ultimately leads to failure. The chain ends

also are unable to carry any of the stress loads so the surrounding chains undergo a larger

stress then being applied to compensate. The low molecular weight additives, introduce

more chain ends from a set volume into the system. However, comparisons and trends

were difficult to declare with the elongation at failure values obtained because of large

standard deviations. This lack of reproducibility of the ultimate mechanical properties of

silicone elastomers were observable in these experiments and also stated by other

researchers.47 As a result, the elastic modulus was calculated and compared in these

experiments as an observance of mechanical properties, along with its role in adhesion

mechanics and rubber elasticity.











3.0
N Low Strain% Modulus N High Strain% Modulus

2.5


S2.0


1.5
O

1.0


0.5


0.0


Silicone Formulation (wt%; cSt)
Figure 3.8: Comparison of elastic moduli values between the different freshly cured
silicone elastomer formulations from both the low strain and high strain regions. All of
the strain measurements were collected from a laser extensometer.



When the low strain elastic moduli data from the laser extensometer and

crosshead displacement are compared, similar trends, which were previously discussed,

are evident. Examination of a graph of the comparison, shown in Figure 3.9, shows that

again an increase in percent weight of silicone oil, significantly decreased the elastic

modulus, except for the 10wt% 5000 cSt formulation which was not detectible as a result

of the larger standard deviation on the 5wt% 5000 cSt formulation. This could be

attributed to the higher molecular weight formulations having a more visibly lubricious

(oily) surface and thus more grip slippage and higher standard deviation. As predicted

earlier concerning the effect of grip slippage on modulus, the crosshead displacement

modulus data, where slippage was an issue, was on average 1.6 ( 0.1) times higher than

the extensometer modulus data. As a result, the extensometer data was closer to the true












1.8
extensometer U crosshead
1.6

1.4

1.2

1.0

2 0.8

0.6

0.4

0.2

0.0



Silicone Formulation (wt%; cSt)
Figure 3.9: Comparison of elastic moduli values between the different freshly cured
silicone elastomer formulations from the two sources of strain values, the laser
extensometer and crosshead displacement. All modulus values are from the low strain
region.



mechanical properties of these formulations. However, on a large-scale view, the

changes seen in modulus between the formulations were miniscule and differences

should have little influence on the biological adhesion.

Aged silicone measurements

Since many of the applications for these silicone elastomers could be over a few

years lifespan, the mechanical properties of aged silicone formulations were examined.

All the formulations were cured at 800C for 2 hours, and then aged in air approximately

128 days at room temperature. The samples were then mechanically tested at room

temperature with the laser extensometer. Again the stress-strain curves exhibited two

distinct linear portions and so both were examined. The moduli calculated for these aged

samples are shown in Figure 3.10. The high strain moduli were 2.7 (+0.3) times higher











3.5
Low Strain 0 High Strain
3.0

0.. 2.5

S2.0 -

0
1.5

01.0

0.5 -

0.0



Silicone Elastomer Formulation (wt%; cSt)

Figure 3.10: Comparison of elastic moduli values between the different 128-day aged
silicone elastomer formulations from both the low strain and high strain regions. All of
the strain measurements were collected from a laser extensometer.



than the moduli at low strain. Just as in 1-day-old samples, a noticeable trend was as the

amount of oil additive was increased, the elastic modulus decreased. However, when the

change in molecular weight (or viscosity) was examined, most formulations were not

significantly different, except for 10, 20wt% 5000 cSt and 5, 20wt% IkSt, which had

lower moduli and happened to be the visibly oily surfaced samples. The decrease in

elastic modulus for these four samples was obviously due to the large migration of the

large chains. It was theorized that as the silicone oils chains totally migrated to the

surface, there was an increase in free volume left in the network elastomer since the

crosslink density should not have changed. Thus the network chains had an increase in

chain mobility, which, as discussed earlier, would lower the modulus.












1.6
SFresh Aged
1.4

1.2

1.0

0.8

.) 0.6

1 0.4

0.2

0.0



Silicone Elastomer Formulation (wt%; cSt)
Figure 3.11: Comparison of the low strain region elastic moduli values between the
different silicone elastomer formulations at two different resting times after cure, 1 day
before testing (fresh) and 128 days before testing (aged).



When compared to the extensometer moduli values of the 1 day old (fresh)

samples, some of the aged samples showed a markedly higher elastic moduli over time at

both low and high strains, as shown in Figure 3.11 and 3.12. The same trends that were

noticed and discussed with the 1-day-old samples were observable with both of the strain

regions. The complete initial curing of the silicone elastomer was previously verified in

Table 3.4 where there was minimal difference in moduli between curing at 800C for 2

hours versus 5 hours. The lower molecular weight silicone oil additives increased the

modulus in the aged samples as compared to the freshly prepared elastomers. This was

attributed to excess curing agent in the network. The slow decomposition of the residual

hydride groups caused a postcuring hydrolysis reaction with water in the system where












3.5
S Fresh MAged
3.0

a 2.5


o
2 1.5
U)
C 1.0

0.5

0.0



Silicone Elastomer Formulation (wt%; cSt)

Figure 3.12: Comparison of the high strain region elastic moduli values between the
different silicone elastomer formulations at two different resting times after cure, 1 day
before testing (fresh) and 128 days before testing (aged).



the hydride groups were converted to silanol groups.79 The increase in silanol groups

increased the modulus by either reacting with the silicone backbone or the production of

more hydrogen bonding, just as in the case of the silica particles. This effect was more

noticeable with the low molecular weight additives than the unmodified elastomer

because of the increase in mobility of the water and curing agent molecules, which thus

decreased the reaction time. For the case of high molecular weight additives, this

postcure reaction was less of an effect than the oil migration, which was just previously

discussed.

Soaked silicone measurements

Since the application for the marine industry would ultimately be in a seawater

environment and many laboratory tests are performed in distilled water, these

environments were examined for their effect on mechanical properties on the silicone









elastomer formulations. For each formulation, 2 approximately 14-day-old dogbone

specimens were soaked for 3 hours at 800C in an environment, DI water or seawater. The

calculated elastic moduli values at high strain are provided in Figure 3.13. Overall, there

was little difference in either environment as compared to the 1-day-old elastomer

samples, previously presented, which was just exposed to air. This led to the conclusion

that there was little uptake of water and the elastomer was hydrolytically stable. The low

strain modulus values also had little water uptake influence observable. J.J. Kennan et al.

also observed little significant change in mechanical properties and advancing contact

angle when a hydrosilylation silicone elastomer was exposed to saline water at elevated

temperatures.80 However, if there was water uptake, it would probably have been more

obvious on the effect of elongation at break. The water molecules would have acted as

defects and decreased the percent elongation at break, just as previously discussed with

the effect of chain ends. However, ultimate tensile properties are not easily reproducible,

as seen in these experiments and noted by other researchers, so little physical mechanics

could be determined from the elongation at break.47

Surface Energetics

Goniometer

Another factor affecting adhesion is the surface energetic at an adhesion

interface. As a result, the surface energy of the silicone elastomer formulations must be

characterized to be able to later compare the adhesion to different formulations. Initially

many of the silicone formulations were examined through contact angle measurements

from a goniometer. Typically a Zisman plot (cos 0 versus yiv) is used to calculate the

critical surface tension (y,) of the solid substrate from contact angle measurements.











3.5
Fresh (Air) DI Water Soaked E Seawater Soaked
3.0

I 2.5 T

2.0

0
S1.5

S1.0

0.5

0.0




Silicone Elastomer Formulations (%; cSt)

Figure 3.13: Comparison of the effect of environment (air, DI water, and seawater) on
elastic moduli at high strain region for the various freshly cured silicone elastomer
formulations.



