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1 FABRICATION OF MICROENGINEERED POLYMERIC FILMS AND INVEST I GATION OF BIORESPONSES TO THE SUBSTRATA By LIWEN JIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010
2 2010 Liwen Jin
3 This work is dedicated to the three most important ladies in my life: m y m other Zhou, Yun my wife Wei, Yuying my daughter Jin, Abbie Siyun
4 ACKNOWLEDGMENTS I must express my sincere gratitude to my advisor, Dr Ronald Baney, and my co advisor, Dr. Anthony Brennan. Not only their rich knowledge in their own fields, but also their keen interest toward all aspects of science as well as life have g reat positive influence on my attitude to the research and life. I appreciate the advice and guidance provided by my doctoral committee consisting of Dr. L aurie Gower, Dr. Christopher Batich, and Dr. Joanna Peris. I must acknowledge and thank my research collaborators Dr John Finley, Dr. Maureen Callow, Dr. James Callow, Dr. Patrick Antonelli, Dr Carol Ojano -Dirain, Ms Qingping Yang, and Ms Edith Sampson. I also acknowledge Ms Jennifer Wrighton, Ms Doris Hallow, Ms Jennifer Holton and Ms Alice Hol t for their administrative assistance throughout my graduate studies. Graduate students, past and present, have been vital for my progression and success throughout this entire process. First, I must thank my mentors, Dr. Soroya Benetez, Dr. Yun Mi Kim an d Dr. Le Song, for getting me started with my research and teaching me the culture of Baney group. I would like to thank former and current group members, Dr Jiwei Wang, Abby Queale, Timothy Gehret, Edward McKenna, Ting Cheng, ChungHao Shih, Ravi Kumar Va sudevan, and Sung Hwan Yeo. I must also acknowledge the senior members of the ONR team, Dr s Leslie Wilson, Cliff Wilson, Michelle Carman, and James Schumacher, who provided their suggestion and support when I started the ONR project. During this time, I have had the pleasure of working with two great colleagues, Kenneth Chung and Christopher Long, who have both been vital in providing experimental as sistance and discussion during the completion of my dissertation research. ONR group members, Chelsea Magin, Angel Ejiesi Jun -Jiun g Chen (Jack), Scott Cooper, Julian Sheats, and David Jackson, helped me with my
5 experiments and discussion. Id like to than k them for their support and friendship. I also sincerely thank the many undergraduate research assistants and technicians that have aided in the preparation of samples for assay analysis, including Felicia S ved l u nd, Andrew Rophie, and Sean Royston. Id l ike to thank all my friends here at UF for their friendship and support. Special thanks to my wife, Yuying Wei. Without her encouragement, support, and endless love, there would not be this work. I want to thank all my family members for their love and sup port.
6 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...................................................................................................... 4 LIST OF TABLES ................................................................................................................ 9 LIST OF FIGURES ............................................................................................................ 10 LIST OF ABBREVIATIONS .............................................................................................. 13 ABSTRACT ........................................................................................................................ 15 CHAPTER 1 INTRODUCTION ........................................................................................................ 17 Scope of R esearch ..................................................................................................... 18 Research Goals and Specific Aims ............................................................................ 21 2 BACKGROUND .......................................................................................................... 26 Introduction ................................................................................................................. 26 Marine Biofouling and Prevention .............................................................................. 27 Factors on Marine Biofouling and Foul Release ................................................. 29 Surface Chemistry/Energy ................................................................................... 29 Surface Charge .................................................................................................... 30 Mechanical Properties ......................................................................................... 31 Surface To pography ............................................................................................ 31 New Coating Strategies ....................................................................................... 32 Polymeric materials ....................................................................................... 32 Surface cata lysis ........................................................................................... 33 Biomimetic measures .................................................................................... 34 Bacterial Biofilms in Medical Environment ................................................................. 36 Bacteria Mechanical Sensing .............................................................................. 38 Bacteria Quorum Sensing .................................................................................... 39 Current Art in Inhibition of Biofilm Formation ...................................................... 41 3 FABRRICATION AND CHARACTERIZATION OF MICROENGINEERED THERMOPLASTIC POLYMERIC FILMS .................................................................. 45 Introduction ................................................................................................................. 45 Materials and Methods ............................................................................................... 48 Materials ............................................................................................................... 48 Surface Treatment of Silicon Wafers ................................................................... 48 Fabrication of Microengineered Thermoplastic Polymeric Films ........................ 49
7 Surface Characterization of Polymer Films ......................................................... 49 Results and Discussion .............................................................................................. 51 PDMS 5K Grafted Si Wafers ............................................................................... 51 Micro engineered Polymer Films ......................................................................... 54 We ttability of the polymer films ............................................................................ 55 Surface characteristics of the polymer films ....................................................... 57 Conclusion .................................................................................................................. 57 4 ULVA ZOOSPORE ATTACHMENT ON THE MICROENGINEERED POLYMERIC FILMS ................................................................................................... 67 Introduction ................................................................................................................. 67 Materials and Methods ............................................................................................... 71 Materials ............................................................................................................... 71 Topographical Replication ................................................................................... 71 Sample Preparation ............................................................................................. 73 Spore Attachment Assay ..................................................................................... 74 Statistical Methods ............................................................................................... 74 Results and Discussion .............................................................................................. 75 Spore Attachment Affected by the Epoxy Glue .................................................. 75 Spore Attachment on Various Surfaces .............................................................. 76 Site Selection by Spores ...................................................................................... 79 Correlation between the Surface Parameters and Ulva Spore Attachment ...... 79 Conclusions ................................................................................................................ 82 5 BIOFILM INHIBITION O N SURFACE MICROENGINEERED POLYMERIC FILMS .......................................................................................................................... 96 Introduction ................................................................................................................. 96 Materials and Methods ............................................................................................... 98 Materials ............................................................................................................... 98 Fabrication of Microengineered Polymeric Films ................................................ 98 Sample Preparation ........................................................................................... 100 Biofilm Form ation Assay .................................................................................... 100 BioTimer Assay .................................................................................................. 101 Statistical Methods ............................................................................................. 102 Results ...................................................................................................................... 102 Characterization of Bacterial Colonies on the PDMSe Samples ..................... 102 Bacterial Biofilm Formation on Various Substrates .......................................... 104 Discussion ................................................................................................................. 105 Development of Bacterial Microcolonies on the Surfaces ................................ 105 Surface Properties on the Development of Bacterial Microcolonies ................ 108 Conclusion ................................................................................................................ 110 6 NEW UNDERCUT SURFACE FEATURE AND BIORESPONSE .......................... 121 Introduction ............................................................................................................... 121