This relationship comes from Young's interfacial equation of equilibrium,

ysv Tsl 71v cos 0 (2)

where ysv, yTs, and y1v are the surface tensions for the surface-vapor, surface-liquid, and

liquid-vapor interfaces, respectively, and 0 is the contact angle. Theoretically, as cos 0

approaches 1 (0 = 0), the liquid completely wets the surface and then ysv -ylv. This

critical surface tension point is determined by extrapolating a linear regression line from

the data of multiple liquids on a Zisman plot.

However, since the solid substrate being examined was poly(dimethylsiloxane)

elastomer, a low surface energy material, a problem was encountered. There was an

induced hydrogen bonding, thus nonlinearity, when polar liquids (usually with a surface









tension greater than 50 mN/m) was introduced to the surface, such as water.81' 82 Young's

equation does not take intermolecular forces into factor. Thus in order to counteract this

nonlinearity, Good proposed a modified Zisman plot of cos 0 versus 1/y1v1/2 for

calculation of critical surface tension of low surface energy materials.53. This

relationship comes from the introduction of a geometric mean combining rule (equation

3) for an i-j interface, which allows ysi to be eliminated from the equation when combined

with Young's equation and thus results in equation 4.53,83

Yij Yi + yj 2 (7iyj)1/2 (3)

cos 0 = 2(D (ys / 71v)1/2 1 (4)

In equation 4, the cos 0 is directly proportional to 1/yiv1/2, which thus promotes the

modified Zisman plot. An example of this plot is provided in Figure 3.14 for an

unmodified silicone elastomer. From this plot, the surface energy of the silicone

elastomer substrate was extracted. There were many assumptions with this type of

surface analysis, which were discussed in Chapter 2. Some of the natural variability in

data points was attributed in part to some of the assumptions not being completely met.

The static contact angles measured with the goniometer for the 5 different liquids

are provided in Table 3.5. Some of these angles were previously reported by Wade

Wilkerson in his University of Florida master's thesis.3 This table also provides the

calculated surface energies of the various elastomer formulations from a modified

Zisman plots. As desired, the surface energies of the formulations were all relatively the

same. All the formulations that were not included in this table were also assumed to have

relatively similar surface energy values. This should be the case, since the elastomer and

the silicone oils all had the same chemical composition. Any small variation of the











1.2

1.0-

0.8 y= 13.52x- 1.916 Prop
R2 = 0.954
0.6
/ACN
DMF
~ 0.4
I-
8 0.2
0 EthGly/
0.0
0. 0 0.05 0.10 / 0.15 0.20 0.5
-0.2

-0.4- /* H0

-0.6
11 (Surface Energy)^1/2

Figure 3.14: A modified Zisman plot for an unmodified silicone elastomer from digital
capture contact angle measurements. The equation for the linear regression and
respective regression correlation are provided on the graph.



surface energies was attributed to either the packing difference of the methyl groups on

the surface, discussed more with the digital capture contact angles, or standard technique

error. Usually, the silicone oils have a tighter packing of the methyl groups on the

surface than the silicone elastomer.84 However, this could not be detected with the

standard deviations from the goniometer technique.

Since all of the elastomer samples were cured against glass, the effect of this

particular cure surface was examined. The other option that was presented during

preparation was curing the silicone elastomer with no glass cover plate, which left the

silicone to cure with an air interface. This technique was rarely performed because the

control over film thickness and uniformity was lost. However, when the surface energies

were compared, given in Table 3.6, there was a slight, noticeable difference. The

elastomer cured facing air had a slightly lower surface energy than the glass surface cured

elastomer. This small difference was again contributed to the packing (density) of the









Table 3.5: Contact angle measurements and calculated critical surface tension values
(modified Zisman plot) from a goniometer of a few silicone elastomer formulations.

Contact Angles (Degrees) y7
Samples Water Mel DMF ACN 1-Prop (mN/m)
Unmodified 109 67 55 48 32 23.0 0.4
5% 50 cSt 110 66 56 44 31 23.3 0.3
20% 50 cSt 109 64 54 46 25 23.7 0.4
5% 5000 cSt 108 65 55 48 26 23.3 0.2
20% 5000 cSt 103 64 52 48 26 23.1 0.3


surface methyl groups due to varying interfacial environments. The closer packing of the

methyl groups on the surface, contributed to a lower contact angle.53 Thus the high

energy of glass enhanced the motility and packing factor of the methyl groups at the

surface during cure.50

Digital capture

Another technique useful for measuring contact angle was taking a digital image

of the liquid droplet on the surface. With ImageTool 2.0 software, the contact angle

could be determined from the digital image. Images could also later be examined and

compared. This technique gave a permanent documentation of the surface energetic of

the substrate, as seen in Figure 3.15. When compared to digital image measurements,

provided in Table 3.7, for an unmodified elastomer substrate, the goniometer surface

energy was slightly higher at 23.0 + 0.4 mN/m, than the digital capture calculated surface

energy, 21.5 0.3 mN/m. This difference was attributed to a larger standard deviation on

the contact angle measurements for all liquids with the goniometer technique. The

goniometer did not have an enlarged view of the surface-liquid interface as the digital

images, so there was decreased accuracy with the goniometer technique. This digital









Table 3.6: The effect of cure surface on the contact angles and calculated critical surface
(from modified Zisman plot) on an unmodified silicone elastomer.

Contact Angles (Degrees) Y
Cure
Cure Water Me DMF ACN 1-Prop (mN/m)
Surface

Glass 103 67 54 45 30 22.9 0.4

Air 105 72 58 48 35 22.0 0.3



technique had the advantage of also being capable of capturing videos in order to

examine the effect of time, which will be important in later experiments. Similar sources

of error with the goniometer were present with the digital capture method, such as dust,

swelling, solvent evaporation, and unsymmetrical droplet, even though they were

minimized.

For permanent documentation, some of the silicone elastomer formulations were

examined with the digital capture system. The contact angles and calculated surface

energies are provided in Table 3.7. There was a clear trend that the linear oil additives,

which had partially migrated to the surface, gave the substrate a slightly higher surface

energy, of approximately 22.6 mN/m, than the unmodified silicone elastomer, which was

measured as 21.5 mN/m. This trend can be attributed to the packing density of the

methyl groups at the surface interface. Silicone oil has a significantly higher density of

methyl groups than the crosslinked silicone elastomer.84 According to Hoernschemeyer,

the increase of surface packing resulted in a higher concentration of attractive centers.50'80

This caused more interactions between the solid and liquid and resulted in a lower contact

angle, which was evident with the contact angles provided in Table 3.7. The lowering of

contact angles resulted in an increase in calculated surface energy. However, this theory



















ACNMF
mom...


Figure 3.15: Digital images of each of the 5 liquids used to measure contact angles and
subsequently calculate surface energy (modified Zisman plot) on an unmodified silicone
elastomer.


Table 3.7: Average contact angle measurements and calculated critical surface tension
(modified Zisman plot) for various silicone elastomer formulations from the digital
capture system.
Contact Angles (Degrees) Y c
Samples Water Ethgly DMF ACN 1-Prop (mN/m)
unmodified 114 88 61 56 38 21.5 0.3
5% 50 107 89 57 52 30 22.5 0.2
20% 50 105 86 57 48 29 22.7 0.2
20% 5000 104 80 55 52 27 22.6 0.2

20% Tetrakis 110 88 64 55 35 21.6 0.2


;;~?
;i;;; ;iiiiiiiii~









contradicts Zisman's original theory that an increase in surface packing results in a larger

force-field around the surface groups, which should repel the liquids and increase the

contact angles (decrease the surface energy).55 All of the data presented here follows

Hoernschemeyers' concepts. In the case of the bulky molecule additive,

tetrakis(trimethylsiloxy)silane, a similar surface energy and contact angles as the

unmodified was observed, evident in Table 3.7. This was attributed to it having a similar

methyl group packing density as the unmodified silicone elastomer. A source of error

that was noticeable in the digital images from the highly oily surface of 20wt% 5000 cSt,

was the deformation of the substrate surface (the silicone oil) by a DMF droplet, as

shown in Figure 3.16. The interface deformation from the DMF droplet looks

comparable to the drawing of Rusanov's model for contact angle-induced deformation in

Figure 3.16.50. This occurrence goes against the assumption that the solid substrate is

rigid thus this was a source of error for this formulation and other oily surfaced

substrates. Another issue was that the excessive oil on the surface meant that the contact

angles were a liquid-liquid interaction as compared to a solid-liquid interaction. Overall,

the surface energies of all of these silicone elastomer formulations only varied minutely

and this factor should have little effect on adhesion between comparisons of the various

formulations with biological organisms.

















Figure 3.16: Digital image of a DMF droplet on a visibly oily surface of a 5wt% 5000
cSt silicone elastomer sample (left). The deformation of the surface caused by the liquid
droplet was observable. This relates to Rusanov's model for contact angle induced
surface deformation (right). (adapted from Andrade)50


t----














CHAPTER 4
ENTEROMORPHA SPORE SETTLEMENT/RELEASE ASSAY


Introduction

Key roles in adhesion to a substrate that must be examined are lubricity and

roughness of the substrate surface. In order to evaluate the influence of roughness on

adhesion, engineered topographies, of various shapes and dimensions, were produced on

silicone elastomer substrates. High fidelity of these patterns was verified during

replication processes with White Light Interference Profilometer (WLIP) or Scanning

Electron Microscope (SEM). The lubricity of the surface was controlled by the

incorporation of various nonfunctional silicone oils into a silicone elastomer substrate.

These chemical modifications were characterized and discussed in the previous chapter.

These additives allowed for similar surface chemistry and bulk properties between every

sample. To evaluate these engineered material factors, Enteromorpha spores were

chosen as the target biological organism to examine settlement and adhesion to the

engineered substrates.


Materials

Silicone Elastomer Substrate

All samples used in this Enteromorpha spores assay were silicone elastomer

substrates prepared from Dow Corning's Silastic T2 formulation. This is a 2-part,

hydrosilylation polymerization reaction with cured properties as discussed in Chapter 3.

This particular elastomer was chosen for its transparency (important for the









hydrodynamic flow cell), low-shrinkage (important for topography reproduction), and

few undisclosed additives (important for chemical modifications). Two linear, trimethyl-

terminated polydimethylsiloxane oils (Gelest, Inc.), of 50 cSt and 5000 cSt viscosity,

were separately added to the elastomer resin during mixing of the base resin and curing

agent. These chemical modifications provided lubricious surfaces, while generally

holding all other factors constant, such as elastic modulus and surface energy. This was

all proven in Chapter 3 with characterization of many different nonbonding silicone oil

additives in the Silastic T2 silicone elastomer.

Epoxy

As part of the replication process, a low shrinkage epoxy was necessary for

reproduction of engineered topographies. The chosen epoxy was EPON 828 Resin,

which is a difuntional bisphenol A/epichlorohydrin derivative. A Jeffamine D-230

hardener (Huntsman), which is a polyoxypropylenediamine with an approximate

molecular weight of 230 g/mol (n = 2-3), was used at a ratio of 9.7 g of the resin to 2.7 g

of the hardener. The two parts were weighed and mixed thoroughly in order to undergo

the reaction shown in Figure 4.1. After completely degassing in vacuum for 30 minutes,

the mixture was cured at 600C for 5 hours in order to increase the reaction rate.

According to Huntsman, the manufacturer, at room temperature the reaction takes 7 days.

The chosen cure temperature was also slightly lower than the deflection temperature of

680C for the cure system (Huntsman product information sheets).

Glass Slide Preparation

All silicone elastomer samples were coupled to borosilicate glass slides (75mm X

25mm X Imm) for rigidity. The coupling agent chosen was allyltriethoxysilane (ATS)













Epon 828 Epoxy

CH3 O

C- O CH CH2

CH3


0

HC-CH2


Jeffamine D230 Hardener

CH3 CH3
H2N-CHCH2 OCH2CH NH2

n=2-3


H OH H

H-N-R'-.- CH Ns
R

Reaction Mechanism


CH3 CH3
N-CCHH2OCH2CH N


CH3

S-C

CH3


Crosslinked Epoxy


CH3 CH3
N-CHCH2 OCH2CH+ N


Figure 4.1: Epoxy cure reaction. Reaction at each of the NH's is possible, which leads to
a crosslinked matrix.




from Gelest, Inc. For the coupling agent solution, a small amount of Aldrich's Glacial


Acetic Acid was needed. In order to prevent silicone elastomer adhesion to the glass


molds and silicon wafers, hexamethyldisilazane (HMDS) from Gelest, Inc. was applied to


the surfaces.


Biological Algae Material

Enteromorpha linza fertile algae plants were collected from Wembury beach, UK.


The zoospores from these plants were then gathered and prepared for experiments as


described by Dr. Maureen Callow in the literature.10


0

H2C CH /









Methods

Unpatterned Elastomer Samples

All of the unpatterned silicone elastomer coated glass slides were prepared as

previously described in Chapter 3. These samples were used for examination of the

effect of silicone oil additives on Enteromorpha spore settlement and allowed for controls

in the topographical studies.

Patterned Elastomer Samples

All of the engineered topographies were prepared through a few replication steps,

beginning with an etched silicon wafer. Standard photolithographic techniques were used

to prepare 5tm wide ridges separated by 5am, 10tm, and 20tm wide valleys or 5tm

wide square pillars separated by 5am, 10m, or 20am wide flat areas on 3in. diameter

silicon wafers in the arrangement shown in Figure 4.2. The length of ridge topographies

was also varied from 60am, 800am, or 10,000am. Each silicon wafer was first coated

with a Clariant AZ 1529 positive photoresist and then exposed to UV light source through

a patterned mask. Two different pattern depths, 1.5am and 5am, were prepared. Chuck

Seegert etched all of the 1.5am deep patterned wafers by a reactive ion etching (RIE)

process at the University of Florida, while Unaxis (Tampa, Fl) etched the 5am deep

patterns through a standard Bosch process.

In order to achieve a positive copy of the topographies from the silicon wafer, 3

intermediate replication steps had to be performed, as shown in Figure 4.3, before the

patterns could be cast into the final silicone elastomer slides. The first step was to make a

direct copy off of the silicon wafer with solvated polystyrene (PS). In a glass flask, 5g of

PS was mixed with 30 mL of chloroform. The mixture was allowed to stir and dissolve








5 pm wide Pillars


df.M


60 pm long ridges


OU


800 mr long ridges


10,000 m long ridges


Figure 4.2: Placement of topography on the etched silicon wafers. Each large quadrant
of patterns had 3 smaller rectangular areas, which corresponded to the 3 different
valley/flat-area widths, 5[tm, 10tm, 20[tm.


for at least 2 hours. Meanwhile, the silicon wafer was cleaned with an ethanol wash,

dried, and treated with hexamethyldisilazane. A small amount of dissolved PS was then

placed on the clean silicon wafer, which was completely leveled for even thickness and

allowed to dry for 24 hours. In order to remove the dried PS, the wafer was placed on a

block of dry ice. This allowed for removal of the PS film due to the difference in thermal

expansion coefficients. The PS was brittle and thin, so attachment to a glass plate for

mechanical support was necessary. However, PS was chemically stable and so the

backside was argon plasma treated in order to increase adhesion to glass with a quick

cure epoxy. The patterned PS film was a negative of the topographies on the wafer, thus

the pillars were now divets.