8 Theory ....................................................................................................................... 122 Materials and Methods ............................................................................................. 124 Materials ............................................................................................................. 124 Process of Making Undercut Micro -features on Polymer Films ....................... 125 Surface Characterization of Polymer Film s ....................................................... 125 Statistical Methods ............................................................................................. 126 Results and Discussion ............................................................................................ 126 Polymer Films with Undercut Micro -Topographical Features .......................... 126 Estimation of Fouling Reduction on the Undercut Surfaces ............................. 127 Underwater Stability of the Undercut Surfaces ................................................. 129 Conclusion ................................................................................................................ 131 7 CONCLUSIONS AND FUTURE WORK .................................................................. 141 Conclusions .............................................................................................................. 141 Fabrication of Micro engineered Polymeric Films ............................................. 141 Marine Antifouling Assay ................................................................................... 141 Bacteria Biofilm Assay ....................................................................................... 142 New Undercut Surface Features ....................................................................... 142 Fu ture Work .............................................................................................................. 143 Marine Bioresponse Assays on the Polymer ic Films ........................................ 143 In vivo Study of the Efficacy of the Micro engineered Polymer Films .............. 144 LIST OF REFERENCES ................................................................................................. 146 BIOGRAPHICAL SKETCH .............................................................................................. 163
9 LIST OF TABLES Table page 1 -1 Force gradient between the adjacent feature elements, assuming 1% bending of the features. ......................................................................................... 25 3 -1 Dynamic water contact angle on the Si wafers. .................................................... 65 3 -2 Root mean square roughness of the flat polymer films via solution casting on PDMS 5K treated silicon wafer .............................................................................. 65 3 -3 Calculated surface parameters for microengineered topographies ..................... 65 3 -4 Predicted wetting regime for the polymer films with varied feature height ........... 65 3 -5 Sessile drop contact angles on polymer films, with water as testing medium. .... 66 4 -1 Static contact angle measurement on various smooth surfaces with different probe liquid. ............................................................................................................ 92 4 -2 The mechanical property and surface free energy of the selected materials. ..... 92 4 -3 Surface parameters in the current and previous spore attachment studies. ....... 93 4 -4 Loadings for the two components generated from principal component analysis using the variables from the five variables data set (Table 43 ). ........... 95 5 -1 Physical properties of the selected materials. ..................................................... 120 6 -1 Softening and melting ranges of some common thermoplastic polymers ......... 139 6 -2 Measured geometry of the Sharklet surface features and the correlation of Ulva spore attachment reduction. ........................................................................ 139 6 -3 Sessile drop water contact angle (CA) measurement and the wetting regime for the topographical surfaces. ............................................................................ 140 6 -4 Estimation of the ERIII values and %Reduction of the attachment density of spores based on the correlation in this study. ..................................................... 140 6 -5 Estimated penetration pressure for the various surface topographies. .............. 140 7 -1 In vivo test groups to evaluate the effectiveness of the proposed treatment strategy.. ............................................................................................................... 145
10 LIST OF FIGURES Figure page 1 -1 Correlation of spore attachment density with ERI values ..................................... 25 2 -1 Time line of marine fouling event sequences ....................................................... 44 2 -2 Illustration of the development of bacterial biofilm. ............................................... 44 3 -1 Schematic description of surface treatment of a Si wafer. ................................... 58 3 -2 SEM images of PS film with +2SK2x2 pattern. ..................................................... 59 3 -3 XPS survey spectra for Si wafer under various treatments. ................................. 60 3 -4 XPS spectra of Si 2p3 peaks for Si under various treatment. ............................... 60 3 -5 (A) Photo of the replicated PMMA film with Sharklet AFTM (+2SK2 x 2) pattern; (B) Light micrograph of the PMMA Sharklet AFTM (+2SK2 x 2) pattern (topdown view). ............................................................................................................. 61 3 -6 SEM images of polymers with Sharklet AFTM (+2SK2 x 2) pattern ........................ 61 3 -7 SEM images of PMMA +2SK2x2 film. ................................................................... 62 3 -8 SEM images of PS +2SK2x2 film. ......................................................................... 62 3 -9 SEM images of P DM S e +2SK2x2 film. ................................................................. 62 3 -10 SEM images of +3SK2x2 polymer film. ................................................................. 63 3 -1 1 AFM images of various smooth polymer surfaces obtained from solution casting against PDMS 5K treated silicon wafer. ................................................... 64 3 -1 2 Photo images of an elliptical water dropl et sitting on +2SK2x2 PMMA film ........ 64 4 -1 The attachment densities of Ulva spores on sharklet patterns. ............................ 83 4 -2 The schematic drawing of +2SK2x2 PS film attached to a glass slide. Microscopic images were taken from each region of the sampleattached glass slide. .............................................................................................................. 84 4 -3 Spores can be observed in the cavity between the PS film and the underneath epoxy glue. .......................................................................................... 85 4 -4 UV -Vis spectra of water extracts from Araldite layer and glass slide stored in D.I water in centrifuge tubes. ................................................................................. 85
11 4 -5 Some representative micro images of topographical features on various substrates. .............................................................................................................. 86 4 -6 Micrograph of +3SK2x2 PDMSe surface after spore attachment assay. Flopped tops of the features were prevailing on the whole sample. .................... 87 4 -7 The attachment densities of Ulva spores on smooth surfaces vs. natural logarithm value of the square root of the product of elastic modulus and surface energ y of the corresponding material. ...................................................... 87 4 -8 The attachment densities of Ulva spores on the Sharklet patterns plotted as a function of nanoforce gradient .............................................................................. 88 4 -9 Images of spore settled on the Sh arklet patterns (fixed sample). ....................... 89 4 -10 Image of spores settled on the +2SK2x2 PS patterns (fixed sample). ................. 90 4 -11 Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII. ................................................................................. 90 4 -12 Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII divided by the square root of ( E ).. ......................... 91 4 -13 Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII divided by the square root of ( E ).. ......................... 91 5 -1 SEM images of Kraton G1650M with +3SK2x2 pattern. ..................................... 111 5 -2 Layout of sampling plan for biofilm formation assay .......................................... 111 5 -3 The correlation of the CFU counts in the BioTimer assays and the time required for color switch (from red to yellow).. .................................................... 112 5 -4 Planktonic -quivalent CFUs counts on PDMSe samples after 7-day culture followed by an antibiotic treatment.. .................................................................... 113 5 -5 SEM images of the smooth surface after 7 -day S. aureus culture ..................... 114 5 -6 SEM images of the Sharklet patterned surface after 7 -day S. aureus culture. .. 115 5 -7 The time required for color switch on smooth and Sharklet surfaces after 7 day culture ............................................................................................................ 116 5 -8 SEM images of the surfaces of smooth and Sharklet polymer films after 7 day S. aureus /TSB culture. ................................................................................. 117 5 -9 Schematic illustration of bacterial microcolonies which developed on the smooth (A) and microengineered (B) surface with time. .................................... 118