Positive Copy
Pillars


Silicon wafer



Silicone K
Elastomer N


Pillars


Negative Copy
Indentations


m/ Polystyrene

i7/


SEpoxy
Glass Slide


Silicone Slide

Figure 4.3: Replication process from patterned silicon wafer to final patterned silicone
elastomer coated glass slides.


The second replication step involved curing Dow Corning's Silastic T2 silicone

elastomer on the patterned polystyrene in order to obtain a positive of the original

topographies. Since the PS was so brittle, continuous replication off of this mold was not

feasible. As a result, additional replication steps were necessary to obtain a durable

mold. Approximately 20g of the silicone base was mixed with 2g of the silicone curing

agent. After degassing, a small amount was poured on top of the secured PS replicate.

This was allowed to cure in air environment at room temperature. After 24 hours, the

silicone elastomer could be demolded. At this point, the patterns could be cut and

rearranged to the desired arrangement, as shown in Figure 4.4. This particular


i ill











o t rKey Feature Width Valley Width
10,000 patterns 10,000 in both spore settlement and release assays were the 1m length
length ridges length ridges 5 sm widewe
orthogonal to parallel to pillers r5 5
Slow nolows
FLOW 5 10
5 20
5 5
5 10
5 20


Figure 4.4: Topography layout on the silicone elastomer coated glass slides as compared
to the direction of hydrodynamic flow.



topography arrangement was chosen for a flow cell apparatus so that the impact of the

hydrodynamic flow as compared to the direction of the ridges could be examined. The

desired patterns in both spore settlement and release assays were the 10,000p m length

ridges and the pillars of the 3 different valley widths. Since 2 ridge pattern sets were

needed, one for each flow direction, two silicone elastomer replications off of the PS

were needed to make one appropriately patterned elastomer mold. The cut patterns were

then placed pattern side down on a clean 4"x 4"x 1/12" glass plate. Cured silicone

elastomer makes a natural seal with the glass so that when additional silicone mixture

was poured on top the patterns were not lost. This glass plate was then set on a larger

(8"x 8"x 3/8") glass plate, which had 4 4mm. thick spacers. Then approximately 120g

of silicone base was mixed with 12 g of silicone curing agent and degassed. This mixture

was poured on top of the inverted elastomer patterns and pressed to the spacer's thickness

with another clean 8"x 8"x 3/8" glass plate. This mold was allowed to cure at 800C for 1

hour. Upon removal of the cured silicone elastomer from the glass plate, a "well" (or

recess) was formed from the thickness of the 4"x 4"x 1/12"glass plate with the patterns

facing upward in the middle.









The next replication step involved the creation of an epoxy mold from the silicone

well just described. The epoxy was first mixed in a weight ratio of 9.7g Epon 828 resin

to 2.7g of Jeffamine D230. The mixture was allowed to degas completely under vacuum.

The silicone well was placed on a large glass plate so that there were minimal air pockets

underneath the silicone elastomer. The silicone elastomer was then placed in a vacuum

oven at 800C for 30 minutes to ensure complete cure and to remove residual moisture.

The epoxy mixture was then slowly poured into the silicone well up to the brim. After

pouring of the epoxy, the mold could not be moved much or ripples would result in the

cured epoxy. A 5"x 5"x 1/8" glass plate, which had a spray thermoset mold release dry

coating (Stoner, Inc), was slowly lowered on top of the silicone well encapsulating the

epoxy. The epoxy does not self-release air bubbles, so care was taken to not introduce

any bubbles in this step. The mold was then placed into an oven at 600C for 3 hours to

cure. The epoxy was allowed to cool to room temperature before removal from the

silicone in order to prevent epoxy deformation. This epoxy had negative patterns, as

compared to the silicon wafer, and was used as the repeatable master mold for patterned

silicone slide production.

In order to reproduce the positive topographies onto silicone elastomer bound to a

glass slide, the general mold setup shown in Figure 4.5 was used. The epoxy mold was

placed on the bottom glass plate with the patterns facing upwards while an ATS treated

glass slide was taped to the upper plate, treated side exposed. The preparation and

application of ATS coupling agent was described in detail in Chapter 3. Appropriate

metal spacers were attached to the bottom plate that would make an approximately

700atm thick elastomer film. The thickness of the epoxy mold, the glass slide, and the










Epoxy Mold Glass slide
Metal (Pattemrns up) (ATS t eated side up)
Spacers





Figure 4.5: Final mold setup for reproduction of a patterned silicone elastomer coated
glass slide from an epoxy master mold.



double-sided tape had to be considered. The silicone elastomer was mixed in a 10:1

weight ratio of base resin to curing agent. After degassing for 25 minutes, the silicone

mixture was poured on top of the epoxy patterns. The top glass plate was then slowly

lowered onto the silicone mixture with the glass slide towards the elastomer and centered

over the patterns. A 5 lb weight was placed on top of the molds to ensure accurate

thickness. This setup was then placed in a 600C oven for 5 hours to increase the

crosslinking reaction rate. After cooling and removing the patterned silicone coated

slides from the epoxy, the excess silicone elastomer around the glass slides was trimmed

off. The film thickness was then measured with a micrometer. The samples that were of

appropriate thickness (600-800tm) and of good pattern fidelity were then individually

placed in polyethylene bags and shipped to Dr. Maureen Callow's group at the University

of Birmingham, UK for Enteromorpha studies.

Adhesion Fidelity

In order to physically endure the hydrodynamic current in a flow cell as a coating,

the silicone elastomer had to be completely adhered to the glass slides. Every sample

was tested for adhesion according to ASTM D3806, which was a quick, qualitative hand









pull-test to measure relative adhesion to the glass slides. If the sample coating failed

adhesively, the slides were not shipped and discarded.

Topography Fidelity

In order to quantify the biological growth or release response to surface

topography, maintaining the fidelity of the patterns were necessary throughout the

replication processes. The first step was to visually examine the smoothness and clarity

of every patterned sample. Images of the engineered topographies were then taken

periodically with either a JEOL 6400 Scanning Electon Microscope (SEM) or a Wyko

NT 1000 (Veeco Metrology software) White Light Interference Profilometer (WLIP). A

Au/Pd coating was applied to the SEM samples and examined under 100x to 10,000x

magnification in order to detect any significant defects. WLIP technique also gave a

nondestructive, noncontact 3-dimensional image of the patterned surfaces.

Spore Settlement Assay

Once the silicone elastomer coated glass slide samples arrived at Dr. Callow's

laboratory, they were incubated in filtered, sterilized artificial seawater (Instant Ocean)

for 24 hours at 200C. The slides were then placed in individual polystyrene petri dishes

and 10 mL of a 2 x 106 spores/mL Enteromorpha zoospore concentration were added to

each dish. The zoospores were then incubated on the substrates for 1 hour at 200C in the

dark. The nonadhered spores were then washed off of the substrates with artificial

seawater and fixed with a 2% glutaraldehyde in seawater solution for 10 minutes.