12 5 -10 Schematic illustration of the microcolonies formed on the topographical and smooth surfaces and the states of the cells after antibiotic treatment.. ............. 119 6 -1 Correlation of spore attachment density and ERIII *.. ........................................... 133 6 -2 Schematic illustration of processing method for fabricating undercut surface topographies. ........................................................................................................ 133 6 -3 Some examples of cross -sectional view (A) and top-down view (B -D) of the undercut surface topography. .............................................................................. 134 6 -4 Experimental setup for processing polymer films to obtain undercut surface topographical features .......................................................................................... 135 6 -5 SEM images of Kraton G1650M undercut +3SK2x2 film .................................... 136 6 -6 SEM images of the PS/G1650M undercut Sharklet +3SK2X2 film. ................... 137 6 -7 SEM images of the under -cut PMMA Sharklet +3SK2X2 film, (A) topdown view, (B) cross -sectional view. ............................................................................. 138 6 -8 (A) a unit cell of the surface features of the Sharklet pattern (topdown view), t he blue area represents the tops of the surface features and the area in the thick black lines is the planar area of the unit cell ( Ac). (B) Schematic illustration of the undercut surface features can increase the critical pressure exerted on the top of th e features . ............................................................. 139
13 LIST OF ABBREVIATION S AF Antifouling AFM Atomic force microscopy APTES 3 aminopropyltriethoxysilane BTM BioTimer medium CPC Cetylpyridinium chloride E Elastic modulus (or Youngs modulus) ECMs Extracellular matrices E. coli Escherichia coli EPSs Extracellular polymeric substances or extracellular polysaccharides ERI Engineered roughness index f The fraction of the rough surface that is in contact with liquid FR Foul release G1650M Kraton SEBS triblock copolymer with styrene block ~30 wt% G1657M Kraton SEBS triblock copolymer with styrene block ~13 wt% HAIs Healthcare associated infections HMDS Hexamethyldisilazane MIC Minimum inhibitory concentration MW Molecular weight MRSA Methicillin resistant S. aureus MS Mechanosens itive MSDS Material safety data sheets n The discrete number of the surface features on a micro engineered surface P. aeruginosa Pseudomonas aeruginosa PBS Ph osphate buffered saline
14 PCA Principal component analysis PDMS 5K Short -chain poly (dimethylsiloxane ) with molecular mass ~5 k g/mol PDMSe Poly (dimethylsiloxane ) elastomer PEG Poly(ethylene glycol) PET poly(ethylene terephthalate) PMMA Poly(methyl methacrylate) PS Polystyrene QS Quorum sensing r Wenzel roughness ratio (total surface area divided by the projected planar surface area) SEBS Styrene ethylene/butadiene-styrene triblock copolymer S. aureus Staphylococcus aureus S. epidermidis Staphylococ cus epidermidis TSB Tryptic soy broth XPS X -ray photoelectron spectroscopy XRR X -ray reflectometry
15 Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy FABRICATION OF MICROENGINEERED POLYMERIC FILMS AND INVESITGATION OF BIORESPONSES TO THE SUBSTRATA By Liwen Jin August 2010 Chair: Ronald H. Baney Major: Materials Science and Engineering Biofouling is the undesired attac hment, accumulation and proliferation of biomass (biopolymers and organisms) on various surfaces. This process impart s adverse influence s on many aspects of human life. Without introducing any bi ocidal agents into the materials, a biomimetic micro -topographical structure replicated onto poly ( dimethysiloxane ) elastomer (PDMSe) has shown antifouling characteristics against marine and medical microorganisms. The purpose of this work was to investigate the efficacy of this pattern on antifouling when it was rep licated onto various polymeric substrata with a wide range of mechanical and energetic properties. A method was develop ed to covalently graft a demolding/antisticking layer short chain poly(dimethylsiloxane), onto a silicon wafer. A series of polymeric ma terials were chosen with varied mechanical properties (Youngs modulus ranging from 1.4 to 3, 3 00 M Pa) and surface free energies (ranging from 21.5 to 42.4 mJ/m2). Polymer films with the micro engineered patterns were then readily fabricated from the treated wafer using solution casting method. The treated silicon wafer was versatile and robust in repli cation of topographical features onto those polymeric materials with high fidelity (>99%)
16 The result ing films were tested against one marine microorganism, U lva linza zoospore The Sharklet textured surfaces with feature heights of 2.1 and 3 .0 microns were tested and corresponding smooth surfaces served as controls. The polymeric films were attached to glass slides with an epoxy glue, which was found to be str ongly attractive to the spores. Assuming the influence of the epoxy glue was the same among all the samples, data were analyzed and s tatistical analys e s were performed. Two groups of the surface properties were important for establishing predictive attachment models However, the results should be interpreted with caution because of the unknown magnitude of the epoxy glue problem. One bacterium Staphylococcus aureus was used to test the antifouling capability for the topographical surfaces The bio activity measured by the metabolic rate (BioTimer assay) showed that there were more bioactive cells on the Sharklet textured surface than on the smooth one, regardless of the chemical nature of the substrate. SEM imaging showed the opposite: mature biofil ms were formed on the smooth surfaces, while disrupted microcolonies were prevailing on the Sharklet textured surfaces. High dose antibiotic treatments suggest ed that the bacterial cells on the Sharklet surface were more easily killed after a 12hr culturi ng. A n ew strategy of treating bacteriaassociated infections for implanted medical devices was proposed. N ew undercut surface topographical structures were proposed and fabricated. It is estimated that t he new surface structures may exhibit higher underwa ter stability and higher reduction of Ulva spore attachment
17 CHAPTER 1 INTRODUCTION Bioadhesion of microfoulers/macrofoulers has attract ed much interest in current research due to environmental and economic concerns. For exampl e, the biomass accumulated on a ship s hull not only caus es higher drag force (and thus higher fuel cost ) [1 3] but also provides a means for spreading invasive species  The chronic infections caused by bacterial biofilms in clinical situ ations, on th e other hand, greatly increase risks during recovery and the cost of treatment [5 -7] Therefore, anti -fouling with minimal side effects has gained more interest during the last two decades. All surfaces in contact with aqueous environments, wheather marine or physiological will be conditioned by organic matter within a short period of time . Th i s conditioning will form a layer which attract s certain microorganisms to the surface. When the se microorganisms establish colonies in the natural environ ment, other species (usually larger in size) will be attracted G radually a complex biolayer consisting of multi ple species (bacteria, plants and animals) will be formed ; t his process is biofouling. To prevent biofouling, the traditional strategy is to kil l the organisms. That means any microorganism attached to or in proximity of the surface will be killed by toxic surfacebound chemicals or leaching agents blended in the substrate. This strategy has limitations due to stringent environmental regulations a nd requirement s on an effective time period. Recently a strategy of repelling the microorganisms has been developed and tested for anti adhesion/antifouling purposes. This strategy, loosely speaking, depends on disguis ing the surface or causing the microorganisms to dislike the surface. This
18 is often realized through manipulati on of the structures and/or surface properties of polymer based coatings [9 11] To effective ly protect the surface during its designed lifespan, the surface should first be resistant to organic matter (such as proteins and polysaccharides). So far, such perfectly resistant surfaces have yet to be dis gned. Th us the effectiveness of such surfaces against biofouling can be significantly reduced once an organic layer is adsorbed onto them. After billions of years of evolution, organisms have already developed various ways to avoid undesired attachment from other species. One example is a chemical defense, in which secreted chemicals (e.g. zosteric acid from some sea weeds) interfere the attachment of microorganisms  Another approach is a physical defense. For example, some slowmoving creatures like shell fish can avoid bioadhesion of foulants on their shell s with micro -topographical features [13, 14] These findings encouraged researchers to explore biomimetic strategies for the antifouling applications. Scope of Research Biomimetic design for a ntifouling applications can also borrow ideas from other creatures. The body of a shark is covered by scales with unique microstructures, which not only help the sharks maintain their superb hydrodynamic characteristic  but also effectively resist adhesion of microorganisms. Based on the characteristic s of shark skin, the Brennan research group at the University of Florida came up with the engineered surface topographical design called Sharklet AFTMBased on the current understanding of the interactions between organisms and surfaces the factors that control the sensing, settling, adhering, and proliferating of [16 -20] These surface topographical features were effective in antifouling against marine organisms [16 -19] and bacteria 
19 microorganisms may include the surface topogr aphy, chemistry, mechanics, torturosity, feature dimension, and surface energetics  Many previous investigations revealed that surface chemistry wettability and energy can affect biofouling process on flat surfaces. Systematic control of surface chemistry through self assembled monolayers (SAMs) showed that the marine microorganism Ulva zoospore can select its settling location on a surface  Though there was no detectable difference in spore attachment density in a narrow range of stiffness (Youngs modulus ranging from 0.2 to 9.8 MPa)  on a flat substrate, mechanical force (interaction between the surface feature and the microorganism) may still play a role in spore sensing and settling on a rough surface  A predictive model was proposed to correlate Ulva zoospore attachment density with the surface topographical features  The parameter in this model, the engineered roughness index (ERI), was further modified to better correlate and predict Ulva spore attachment behavior  The biofouling investigations were performed on various topographical features replicated on a poly(dimethyl siloxane) (PDMSe) material (Silastic T2TM). Although the concept of a nanoforce gradient was introduced and explained how that mechanical force may influence spore attachment (Figure 1-1)  the influence of factors such as surface chemistry, mechanics, and energetics were not fully explored. One objective of this work was to explore the influence of the various surface properties on the attachment of Ulva spore s to help further understand i ng of the biofouling process. Th is understanding is hoped to lead to the development of more efficient surface designs for antifouling applications.
20 In the field of bacterial adhesion to various substrata, surface properties also affect the quantity and pattern of the adhered cells. Short period (usually within several hours) of bacterial adhesion studies showed that the difference of surface chemistry /energy can influence the initial adhesion of the bacterial cells  Surface roughness in the nanometer to submicrometer range is another factor found to affect a bacterial cells adhesion and attachment pattern on various substrata [32, 33] However, the surface properties, including surface chemistry, energy, wettability, charge, mechanics, and nano /micro -struc ture have not been fully explored in the long-term biofilm development. Micro -sized Sharklet AFTMBased on the application of inhibiting biofilm formation, another objective of this research w as to try to answer the following questions regarding bacteria attaching to a surface: surface features blocked the connection of the bacterial colonies on the surface s deterring the formation of biofilms for up to 21 days  This type of micro -sized surface topographical features may thus provide physical defense against bacterial biofilm formation for more general applications Does the Sharklet topographical feature have the same effectiveness against bacterial biofilm formation on other polymeric materials than PDMSe ? In other words, do other surface properties such as mechanical stiffness and surface energy affect the ability of inhibiting the biofilm formation on a topographical surface? In a long-term (7 -day) cell culture, d o es th e surface topography affect the morphology of the microcolonies? Are the bacterial cells attached to the topographical surface affected in terms of metabolic rate and defense mechanism ? For these two objectives, an engineered topography was replicated onto the surface of nontoxic polymeric materials with systematically varied elastic modul i and surface energy Simple and cost effective process es w ere explored to fabricate the micro engineered polymer films. Statistical methods (e.g. principal component
21 analysis) were employed to determine the influence of various surface properties on the marine biofouling process. O ptimized surface design could then be performed for potential marine antifouling applications. The marine biological assays were carried out by Dr John Finley, Dr. Maureen Callow and Professo r James Callow at the University of Birmingham, U K; all three are biologists specializing in ship fouling species such as algae and diatom s Bioassays for biofilm formation and characterization were developed in collaboration with Ms. Qingping Yang, Ms Ed ith Simpson, and Dr. Patrick Antonelli of the Department of Otolaryngology at the University of Florida. Research Goals and Specific Aims The overall goals of this research were (1) to determine the influence of various surface properties ( i.e., surface m echanics, chemistry /energy and topography) on the attachment behavior of one marine biofouling species Ulva linza zoospore, and (2) to understand t he effect surface microstructure s have on the inhibition of bacterial biofilm formation. Completion of these goals required the successful fabrication of microtopographical polymeric films while maintaining the fidelity of the surface topography. These propertie s, correlated with the bioresponse data, could allow for the further development of the predictive ERI model. Novel engineered topographies were designed based on this model. At the start of the project, the s pecific aims listed below were chosen with special emphasis on the use of the Sharklet AFTMSpecific Aim 1: Fabrication of Micro-engineered Polymeric Films with High Fidelity topography for biofouling applications. The objective of this aim was to replicate high fidelity (> 95% feature replication), micrometer -scale structures onto the surface of various polymers ; the polymers tested
22 include PDMSe, styrene ethlyene/ but a diene -stryene (SEBS) tri block copolymer, poly (methyl methacrylate) (PMMA), and polystyrene (PS). These materials have a large span of elastic muduli (from ~1 MPa to ~3 GPa) and low to medium surface energy Thus the micro -patterned polymer films can be tested against fouling species. A solution casting method was chosen for the se thermoplastic polymers. An anti sticking layer was covalently grafted onto t he silicon wafer patterns and fabrication processes were developed for each poly mer Pattern fidelity was evaluated by light microscopy and scanning electron microscopy (SEM) of the finished, microengineered polymer surfaces. Successful completion of this aim was essential to the following bioassay tests. Specific Aim 2: Investigate the Effects of Mechanics and Surface Chemistry/Energy of Substrata on Algal Spore Attachment on the Sharklet AFTMThe objective of this aim was to determine the effect of mechanical property and surface chemistry/energetics of a substrate on the attachment of a model marine microfouler, Ulva zoospores. The polymer films fabricated while researchin g Specific Aim 1 were tested using Ulva spores. It was assumed that the unique Sharklet AF Surfaces Fabricated with Various Polymeric Materials TMThe spore attachment assay protocol and collection of the raw data of these tests were performed by Dr. Maureen Callow and Dr. J ohn F inlay at the University of Birmingham, UK. Statistical analyses of the spore attachment data were carried out to pattern was the primary factor in prohibiting the attachment of Ulva spores based on current research carried out using PDMSe material. Thus the hypothesis was that the spore attachment reduction would be the same for all tested materials with the same micropattern using the corresponding smooth films as controls, after taking into account other factors such as surface chemistry and energy.
23 determine the influence of the mechanics, surface chemistry, surface energetics and topography of the base materials on the marine biofouling process. Th os e spores successfully settl ing on Sharklet surface s usually squeeze between the features, as can be seen in previous work [17, 22] The nanoforce gradient on various surface patterns shows significant v ariation on different base materials (see Table 1 1). If the spore attachment data showed any difference among various substrata, a comprehensive model may be proposed to include contributions from other factors, such as mechanical properties and surface energy. Specific Aim 3: Investigate the Mechanism of Inhibiting Bacterial Biofilm Formation on the Microengineered Topography There were two objectives for this aim: (1) to test the effect of mechanical and chemical properties of polymeric substrata on lo ng -term bacterial biofilm formation on the topographical surfaces, and (2) to test the effect of antibiotic treatments on the bioactivity of the bacterial cells in the microcolonies formed on the topographical surfaces. It was hypothesized that the Sharkle t AFTMAn analytical technique utiliz ing metabolic products (carbon dioxide in this case) to trigger the color change of a pH indicator in the culture medium was us ed to quantify and statistically compare the amount of active bacterial cells attached on the tested samples. T he biofilms were cultured with help from Ms Qingping Yang i n Professor Patrick Antonellis lab in the Department of Otolaryngology at the University of Florida. An engineered topography originally designed for algal spores ( Ulva ) was shown to topography replicated on the surfaces of PDMSe, SEBS, and PMMA will show the same inhibition of bacteria attachment and biofilm formation for Staphylococcus a ureus (S. aureus ).
24 have a significant inhibitory effect on the formation and growth of biof ilm from Staphylococcus aureus up to 7 days. Specific Aim 4: Prepare Undercut Surface Topographical Features on Various Substrata with Estimated Higher Reduction Targeted at Spore Attachment There were two objectives for this aim: (1) to explore the proc ess of fabricating new surface features and (2) to predi ct the improve ment of the antifouling properties of the new design based on the predictive model. A therm al press ing method was used to flatten the top of the protruding features to increase the size of the feature top Suitable processing temperature s and pressure s were found for various materials and new surface features were characterized by SEM imaging to obtain the feature size data. Water contact angle measurement s w ere taken to verify that the novel surface structure could help maintain a stable air pocket underneath the water layer and thus higher underwater stability than that on the normal protruding features. The new design resulted in higher ERI value, and therefo re a higher antifouling capability was expected.