Settlement counts were then taken from images examining the autoflorescence of the

chlorophyll.61 On the unpatterned elastomer slides, spores were examined and counted at

1mm intervals down the center of the slide's long-axis for a total of 30 measurements for

each slide. Three replicate slides were run for each formulation. For the flat areas in









between the patterns and on the actual topographies, the spores were counted at 0.5mm

intervals along the 10mm length of the pattern squares. Exact counting methods and

calculations have been previously described by Dr. Callow.61

Spore Removal Assay

For examining the adhesion of the Enteromorpha zoospores, the same

experimental methods for settlement were performed, as just described, but the elastomer

substrates were additionally placed into a flow cell after spore settlement counts were

taken. One difference in methods from before was that the spores were only allowed to

settle for 45 minutes as compared to the hour-long immersion time. This change was due

to the inability of the flow cell to detach the spores if they were allowed to settle more

than an hour on any substrate. After settlement, the elastomer coated slides were placed

into a flow cell and run at a 55Pa maximum wall velocity for 15 minutes.86 There was

obviously no spore fixation step in this experiment. The number of spores that remained

attached after exposure to the hydrodynamic flow was then counted as described for the

spore settlement assay. The spore densities from before and after the flow cell exposure

on the different topographical areas were then compared and presented as percent spore

release.


Results and Discussion

Silicone Elastomer Fidelity

Before any topographically modified silicone elastomer samples were sent to Dr.

Callow for Enteromorpha spore assays, the fidelity of the patterns through the many

replication steps from the original silicon wafer was verified. All samples where the

topographies and flat areas were not visually acceptable were immediately discarded.






62




























Figure 4.6: SEM and WLIP images of an epoxy mold and silicone elastomer coated glass
slide. A) SEM at 800X magnification of patterned epoxy with the negative topographical
features (10,000tm length ridges at the transition point from 5[tm wide ridges to 10tm
wide ridges) replicated from a silicone well. B) SEM (350X magnification) of silicone
elastomer with 10,000atm length ridges at the transition from 5atm wide valleys to 10atm
wide valleys that was replicated off of an epoxy mold from the last step in the replication
process. C) WLIP image of the 10,000atm length ridges with 5atm valleys from the
silicon wafer. D) WLIP image of the 60atm length ridges with 5atm wide ridges from an
epoxy replicate.



Then random samples were periodically examined with either SEM or WLIP. A few of

these images are shown in Figure 4.6. These images showed that there were minimal

defects in the samples. It was also noticeable, both visually and microscopically, that

some of the silicone oils were partially filling in the patterns as they migrated to the

surface. The 20wt% 5000cSt elastomer formulation filled in the patterns to the extent

that the patterns disappeared. As a result, an examination of the spores on the patterns









was not feasible and so this formulation was not included in the topographical

experiments.

Each sample was also examined for suitable adhesion between the silicone

elastomer and the glass slide. A hand pull-test was performed on every sample to ensure

that it would withstand the shear stress exerted by the flow cell. Only samples that

passed were sent to Dr. Callow's laboratory for testing. The rest were discarded as

unusable.

Another factor of the process that was examined before samples were sent was

that the polyethylene (PE) bags, which every sample was individually stored and shipped

in, had little effect on the surface properties of the elastomer. This factor was examined

by measuring contact angles and calculating the surface energy of unpatterned,

unmodified silicone elastomer samples that were both stored and not stored in the PE

water for an hour to see if their surfaces reoriented differently after exposure to the PE

bags. The results of this study are shown in Table 4.1. The contact angles were

measured from 5 different liquids by the goniometer technique discussed in Chapter 3.

The surface energy was then calculated from a modified Zisman plot (cos 0 versus l/yll/2)

as also previously described in Chapter 3. From comparison of the measurements, there

was no significant alteration of the silicone elastomer surface from storage in the PE

bags.









Table 4.1: Measured contact angles and calculated surface energies examining the effect
of storage and shipment of unmodified silicone elastomer samples in polyethylene bags.


Static Contact Angle (degrees) Y c


Samples Water Mel DMF ACN 1-Prop (mN/m)

23.0
PE Bag 108 69 54 48 30
(+ 0.2)
23.0
No bag 112 67 53 50 31
( 0.1)
Bag 23.2
Bg 110 66 52 48 30
(Ihr soak) ( 0.2)
No bag 23.0
Nobag 102 63 51 49 25 23.0
(Ihr soak) (+ 0.4)


Spore Settlement Assay

The unpatterned samples of the various elastomer formulations were examined to

distinguish whether the oil additives influenced spore settlement. A trend that was

slightly observed, from the collected data shown in Figure 4.7, was that as the wt% or

molecular weight (or viscosity) increased, the amount of spores that settled was

increased. The 5wt% 5000 cSt formulation was the most significantly different from the

unmodified elastomer sample. An uncoated glass slide was included in the experiment as

a reference point. The glass had noticeably higher settlement than any of the elastomer

formulations. Dr. Maureen Callow noted that the spores that settled on the 5000 cSt

elastomer samples were visually "embedded" into the oily surface film. This introduced

an additional adhesion mechanism than on the unmodified elastomer samples. Even if

the spores were not cued to adhere, they got trapped to the surface. This was an influence











1400

1200

g 1000
E

800

E
E 600

O 400

200

0
glass UM 5% 50 20% 50 5% 5000 20% 5000
Silicone Elastomer Formulation (wt%; cSt)

Figure 4.7: The settlement of Enteromorpha spores on the different unpatterned silicone
elastomer formulations.



and contributed to the small differences in settlement seen between the different

formulations rather than chemical settlement cue variations. As a result, the trend

observed correlated with the amount of excess silicone oil on the surface.

The topographical and silicone oil additive influences were examined with the

spores in order to determine if there was an influence on their settlement cues. The spore

settlement densities observed on the different samples are shown in the 3 graphs in

Figures 4.8 4.10 according to the dimension of the spacings between patterns. The

settlement data for the flat areas between both the 1.5 and 5tm deep patterns are shown

on each graph for comparison. The 5tm deep valley patterns had noticeably higher spore

settlement for the unmodified, 20% 50cSt, and 5% 5000cSt elastomer formulations at all

three valley widths (5, 10, and 20tm). However, the 5% 50 cSt formulation at 5tm

depth valley pattern was not significantly different from the flat, 1.5am deep, or pillar












2000
5 iLm wide spacing
1800


1600

4n -\


-5 micron flat
--1.5 micron flat
--5 micron deep valleys
S-5 micron deep pillars
- 1.5 micron deep valleys
- 1.5 micron deep pillars


I- -

1000

800

600A nn6 A


200


Unmod. 5% 50 20% 50 5% 5000


Silicone Elastomer Formulation (wt%; cSt)

Figure 4.8: The settlement of Enteromorpha spores on 5pm wide spaced features on
both the topographies of the 2 depths and 2 types of features and on the flat areas
between the features.


-5 micron flat
-*-1.5 micron flat
S--5 micron deep valleys
- -5 micron deep pillars
- 1.5 micron deep valleys
- -1.5 micron deep pillars


*&


10 pm wide spacing


Unmod.


5% 50 20% 50 5% 5000


Silicone Elastomer Formulation (wt%; cSt)

Figure 4.9: The settlement of Enteromorpha spores on 10pm wide spaced features on
both the topographies of the 2 depths and 2 types of features and on the flat areas
between the features.


E

ci
Cl,
E
E


0
C,


1200


1000


I I


40nn











900
20 lim wide spacing
800

700

E 600

500 -
E -
E 400
--5 micron flat
300
o --1.5 micron flat
S200 *-5 micron deep valleys
-5 micron deep pillars
100 1.5 micron deep valleys
-1.5 micron deep pillars
0
Unmod. 5% 50 20% 50 5% 5000
Silicone Elastomer Formulation (wt%; cSt)

Figure 4.10: The settlement ofEnteromorpha spores on 20pm wide spaced features on
both the topographies of the 2 depths and 2 types of features and on the flat areas
between the features.



samples at all three valley widths. When the unmodified sample was specifically

examined, the 5[tm deep valleys had approximately 3 times as much spore settlement as

the flat area in between the patterns for the 5[tm wide valleys. The two trends observed

previously with the increased settlement on the unpatterned silicone oil modified samples

were not as evident in the data from the flat areas in between the patterns in this

experiment. Perhaps the topographies slightly influenced the settlement on the flat areas

or the topographies highly dominated the settlement cues of the spores. However, this

aspect could not be extracted from the data collected as a result of different spore batches

being incomparable between experiments because of large statistical differences between

each batch from different seasons and tidal cycles.10, 57 Another experiment would need

to be designed in order to look at this particular factor.



































Figure 4.11: Fluorescent microscope images of Enteromorpha spores settled on 3
different 5[tm deep patterned areas on unmodified silicone elastomer. (A) 5[tm wide
ridges with 5[tm wide valleys. (B) 5atm wide ridges with 10atm wide valleys. (C) 5atm
wide pillars with 5atm spacing. (Images courtesy of Dr. Maureen Callow)



The spores did, however, choose to settle in the most energetically favorable areas

on the samples. The spores settled preferentially in valleys, rather than on top of the 5atm

ridges, and against the walls pillars, rather than on top of the pillars, as evident in

Figure 4.11. Furthermore, the spores settled in the bottom covers of the valleys, between

the valley floor and ridge sidewall, rather than on the flat space of the valleys between the

ridges like on the unpatterned samples. Thus there was an active topographical settlement

cue for the spores. Originally the 5atm topography dimensions were established to be

smaller than the size of the spores, which were thought to be approximately 10atm, so that









they would be unable to securely attach to the bottom of the valleys. However, from the

images in Figure 4.11, the spores were obviously still able to migrate completely into the

valleys. Thus studies must progress to smaller dimensions in order to get the unstable

and incomplete adhesion underneath the spores.

Spore Removal Assay

The significance of the silicone oil additives was to increase the lubricity of the

surface and thus decrease the force required for removal of biological organisms. This

factor was examined by placing the samples in a flow cell with a hydrodynamic wall

shear stress of 55MPa for 15 minutes and obtaining the percent spore release as compared

to settlement counts on the same samples. The more densely populated patterns (5tm

wide pillars/ridges with 5tm wide spacing/valleys) were the patterns examined in this

experiment because they seemed to influence settlement the most in the previous study.

When the flat areas between the 5tm deep patterns were examined, most of the elastomer

formulations had the same relative spore settlement and removal, as shown in Figure

4.12. However, the 5wt% 5000cSt formulation did show a slightly higher settlement than

the other formulations, which followed the trend seen in the previous spore settlement

assay. It also had a slightly lower spore density after flow, which demonstrated a slightly

higher percent spore removal of 79% than the other formulations that had approximately

67% as compared to the flat areas and pillars for all elastomer formulations. The 5tm

deep and wide valley pattern spore density data both before and after flow in the parallel

direction is shown in Figure 4.13. In this graph, all the silicone oil modified samples had

lower initial spore settlement and lower spore densities than the unmodified elastomer

formulation of this pattern type. However, the spore removal for all of the formulations

was roughly the same. As a result, the percent spore removal was mainly a factor







70




2000

1800 Before Flow
0 After Flow
1600

1400

E 1200
E
; 1000
0. 800

600

400

200

0
Unmod 5% 50 20% 50 5% 5000
Silicone Elastomer Formulations (wt%; cSt)

Figure 4.12: Spore density measurements before and after immersion in a flow cell for
the flat regions between the 5ptm deep patterns of the different elastomer formulations.





1000
900 Before Flow
9 After Flow
800

700

E 600
E
500
O

300

200

100

0
Unmod 5% 50 20% 50 5% 5000
Silicone Elastomer Formulation (wt%; cSt)

Figure 4.13: Spore density measurements before and after immersion in a flow cell for
the 5[tm deep and wide valley pattern set parallel to flow direction of the different
elastomer formulations









of the pattern type rather than the oil additives. The increased settlement trend with oil

additive seen previously was not observed in this data set. The flat area between the 5tpm

pattern sets definitely had a lower spore density of approximately 200 spores/mm2 on the

surface than the pillars and valleys after hydrodynamic flow conditions. The pillars had a

much lower spore density after flow than the valleys though.

When the percent spore removal measurements were compared for all of the

different pattern types and silicone elastomer formulations, additional trends were

observed, as seen in Figure 4.14. The flat areas between the patterns had on average a

higher spore removal than the other patterns. However, the pillars did have similar

removal as the flat areas for the unmodified elastomer and the 20wt% 50cSt formulation.

There was also a slightly higher percent removal for the 5wt% 5000cSt formulation with

the different pattern types except for the pillars, which had erratic measurements. When

the direction of flow with respect to the valleys was examined, the valleys parallel to the

direction of flow had a slightly higher percent spore removal than the orthogonal flow

direction for all the elastomer formulations. This was expected due to the laminar

hydrodynamic flow through the parallel valleys. However, both valley direction sets

were significantly lower than the flat and pillar regions for all the formulations.

In the same experiment, patterned silicone elastomer coated slides with 1.5jpm

depth features were also examined for spore settlement and release. As shown in Figure

4.15, there was no significant difference between the two depths on pillar patterns or on

the flat area between patterns for most of the silicone elastomer formulations. However,

there was an increase in settlement for the 5wt% 5000cSt as compared to the other












- -flat --parallel valleys
--orthoganol valleys -cpillars




i


Unmod.


5% 50


20% 50 5% 5000


Silicone Elastomer Formulation (wt%; cSt)

Figure 4.14: Comparison of the percent spore removal for the different 5pm deep pattern
sets and the different silicone elastomer formulations.


TT T


Unmod. 5% 50 20% 50 5% 5000 Unmod. 5% 50 20% 50 5% 5000
*-------------* ------------*
Flat Area Pillars Area
Silicone Elastomer Formulation (wt%; cSt)


Figure 4.15: Enteromorpha spore settlement densities on the flat areas between the
patterns and on the pillars for the different elastomer formulation samples with either
1.5pm or 5pm deep pattern features.


700

S600
500
E
S400

S300
0
200

100

0









formulations on both flat and pillar regions when the pattern depth was increased from

1.5am to 5[tm. This increase was contributed to the infill of the patterns by the viscous

silicone oil. On the 1.5am patterns, the oil provided a relatively flat surface by almost

completely filling the patterns, where as the oil did not completely fill in the 5[tm deep

patterns. Thus the impact of the patterns on settlement was not significantly noticeable

on the 1.5[tm deep patterns. When the samples were then exposed to a hydrodynamic

flow, additional trends were noticed, as seen in Figure 4.16. The flat area between the

5[tm deep patterns had consistently more spore release as compared to the flat area

between the 1.5am deep patterns for all formulations. It was also evident that the

elastomer samples with the oil additives had more removal from the 1.5[jm deep pillars

than the flat areas between the 1.5am deep patterns. The percent spore removal from the

flat areas between the 5[tm deep patterns was always equal to or greater than the 5[jm

pillars and always greater than the 1.5am deep patterned elastomer slides for all

formulations. Overall, the 5[tm deep areas had more spore removal with flow except that

some of the data from the pillars was somewhat disordered. The spore removal for the

5[tm pillars was significantly lower than all the other pattern areas and depths for the

5wt% 50cSt and 5wt% 5000cSt elastomer formulations. Perhaps there was another

mechanism interacting with these formulations. These trends could not be compared to

the settlement assay on the previous study, unfortunately, due to uncontrollable variations

in Enteromorpha spore aggression and motility. Overall, even though some trends were

seen in each experiment, no overall statistically significant trends could be claimed

possibly due to low spore densities on the samples.
