25 Figure 11. Correlation of spore attachment density with ERI values (D ata adapted from Schumachers work [20, 22] ) Table 1 1. Force gradient between the adjacent feature elements, assuming 1% bending of the features. Material Youngs Modulus, MPa +2SK2x2 nN +3SK2x2 ** nN DMSe 1.4 28 12.5 Kraton G1657M 6.3 126 56 Kraton G1650M 3 4 6 8 0 300 G1650M/PS blend 2. 4 0 2 4. 8 0 3 2. 1 0 3 PMMA 3.3 0 3 6.6 0 4 2. 9 0 4 *: +2SK2x2 means Sharklet AFTM**: +3SK2x2 means Sharklet AF pattern with feature height 2 m, feature spacing 2 m, and feature width 2 m; TM pattern with feature height 3 m, feature spacing 2 m, and feature width 2 m. y = -25.6x + 466 R = 0.782 0 100 200 300 400 500 600 0 4 8 12 16Attachement density (spores/mm2)ERIII GR4: 12.5 nN GR2: 24.9 nN GR1: 12.5 nN GR3: 37.4 nN GR5: 0 nN GR0: 0 nN
26 CHAPTER 2 BACKGROUND Introduction Biofouling is the undesired attachment and development of cell layers on a substrate  This phenom enon can be observed almost everywhere: from bath tubs to kitchen sinks, from our teeth  to various medical devices  from pipelines and reservoirs to ship hulls. Biofouling most easily occurs at the interfaces of a solid and an aqueous solution, or a solid and humid air [8, 34] Many c onsequences are associated with biofouling in various forms. Examples are the plaques and corrosion caused by bacterial biofilms  the chronic release of pathogens from bacterial biofilms  the blockage of water intakes by biomass accumulation  and the increased drag imposed to ship hulls by the multi ple layers of marine biofoulants [2, 3]. These examples show that biofouling not only causes lar ge economic losses but also threat ens human health. As biofouling has had these negative impact s antifouling strategies and technologies have been developed to battle those unwanted effects. Previous strategies have focused on damaging or killing the adhered fouling organisms with toxic agents. However, t here are two main drawbacks to this approach. First, the toxic agents can be accumulated and passed on through the food chain, eventually threat ening human lives S econd, the target organisms may develop various resistance to the toxic agents; this evolutionary process is thus accelerated by repeated biocide application Th us d ifferent and more environmentally friendly strategies are called for Biomimetics is an interdisciplinary field which employs the chemical and phy sical methods and concepts of living organisms in the design of devices and
27 systems [39, 40] As a result of evolution, organisms have developed superior properties relative to the peer man-made products when dealing with biological environment s In the field of antifouling, these biom imetic principles can also be discovered and employed effectively. In this chapter marine biofouling and medical biofouling will be reviewed; the current state of antifouling technologies will also be summarized. Marine Biofouling and Prevention Marine b iofouling is a complex process, which in most cases can be described by a basic sequence of event s : biochemical conditioning, bacterial attachment and colonization, and unicellular and then multicellular eukaryotic adhesion and proliferation  When any solid substance is immersed in seawater, the surface will adsorb dissolved chemicals (usually macromolecules) in a short period of time. This process, biochemical conditi oning, sta rts immediately after immersion and reaches equilibrium in a few hours . Then, f ollowing chemical cues from the surface, bacteria, unicellular algae, cyanobacteria (blue green algae) protozoa and fungi, attach and begin to colonize on the conditioned surface within hours  These colonies, or biofilms, are often referred to as microfouling or slime and provide an anchoring layer for the so called macrofouling species such as higher algae, sof t -bodied invertebrates (sponges and tunicates) and calcified invertebrates (barnacles and tubeworms) [8, 41, 42] The time scale of the fouling stages is shown in Figure 21  Biofouling is undesirable for many reasons Microbial biofilm can cause local fluctuations in the concentration of various chemical species such as oxygen and metal ions, therefore accelerating corrosion of the metal substrata  The metaboli c process or products (e.g. sulf ides) may also be corrosive to steel surface [43, 44] Biofouling can
28 increase the roughness on ship hulls, resulting in an increased hydrodynamic drag [1, 2] The increased fuel consumption, toget her with maintenance cost s (such as dry dock cleaning, paint removal and repainting), may cost the US N avy more than one billion dollars annually  In addition to economic losses, a more urgent threat associated with marine biofouling is that it can spread invasive species globally endangering local biodiversity  The strategies in marine biofouling control include antifouling (AF), in which the surface is capable of killing or repelling the fouling species, and foul release (FR), where, due to the non -stick property of the coatings, accumulated foulers are removed by gravity or shear ing forces from the water Various biocides have been used for antifouling, with the most effective being tributyltin (TBT) compounds. Due to the adverse effects on the food chain [45, 46] TBT based coatings were banned by the International Maritime Organization  and was supposed to be phased out by the year 2008. Meanwhile, other metal containing antifouling coatings, e.g. Cu2In the following sections, the factors affecting marine biofouling and foul release are reviewed. New coating technologies, using both strategies for biofouling control, a re the main focus o f this literature review. O, have also raised concern of environmental impacts. Foul release measures, on the other hand, depend on the coating materials (e.g. polydimethylsiloxane) with a low su rface energy and a low elastic modulus  The macrofoulers could be removed under normal operating speed, b ut the slime layer is difficult to detach. Thus environmental -friendly coating materials and strategies are needed to address modern marine antifouling applications.
29 Factors on Marine Biofouling and Foul Release The effect that s urface properties (e.g., topography, chemistry, mechanics, and surface energy ) have on the attachment adhesion, and growth of living organisms was researched by the B rennan research group at the University of Florida [1719, 21, 48] Als o surface charge has recently a ttracted more interest [49, 50] Surface Chemistry/Energy Surface chemistry and/or surface energy certainly play a role when a microorganism approaches and settle on a surface. Finlay et al.  found that Ulva spores tended to settle on hydrophobic rather than hydrophilic surfaces The spores that settled on hydrophobic surfaces showed higher adhesion strength than those on hydrophilic surfaces, tested by water jet removal. Ista et al.  changed surface chemistry (and therefore surface energy) systematically via a self assembled monolayers (SAMs) technique and tested the surfaces with marine bacteria and Ulva zoospores. T hey prepared two series of mixed SAMs on a gold surface: one was alkyl chains with mixed end groups of COOH and CH3; the other was alkyl chains w ith mixed end groups of OH and CH3. Although the adhesion of bacteria agre ed with the prediction of the thermodynamic model, a spore test yielded more complicated bioresponses, indicating that a simple model cannot fully explain the interactions between the somewhat complex microorganisms and the surface. In a recent paper, Sch il p et al.  studied Ulva spore and diatom attachment and removal on a hexa(ethylene glycol) (HEG) SAMs with various end groups (hydroxyl, methyl, ethyl, and propyl). The water sessile contact angles varied from ~30o to ~90o as the end groups varied. Ulva spores tended to settle on the hydrophilic surfaces (hydroxyl and methyl ended HEG), yet the adhesion strength was very weak : the surface attached
30 rafts were easily removed by the operational procedures at the end of attachment assay and the subsequent flow test. As the diatom does not have any motile capability, its attachment depends on gravity (and Brownian motion when close to a surface). The adhesion between the diatom and the surfaces also increased with the hydrophobicity after rinsing. Therefore, the overall apparent attachment density increased with the hydrophobicity o f the surface for both spores and diatoms. The recent work by Schilp et al.  showed that surfaces covered with poly(ethylene glycol) chains (MW ~2 and ~ 5 kg/mole respectively) were resistant to spore and diatom attachment Surface chemical patterns, result ing from the self assembled monolayers ( SAMs ) technique, also showed an influence on the attachment of motile spores. As Ulva zoospores tend to settle on completely fluorinated surfaces rather than fully PEGylated surfaces, Finlay et al.  first showed that spores could not distinguish between patterned SAMs with alternating fluorinated stripes and PEGylated stripes when the width of the stripes w as less than ~20 m. In that case, the spores acted as if the alternating p atterns were purely PEGylated surfaces. Gradient surface chemical patterns influenced the attachment characteristics of zoospores  Unlike the sole hydrophilic or hydrophobic surfaces, the attachment of Ulva zoospores on this gradient pattern exhibited the opposite trend. Excluding the migration of the settled spo res, the authors speculated that the gradient surface sent long -range signal s to disturb the swimming directionality of the spores. Surface Charge Ederth et al.  found that Ulva spores were attracted to a cationi c oligopeptide (aginine) self -assembled monolayer but were easily removed. In a recent ly published work, the zeta potential of the motile Ulva spores was determined to be 19.31.1 mV
31  This measurement could explain why spores tend to adhere to neutral or positively charged surfaces rather than negatively charged surfaces. Mechanical Properties Generalizing Kendalls model  Brady [59, 60] declared that the relative adhesion of marine organisms onto the surface of a polymer substrate is proportional to the square root of the product of the elastic modulus and the critical surface energy. Polydimethylsiloxane (PDMS), with low surface energy and elast ic modulus, is therefore an ideal foul release coating material. The mechanical properties of PDMS on spore attachment and removal was studied by Chaudhury et al.  In their study, t he el astic modul us of PDMS ranged from 0.2 to 9.4 MPa. These smooth samples showed no significant difference i n spore attachment while spore and sporeling removal were highest on the lowest modulus sample  When the mechanical property is fixed (modulus 0.8 MPa), the film thickness (16, 100, and 430 m respectively) showed no significant effect on spore attachment while spore and sporeling removal were more easily achieved on thicker films (430 m film for spore and > 100 m films for sporeling)  The effect of the substrate thickness on barnacle adhesion was studied by Sun et al.  It has been known that barnacles can feel and penetrate through the upper layer to the underlying coats and thus settle firmly when the surface film thickness is below ~100 m (dry film thickness)  Surface Topography The effect of surface roughness on the attachment of animals such as barnacle cyprids was extensively investigated [59 61]. Andersson et al.  prepared a microtextured P DMS surface by casting a PDMS prepolymer against metal mesh with
32 various sieve sizes. Their field test showed significant reduction of barnacle fouling  Petronis et al.  replicated micron-sized pyramids and grooves onto PDMS from silicon wafers which were treated by photolithography and etching to define the sizes and shapes of the surface structures. They claimed that the groove structure could effecti vely reduce barnacle attachment while the pyramid structure was not able to do so (both compared with smooth surface)  Berntsson et al.  carried out an investigation of surface microtexture on the exploration and attachment by barnacle cyprids. Their results showed a remarkable 92% reduction of cyprid attachment compared to smooth controls. It is important to note that they used poly (methyl methacryla te ) (PMMA) and poly ( vinylidene fluoride ) (PVDF), which have higher surface energ ies and elastic moduli than PDMS. Surface topography can also affect Ulva zoospore attachment Callow et al.  found spores preferentially settled against the surface features when the spacings between the features were similar to or larger than the size of the spores. From these findings a narrower spacing between the surface f eatures could prevent spore attachment This result was later demonstrated by Carman et al.  New Coating Strategies Polymeric m aterials Many new polymeric coating materials are under investigation to replace the toxic organometal based coatings which currently are in extensive use Among all polymer materials, zwitterionic polymers showed promis e in antifouling toward both protein adsorption and bacteria adhesion [11, 66, 67] Ongoing research with this type of material also showed excellent resistance to marine micro organisms  In order to combine the antifouling (AF) property of oligoethylene glycol and the foul release (FR)
33 property from perfrluoroalkanes, Krishnan et al  synthesized an amphiphilic copolymer. O ligoethylene glycol and perfluoroalkyl moieties were pr esent in the same side chains of their synthesized polymers. In an aqueous environment, the side chains bent with oligoethylene glycol segments sticking toward water and perfluoroalkyl segments bowing toward the coating surface. The obtained surface show ed improvement against attachment s of Ulva and Navicula and a higher removal rate of sporelings compared with glass and PDMS surfaces. Linear -chain poly ( ethylene glycol ) (PEG) grafted surfaces have long been shown to be effective against protein adsorption and cell adhesion [10, 70, 71] Statz et al  coated a titanium surface with PEG chains via conjugation with L 3,4-dihydroxyphenylalanine and the result ing surface showed AF/FR against spore and diatom Furthermore, an almost 100% removal rate was achieved when a 20 -Pa shear stress was applied. Other polymeric materials, such as perfluoropolyeth ers (PFPE) and their PEG blends  and siloxane polyurathane copolymers  also showed improved AF properties. There are still potential issues to be addressed with these polymeric materials. For zwitterionic polymers  hydrolysis may occur in the physiological environment with the help of enzymes and the result ing products may contain quaternary ammonium salts, which are undesired leachants. For amphiphilic copolymer  and PFPE  the synthetic/processing procedures are time consuming and thus inefficient in terms of production. A disadvantage of PEGylated polymer coatings is that PEG can gradually degrade [9, 75], maki ng it unfavorable for long term applications. Surface c atalysis The Detty group  explored an innovative approach for marine antifouling applications They used se lenoxide or telluride as a catalyst and xerogel film as a carrier
34 to convert the low concentration of H2O2Biomimetic m easures (in the natural marine environment) into active oxygen atoms, in order to expel the unwelcome species. The initial lab test results showed an effect ive reduction of attachment from barnacle larva, tubeworm larva, and Ulva spores. A potential obstacle with this novel approach is the adsorption of organic matter onto the xerogel film. The contamination would gradually deactivate the catalyst. Another po ssible disadvantage of the xerogel film is its mechanical strength, which may not endure the shear forces arising from vessel at normal cruising speed Marine bacteria communicate via signaling chemicals to form biofilms and this cue c an strongly attract algal spore to deposit on the biofilms  Therefore a strategy of quenching these signaling chemicals was proposed by biomimetic routes. Zosteric acid ( p -(sulfo oxy) cinnamic acid), a naturally occurring phenolic acid in eelgrass (Zostera marina L.) plants, was found to have antifouling activity toward a wide spectrum of species such as Ulva zoospore, marine bacteria and barnacle larvae  This compound has also been used in antifouling against fresh water microorganisms  and fungal spores on plant leaves  Recently, the search of effectiv e antifoulants has been expanded to the extracts from some marine organisms, including plants [82 -84] and animals [83, 85] When th ese type s of leachants are blended with coating materials for antifouling applications, there is still a concern that a higher leaching rate is needed so as to reach comparable antifouling efficacy as traditional organometallic agents  Researchers have found that some marine animals are resistant to biofouling without secreting chem ical signals. Instead, these animals grow complex outer surfaces with micro or even nano-sized topographies [87, 88] A nother biomimetic route is thus
35 to explore the antifouling properties from these naturally evolved surface topographies. Direct replicas of rough surfaces from foul -resistant species such as tropic al sea stars onto an epoxy resin did n o t lead to antifouling in a field test  However, a further comparison of the surface properties of the two (sea stars and epoxy) in terms of chemistry and mechanics c ould have explained the difference in antifouling performance. Meanwhile, the direct deployment of the shells of bivalve molluscs  which can have regular or irregular surface microtopogaphies showed resistance to the fouling species in a field test. Researchers at the University of Florida developed surface micro patterns for antifouling applications. One biomimetic pattern, named Sharklet AFTMIn summary, there are many factors controlling the initial attachment o f marine organism s onto a surface. From the point of view of long term application, coatings with various surface chemical compositions will not permanently resist the deposition of organic matter in natural marine environment. Therefore the surface will eventually attract microorganism s to grow on it, causing the subsequent accumulation of various larger plants and animals. On the other hand, a physical surface defense inspire d from biomimetics may work effectively in the long run. Surface hierarchical str uctures (in both showed an up to 86% reduction of Ulva zoospore attachment [17 ] and 97% reduction of barnacle cyprids attachment  By correlating spore attachment with various surface patterns, an engineering roughness index (ERI) model was proposed  This model was further refined and showed predictive capability  Engineered surface micro -topographies were mostly replicated onto PDMSe by the group, and other materials and surface chemistries are under investigation by the group.