80

70

5 60
0
E
50

10
S40
Q.
U)
6 30

20

10

0


S1.5 micron deep
*5 micron deep


Unmod. 5% 50 20% 50 5% 5000 Unmod. 5% 50 20% 50 5% 5000


Flat Area Pillars Area
Silicone Elastomer Formulation (wt%; cSt)

Figure 4.16: Percent Enteromorpha spore removal on the flat areas between the patterns
and on the pillars for the different elastomer formulation samples with either 1.5pm or
5pam deep pattern features after exposure to hydrodynamic flow.














CHAPTER 5
QUATERNARY AMMONIUM SALT CHEMICAL MODIFICATIONS


Introduction

The chemical species on the surface of a substrate is crucial to the surface

energetic and biological response. The ability to control the biological response,

depending on the application, would be crucial to many fields. In the case of marine

structures, which need a designed coating to prevent biological growth, an antimicrobial

chemical species on the surface could greatly influence biological settlement. Biofilm

formation is the first step to adhesion of biological organism whether in the body or

marine environment. Thus in order to prevent larger organisms from adhering to a

surface, the initial stage needs to be blocked. However, it is also necessary for only the

targeted organisms to be influenced by the antimicrobial action. Thus the biocide cannot

be incorporated freely into a release coating. This release mechanism also provides a

short life for the activity and productivity of the coating. As a result, binding the

antimicrobial molecule to the surface both prevents toxic release and provides a longer

productivity time and is a reasonable solution. Thus, the chemistry behind coupling a

quaternary ammonium salt to various substrates, glass, silicone elastomer, and

polysulfone, is presented and analyzed as a feasible solution to preventing microbial

fouling on a surface.










Materials

Biocide

The chosen quaternary ammonium salt biocide compound was Dow Corning's

5700 Antimicrobial Agent (now sold by Aegis Environments). This compound is

octadecylaminodimethyltrimethoxysilylpropyl ammonium chloride, structure shown in

Figure 5.1, and comes in a 50wt% methanol solution. Some experimental protocols

required that this biocidal silane be coupled to a borosilicate glass slide (75mm x 25mm

xlmm). For comparison, a long alkyl silane molecule, octadecyltrimethoxysilane, was

also coupled to a glass slide. This silane has the chemical structure shown also in Figure

5.1 and was obtained from Gelest, Inc.

Silicone Elastomer Substrate

As described in Chapter 3, the silicone elastomer chosen was Dow Corning's

Silastic T2 resin. This elastomer is a hydrosilylation cure reaction with platinum



Octadecylaminodimethyltrimethoxysilylpropy
ammonium chloride

OCH3 CH3 Cl
H3CO -Si-CH2CH2CH2-N-C17H34CH3
OCH3 CH3


n-Octadecyltrimethoxysilane

OCH3
H3CO--Si --C H34CH
OCH3

Figure 5.1: Chemical structures of Dow Corning's 5700 Antimicrobial Agent (top) and a
trifunctional alkoxy silane (bottom) with same length unreactive alkyl chain.









coordination catalyst. The elastomer was mixed as 10 parts of the base resin with 1 part

of the curing agent by weight and then degassed. The mixture was cured on glass slides

and adhered with allyltriethoxysilane coupling agent by the methods described in Chapter

3 for the preparation of unpatterned elastomer samples. The elastomer was allowed to

cure for 24 hours at room temperature. For coupling of the biocide to the silicone

elastomer surface, tetraethoxysilane (TEOS) from Gelest, Inc. was used.

Polysulfone Substrate

For the coupling of the biocide to a more rigid polymer substrate, UdelTM

polysulfone from Union Carbide was used, structure shown in Figure 5.2. The typical

properties designated by the manufacturer are shown in Table 5.1. The addition of

sulfonate groups to the polysulfone used Fisher's purified grade chlorosulfonic acid, 1,2-

dichloroethane (DCE), and methanol. In order to redissolve the sulfonated polysulfone,

HPLC grade N,N, dimethylformamide (DMF) from Aldrich was used as the solvent. A

3-glycidoxypropyltrimethoxysilane (GPS) coupling agent (from Aldrich) was used to

adhere the quaternary ammonium salt to the polymer backbone. All densities and

molecular weights necessary for molar ratio calculations are provided in Table 5.2.




CH3 O

0 -C O -S
CH3 O


Figure 5.2: Chemical structure of UdelTM poly(ether sulfone), which was used as a more
durable substrate as compared to silicone elastomer for chemical modifications.









Table 5.1: Typical material properties for poly(ether sulfone) as provided by the
distributor, Amoco Products.


Test
Elastic Modulus (23 C)
Tensile Strength (23 C)
Elongation at Break (23 C)
Hardness (Rockwell) (23 C)
Deflection Temperature
Glass Transition Temp.
Linear Shrinkage


Result
2.7 GPa
79 MPa
40-100%
69
171C
1900C
0.5-0.7%


Table 5.2: Density and molecular weight values for various chemicals and polymers
necessary for the molar ratio calculations in the binding of the quaternary ammonium salt
to the polysulfone backbone.

Che l Density Molecular weight
Chemical
(g/mL) (g/mol)
Polysulfone
1.24 442.53
(repeat unit)

1,2 Dichloroethane 1.25 98.96


Chlorosulfonic acid 1.75 116.52

N,N,
NN, 0.95 73.09
Dimethylformamide
(3-Glycidoxypropyl)
1.07 236.34
trimethoxysilane









Methods

Glass Slide Grafting

In order to examine the effect of the two different silanes on interfacial properties,

they were adhered to a glass slide. The glass surface was first ultra-cleaned to clear any

residue or contaminates from the surface by burning the slide through a clean flame on

the Bunsen burner and then soaking them in a 10Normal acetic acid solution for 5

minutes. The slides were allowed to drip dry and then immediately placed in the silane

solution. A Ivol% quaternary ammonium silane solution was prepared in DI water and

stirred vigorously for 5 minutes. A Ivol% octadecyltrimethoxysilane was prepared in a

95% ethanol aqueous solution, which had been adjusted to a pH of 4-5 with acetic acid.

This solution was allowed to mix for 10 minutes to undergo hydrolysis. After the glass

slide soaked in the appropriate silane solution for 10 minutes, they were dried at 600C for

1 hour. This method results in a chemical reaction between the silanol groups on the

glass surface and the hydrolyzed methoxy groups on the silane molecule for both

solutions.

Silicone Grafting

Since many of the current research for foul-release coatings are with a silicone

elastomer substrate, the quaternary ammonium antimicrobial was grafted to the surface of

the elastomer. The surfaces of silicone elastomers are known to have a higher

concentration of silica particles than the bulk so the biocide was grafted to the surface

particles with tetraethoxysilane (TEOS) coupling agent. The TEOS was prepared in a

10vol% solution in 200-proof ethanol. The pH of this solution was reduced to 4-5 with

acetic acid in order to accelerate the hydrolysis reaction. The solution was then allowed

to mix for 10 minutes. Meanwhile, the cured silicone elastomer (adhered on a glass









slide) was wiped intently with toluene in order to clear the surface of residual silicone oil.

The elastomer was then immersed in the TEOS solution for 10 minutes. A Ivol %

quaternary ammonium solution was prepared in ethanol and allowed to mix for 20

minutes. The silicone elastomer was then removed from the TEOS solution and

immersed in the antimicrobial silane solution for 10 minutes. Afterwards, the slide was

rinsed with ethanol to remove any excess agents and heated at 600C for 1 hour. The

modified elastomer was then immediately cooled to room temperature and tested with

digital contact angle images.

Polysulfone grafting

Two methods were performed for binding the quaternary ammonium compound

to the polysulfone, solution and surface reactions. By performing the chemical reactions

in solution, the quaternary ammonium biocide was introduced into the bulk polymer,

rather than just on the surface. This treatment method could affect the mechanical and

morphological properties of the polysulfone.

The initial step in this chemical modification was to sulfonate the polysulfone in

order to increase its hydrophilicity and directionalize the silanating reaction. First 10wt%

of the unmodified polysulfone was dissolved in 1,2-dichloroethane. Argon gas was

bubbled though the mixture for 2 minutes before sealing the tri-neck flask in order to

force any oxygen and moisture out. The polysulfone was allowed to dissolve while

stirring for 24 hours. The argon gas line was then replaced back onto the flask creating a

continuous gas flow. Chlorosulfonic acid was quickly added in a 1:1 mol ratio to the

polysulfone repeat unit. After stirring for 30 minutes, the rest of the polymer was crashed

out with methanol and then rinsed 3 more times with a methanol wash. The resulting

polymer was then quickly sliced into small pieces, in order to increase the surface area









for solvent to diffuse from, and placed in a vacuum oven at 75C for 24 hours. The

obtained polymer is shown in Figure 5.4.

The next step was to use a GPS coupling agent to adhere the biocide to the

sulfonated polysulfone (SPSF). The SPSF was redissolved (5wt%) in DMF and allowed

to stir for 24 hours. Since the sulfonation reaction would have left theoretically one

sulfonate group on every polysulfone repeat unit, proven by previous researchers, where

the silane was also added in a 1:1 mol ratio to the polysulfone repeat unit.87 The silane

was allowed to evenly distribute in the solution by stirring for 5 minutes. Then to

accelerate the hydrolysis reaction, water adjusted to a pH of 1.5 with nitric acid was

added in a 3:1 molar ratio to the silane. The solution was again allowed to stir for 5

minutes. The quaternary ammonium salt was then added in a 1:1 mol ratio with the

glycidoxypropyltrimethoxysilane. This final solution was allowed to stir for 1 hour at

which point the polymer was dried on a glass slide or polyimide film at 600C for 24

hours. Before characterization, the films were dried again at 1500C for 1 hour in order to

prove that all of the DMF solvent was completely evaporated from the polymer.

The biocide was also surface grafted onto a dried sulfonated polysulfone (SPSF)

film. First the dried sulfonated polysulfone, prepared as previously described, was

resolvated into DMF. This solution was then dried at 600C for 4 hours and then 120C

under vacuum for 1 hour onto either a section of polyimide (Kapton) film or glass

coverslip. Meanwhile, a lvol% aqueous solution of glycidoxypropyltrimethoxysilane

was prepared. The DI water was dropped to a pH of 4-5 by the addition of acetic acid

and thoroughly mixed before the silane was added. This silane solution was allowed to

mix for 10 minutes before the SPSF film was introduced. Subsequently, the film was









soaked for 5 minutes in the GPS silane solution and then dried for 20 minutes at 600C. A

Ivol% aqueous solution of the quaternary ammonium salt was then prepared as

previously described with the glass slide grafting. The GPS silanated SPSF film was then

allowed to soak in the biocide solution for 10 minutes. The film was then dried at 600C

for 20 minutes and stored under dry conditions until analysis.

SEM/EDS

In order to examine the morphological properties of the polysulfone chemical

modifications, the dry films were examined with a JEOL 6400 Scanning Electron

Microscope (SEM). These images were obtained with a 10kV accelerating voltage at

magnifications ranging from 100x to 10000x of Au/Pd coated specimens. In order to

examine the chemical species exposed on the surface, an Electron Density Spectrum

(EDS) analysis was also performed on the modified polysulfone films.

FTIR

In an attempt to examine the chemical bonds present after each modification step

with the solution grafting of the polysulfone, Fourier-transform infra-red (FTIR)

spectroscopy analysis was performed. Dry films were prepared on NaCl crystals by

drying each solution at 1500C for 30 minutes. Each film was analyzed with a Nicolet

20SX spectrometer in transmission mode using 64 scans at a 4 cm-1 resolution. Peaks

were analyzed using ACDLabs Freeware 5.0 SpecViewer software.

Contact Angle

In order to examine the molecular compounds on the surface of the various

modified substrates, glass, silicone elastomer, and polysulfone, the technique of contact

angle was used. Both static contact angle techniques, sessile drop and captive air bubble

were used with Ultrapure water as the liquid interface. These techniques have been









described in detail in previous chapters. An approximate 24L bubble or droplet was used

depending on the technique. From the digital capture goniometer setup, the surface

response to a water/air/solid interface was obtained by either still pictures or video

through the Viviewer software. The contact angles were measured from the still images

with UTHSCSA ImageTool 2.0 software. Still frames from the captured digital videos

were seized with Adobe Premiere software.


Results and Discussion

Glass Slide Grafting

The interfacial properties of a substrate have shown to have a large impact on

biological adhesion. As a result, the surface energetic of the quaternary ammonium salt

on a glass surface was examined. First, an untreated glass slide's effect on contact angle

measurements was examined for a comparison. From the captive air bubble technique

with ultrapure water, it was evident, as shown from the captured video frames in Figure

5.3, that the air bubble immediately attached to the untreated glass surface. However,

when an air bubble was introduced to the water-quatemized glass substrate, by the same

method, an interesting phenomenon occurred as compared to an unmodified glass

substrate. Trying to press the air pocket to the surface did not induce the bubble to settle,

as was the case for the untreated glass surface. As seen in Figure 5.4, the air pocket

underwent large deformation when pressed upon the quatemized glass substrate and

would not leave the micropipet tip to settle on the surface. After gentle tapping of the

micropipet, the air droplet jumped to the surface, but did not immediately settle to the

glass surface. Instead the bubble glided across the surface until a favorable location for

settlement was found. This observance was captured by a digital video. Some time









progressive still frames of this movement and settlement behavior are shown in Figure

5.5. Sometimes the air bubble would move only a short distance and settle to the surface,

as shown in Figure 5.5, and other times it would progress all the way to the edge of the

sample where another interface was introduced, as is observed in Figure 5.6. This

observation was theorized to be a factor of the cationic ammonium atom strong attraction

to the water molecules to the extent that the air droplet did not have enough energy to

displace the water molecules. This meant that there was always a minuscule water layer

between the air bubble and the substrate so that the air bubble could not settle on the

surface. However, due to inconsistencies of the surface silane monolayer, the air pocket

was able to eventually find a weakness in the water adhesion strength and settle on the

glass surface. While unstable, the air bubble was pulled along the surface by the

extremely hydrophobic long alkyl chains, which were attracted to the air rather than the

water. The alkyl chains were so long and mobile that they would grab for the air pocket

and cause it to move. The interfacial effects of the hydrophilic quaternary ammonium

and hydrophobic alkyl chains were noted by Vaidya et al., but they only reported the

lowering of water's surface tension.88

In order to verify that the lack of settlement of the air bubble was due to the

cationic nitrogen, a silane with the same long alkyl chain, octadecyltrimethoxysilane

(ODS), was also coupled to a glass slide and the interface examined. Harder et al. have

previously shown that a self-assembled monolayer of long polyethylene glycol

terminated chains on a surface can prevent the absorption of proteins to the surface. 89

They contributed this to a steric repulsion effect that was due to the mobility, otherwords

compressibility, of the monolayer chains and the strong bonding of water to the surface,