36 chemical and physical patterns) may be needed to repel organisms with different sizes and attachment mechanisms. Bacterial Biofilms in Medical Environment Healthcare associated infections (HAIs) have become familiar in the last decade. In the United States alone, more than 1.7 million people are affected by HAIs and approximately 100,000 people lose their lives due to the complications from HAIs annually  HAIs also result in $4.5 billion of additional healthcare exp e ns es  As one imp ortant part of the healthcare industry, invasive medical devices from simple cath eters to sophisticated artificial heart valves are improving human health and life quality while at the same time imposing substantial risk of infections. For example, it i s estimated that up to 20 000 patients died annually of central line associated bloodstream infections in U.S. intensive care units . The annual cost of caring for patients with central venous catheter (CVC) associated bloodstream infections could be as high as $2.3 billion. In natural environment s bacteria tend to attach to surfaces and form colonies/biofi lms for an increased rate of survival and proliferation  According to Costerton et al.  a bacterial biofilm is defined as matrix enclosed bacterial populations adherent to each other and/or to surfaces or interfaces. The process of bacterial adhesion to a surface occurs in five stages as shown in Figure 2 2  During the initial stage of attachment, there are specific interactions (e.g. cell -protein film or cell -sugar receptors interactions ) and non-specific interactions (e.g. electrostatic or hydrophobic interactions ) between the cell and the sur face [95, 96] Once irreversibly attached to the surface, the bacterial cells will activate specific genes which lead to the synthesis of ex tracellular poly meric s ubstanc es (EPSs) or extracellular matri ces (ECMs)
37 to encase the bacteria [97, 98] A recent report on the adhesion of Staphylococcus a ureus (S. a ureus ) to engineered PDMSe films clearly demonstrated the cycle of bacterial adhesion  Due to the pro tection of the EPSs/ECMs secreted by the adhered bacteria, the colonies/biofilms are 500 times more resistant to common biocides, antimicrobial treatments and the antibodies of the host, when compared with the planktonic bacteria [93, 94] The pathogens released from the bacterial biofilms can cause chronic infections in the host and thus the formation of bacterial biofilms on the surface of biomedical devices is undesirable To avoid bacterial ad hesion to biomedical devices, two primary strategies have been proposed and utiliz ed for biomaterials: bioadhesion -free coatings and incorporated antimicrobial agents  Due to the complex physiological environment, the surface of an inserted device will eventually be cover ed by biomatter Therefore the chemical cues will favor the recruitment of pathogens, if they are present On the other hand, antimicrobial agents face the challenge of multi drug resistant bacterial strains, which are increasingly common in current medic al facilities world wide  From the standpoint of materials science and engineering, we may achieve antifouling by (1) carefully selecting the desired materials, (2) further adjusting the chemistry at the interfaces, and (3) biomimetically engineering the interfacial characteristi cs. The initial adhesion of bacterial cells onto the surface is the critical step for biofilm formation. The types of interactions in bioadhesion were summarized by Glantz et al  Many factors, such as a protein conditioning film, surface charge, surface hydrophobicity, and surface micro topography, can affect this step. A recent review  summarized the effects of th e se factors and suggested that the
38 attachment of bacterial cells to the solid surface is not only affected by the culture medium and surface properties, but also by the dynamic change of bacterial membrane structures. In the following section some other factors involving the characteristics of bacterial cells are reviewed and recent advances in the prevention of biofilms ar e summarized. Bacteria Mechanical Sensing Mechanical senses originate from sensing such forces as osmotic force, thirst, touch, vibration and texture  It was postulated that early cells developed two basic types of sensing mechanisms to ensure survival  One is based on solute sensing with a key lock type interaction at the cell wall. Another is based on solvent (i.e. water) sensing with ion-channel type gauges at the cell wall. After billions of years of evolution, c ells living in an aqueous environment are usually equipped with m echanosensitive (MS) ion channels which are embedded in the cell membranes and which could respond to tension in proportion to the concentration of water  ; i t is believed that the se channels are critical to osmotic pressure regulation in bacterial cells  The b acterial MS ion channels were first found in Escherichia coli (E. coli)  and were well characterized. The MS ion channels are divided into three categories, i.e. MscL, MscS, and MscM, representing MS channel of large, small, and mini conductance, respectively  MscL is non -selective, whereas MscS is selective to anions over cations and MscM slightly prefers cations over anions  Tissue cells were reported to respond to the stiffness of the substrate [106, 107] The c ell membrane not only protects the inner environment, but also plays a critical role i n exchanging materials and information (e. g. chemical and mechanical cues). The bioactivities occurr ing at the cell membrane certain ly involve the energy: the biomotor
39 proteins (e.g. myosins) are powered by adenosine triphosphate (ATP). Recently, atomic force microscopy (AFM) has been used to reveal the characteristic vibrations of cell membrane s In an early report, Pelling e t al  determined the periodicity of the membrane motion of singe yeast cell s ranging from 0.8 to 1.6 kHz depending on temperature, and they associated this vibration with membrane motor protein activity. A l ater investigation of body cells (rat cardiomyocytes (CM) and human foreskin fibroblasts (HFF))  revealed that the vibrational frequency was 4.2 Hz for CM and 0.06~0.34 Hz for HFF. There a re few publications on the effect of the substrate mechanical properties on the adhesion of bacteria. Lichter et al  investigated the adhesion of Staphylococcus epidermidis ( S. epidermidis ) and E. coli on a polymeric thin film with varying mechanical properties. They found a positive correlation between surface stiffness and adhesion for the two bacterial species. Bacteria Quorum Sensing Cell communication and activity coordination have long been consider ed a characteristic of advanced creatures. However, research in the past two decades has revealed that bacteria can also establish communication systems to synchronize biological activities such as biofilm formation  As this communication is population (and signal concentration) dependent, the process of adjusting bacterial social activities is referred to as quorum sensing  Q uorum sensing may be divided into 4 steps  : (1) bacterial cells produce small signaling chemicals (2) the chemicals are released into the local environment by active transport or passive diffusion (3) the chemicals are picked up by the receptors on the cell wall of other bacterial cells and (4) specific gene expressions are induced by
40 certain concentrations of signaling chemicals T his process is a positive feedback loop, which means that the concentration of signaling chemicals can increase exponentially above a threshold (Thus quorum sensing signals are also referred to as autoinducers. ) Various bacteria species utilize different chemical signal molecules for quorum sensing. In general, Gram -negative bacteria use acylated homoserine lactones as autoinducers while Gram -positive bacteria use processed oligo peptides to communicate  Recent advances indicate that cell -to -cell communication via autoinducers occurs both within and between bacterial species  Furthermore, evidence suggests that bacterial autoinducers can elicit specific responses from host organisms  The effects of quorum sensing on biofilm formation vary with different species. Pseudomonas aeruginosa (P. aeruginosa), a Gram negative bacterium, show s less tendency toward biofilm formation with one lasI mutant strain compared with a wild type  This finding led to a new treatment method for biofilm associated infections using quorum sensing inhibitors  Unlike P. aeruginosa, the quorum sensing in the Gram positive Staphylococc al species does not facilitate biofilm formation. The quorum sensing system in Staphylococcus aureus (S. aureus ), which is regulated by an accessory gene regulator (agr), suppresses adhesin production (hindering colony formation) and induces secreted exoprotein production. On the other hand, agr dysfunction or inhibition may increase the adhesive properties of S. aureus Studies using fluorescent S. aureus confirmed that agr is not activated in all areas of the biofilm except for the cluster s that detached from the biofilm in a later stage 
41 Current Art in Inhibition of Biofilm Formation The relationship between chronic infection and bacterial or fungal biofilm s, and also the strategies to inhibit biofilm formation were summarized in several reviews [118, 119] Surface grafted polymeric/oligomeric ethylene glycol brushes (PEG) have attracted much attention in the past two decades since the PEG layer is resistant to protein and cell adhesion. Though early investigation suggested longer chain PEG graft s (MW 18 5 kg/mole ) on biopolymers were effective in protein and cell repelling  recent reports on PEG grafted surfaces for prevention of cell adhesion [10, 120123] mainly focus ed on relatively shorter chains (MW 2 to 5 kg/mole ) and were based on simulation and experimental data of protein adsorption. Du et al  d eposited a PEG layer onto a lipid film by conjugation, which is relatively weak compared with covalent bonding. The brushes ( with chain length of MW 5 kg/mole) provided sufficient protection to the underlying lipid layer from protein adsor ption and cell ad hesion after two hours of animal cell/protein-surface attachment. Cunliffe et al  covalently bonded alkyl, perfluoroalkyl, alkylamide, or PEG (MW 5 kg/mole) chains onto a glass substrate and f ound that, after a 24 hr incubation, the PEG modified surface wa s the least fouled by the four bacteria types tested. In a later report  s ign ificant reduction of bacterial adhesion (o n the order of 2 -4 magnitudes) was achieved during five hour experiments on a PEG (MW 5 kg/mole ) grafted poly ( ethylene terephthalate) (PET). Other surface modifying agents were also proposed and tested for anti -fo uling purposes. Among them, the charged polymeric coatings/grafts seem to be very promising. Haldar et al  tested branched and linear N,N -dodecy l methyl-
42 polyethylenimines (PEIs) and their derivatives against the influenza virus and bacteria. Four -log reduction was achieved for the virus test and complete bactericidal activity was obtained when E coli and S. aureus were tested. The b acteicidal act ivity was not due to leaching from the coating, but rather due to the rupture of the bacterial cell membranes caused by sticking side chains  Chen et al  showed that a surface grafted poly(sulfobetaine methacrylate) layer (zwitterionic polymer) was r esistant to biofilm formation for 48 hours against S epidermidis (Gram positive) and P. aeruginosa (Gram negative). Due to thermodynamic reasons, i t may prove impossible to create a surface that is perfectly non-fouling. In fact, the surface of any inserted medical device in vivo is rapidly covered by plasma and connective tissue proteins. Thus the efficacy of the surface grafts may not last a required period of time for practical applications. Therefore, another concept for the prevention of implant associated infections involves the impregnation of devices with various antimicrobial substances such as antibiotics, antiseptics, and/or metals. In fact, materials for clinical use (such as antimicrobial catheters ) are commercially available with considerable impact on subsequent inf ections  Several quorum sensing (QS) modulating therapies, such as macrolide antibiotics, QS vaccines, and competitive QS inhibitors, have been investigated. These therapies may prove to be helpful in diminishing the translation of QS -directed toxins or can prematurely activat e the QS response so as to alert the immune system to bacteria hid in a low cell density. QS represents a recently discovered method of bacterial
43 communication and population control, which may prove to be a unique mechanism to prevent, suppress, and treat infectious diseases  Biomimetic strategies have been adopted to develop antifouling coatings to prevent adhesion of marine microorganisms [17, 128] Recent work by Chung et al  showed the promising application of engineered PDMS e surface features in preventing biofilm formation. Since the first stage (reversible attachment of the cells on a surface) is the most critical in bacterial adhesion, surface engineering strategies should be developed to interrupt the cells sensing of the surface and hinder the initial attachment. As surface -immobilized linear PEG chains and zwitterionic short chains show excellent perf ormance in preventing protein adsorption and cell adhesion, a combination of an engineered surface covered with a tethered oligomeric layer may have a synergetic effect and therefore act as antifouling coatings for biomedical devices over an extended perio d of time in vivo
44 Figure 21. Time line of marine fouling event sequence s  Figure 22. Illustration of the development of bacterial biofilm: (1) individual bacterial cells attach reversibly to the surface; (2) the cells anchor to the surface irreversibly by secreting exopolymeric substances, and the cells lose their flagella-driven motility; (3) early development of bacterial colonies indicates the first matur ation phase is reached; (4) the fully grown and developed biofilm shows the second maturation phase is reached; (5) single motile cells (dark cells on the figure) disperse from the colonies to start another cycle  1 0 2 4 5 6 3 7 8 Macromolecular Film Bacteria Diatoms Larvae SporesTime ( 10xsec)1 min 1 hr 1 d 1 wk 1 0 2 4 5 6 3 7 8 Macromolecular Film Bacteria Diatoms Larvae SporesTime ( 10xsec)1 min 1 hr 1 d 1 wk
45 CHAPTER 3 FABRRICATION AND CHA RACTERIZATION OF MIC ROENGINEERED THERMOPLASTIC POLYME RIC FILMS Introduction One objective of this work w as to establish an easy and efficient technique to produce various polymeric films with microengineered, surface topographic features. There are basically two ways of producing micropatterns on polymers: direct patterning (such as photolithography and reacti ve ion etching) and molding (such as hot embossing). Direct patterning can produce a desired surface pattern in a short period of time. However, there are several drawbacks associated with this method, such as high processing cost, limited size of the prod ucts, and restrictions on the substrates. Molding on the other hand, is and attractive alternative due to its flexible processing choices low operation al costs, and highquality and high -resolution products  Molding technique involves mold f abrication and mold treatment. The mold fabrication was thoroughly reviewed by Campo and Arzt  De molding is the last step for surface patterning in micro -fabrication using the molding technique. This step determines the quality (fidelity and resolution) of the final products. Therefore an antisticking (or antiadhesive) surface treatment is usually perform ed for easy demolding Previously, researchers deposited fluoropolymers onto the stamps with the help of a CF4/H2 or CHF3 plasma treatment. This layer was not strongly bonded to the surface and therefore repeated coatings w ere needed for multiple fabricat ions. Another antisticking coating was developed by applying s elf assembled monolayers (SAMs) Perfluorosilanes have been used to treat silicon wafers for antisticking coatings [130, 131] The SAMs coatings are strongly bonded to the substrate, but the perfluorosilanes are usually highly reactive with water. T hus t he surface coverage and
46 stability of the SAMs are adversely affected by the water content in the processing en vironment and t herefore the operation cost is increased for this technique. When characterizing the thickness of thin films grafted onto surfaces, several methods, including x -ray reflectivity (XRR) [132-138] neutron reflectometry (NR) [134, 135, 139] x -ray photoelectron spectroscopy (XPS)  atomic force microscopy (AFM) [139 -141] and ellipsometry [139, 142] were explored. XRR is an absolute technique for measuring film thickness [133, 137] and has been used to calibrate XPS and ellipsometry measurements [137, 139] After preparation and characterization of the antisticking layer on a mold, polymer films with nano or micro -sized topographical structures can be replicated with ease. C ommon replication techniques include solution casting and thermal embossing. One important phenom enon associated with t he surface roughness is that it influences the wetting and dewetting of the surface An example from nature is the lotus leaf, which acquires superhydrophobicity through hierarchical nano and micro -sized features, although the Young s contact angle is only ~74oWenzel s model assume s the liquid fully wets the total available surface of the substrate, giving the following expression for the apparent contact angle: for the coating wax on the surface  There are t wo generally accepted models to explain the effect of roughness: Wenzel s model  and the Cassie and Baxter  (C -B) model cos = cos (3 -1 ) where is the apparent contact angle on the rough surface, r is the Wenzel roughness factor (defined by the total surface area divided by the projected plan a r surface area),
47 and is the equilibrium contact angle on a smooth surface of the same material, and is determined by Youngs equation: cos = (3 -2 ) where refers to the interfacial tension and the subscripts s l and v refer to the solid, liquid, and vapor phases, respectively. Equation 3 shows that a rough surface will promote hydrophilicity (or hydrophobicity) if the material is hydrophilic (or hydrophobic). The C -B model attributes the change of the contact angle on a rough surface to the composite solidliquid vapor interfaces. In other words, the rough surface is not fully wet as there are entrapped air pockets underneath the liquid. If f is the fraction of the solid in contact with the liquid, the C -B equation is cos = 1 + ( 1 + cos ) (3 -3 ) T he C -B model predicts that a hydrophobic state can be reached even with intrinsically hydrophilic materials. Thermodynamic analysis support s this phenomenon : examples include re entrant micro -structur e  and hierarchical nano micro sized surface features  Here, the process of producing high -fidelity surface micro -patterns was developed. An anti -sticking layer short -chain poly(dimethyl siloxane) (MW 5 kg/mole) was covalently grafted onto a mold surface with relatively simple treatment procedures Solution casting was chosen for the fabrication of polymer films as it was simple and reproducible. Solvent was slowly removed so that the polymer film could fully replicate the surface nano and micro -structures from the mold. Surface morphology, topogr aphy and wettability were characterized for better understanding the subsequent bioresponse studies.
48 Materials and Methods Materials Polystyrene (PS, Mw ~350 kg/mole) and poly methylmethacrylate (PMMA, Mw ~350 kg/mole) were purchased from Sigma -Aldrich (St Louis, MO). Kraton G1657M styrene ethylene butadiene-styrene triblock copolymer (SEBS, mass fraction of styrene block ~ 0.13) and G1650M (mass fraction of styrene block ~ 0.30) were purchased from Kraton (Houston, TX). 3-(aminopropyl triethoxysilane) (APTES ), and monoglycidyl ether terminated PDMS (Mn 5 k g/mole, abbreviated as PDMS 5K) were purchased from Sigma -Aldrich (St. Louis, MO). Anhydrous ethanol, 1propanol, and 2 -propanol, hydrogen peroxide (30 wt%), and concentrated sulfuric acid (95%) were purchas ed from Fisher Scientific (Pittsburgh, PA). All chemicals were used without further p urification. cm) was prepared in the lab with a Barnstead Nanopure DiamondTM Surface Treatment of Silicon Wafers lab water system from M illipore (Billerica, MA). The grafting of PDMS -5K onto Si wafer was carried out in three steps : (1) a silicon (Si) wafer was first soaked in xylenes overnight to remove possible organic matter, followed by immers ion in a freshly mixed concentrated sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) water solution (30 wt%) (1:1 volume ratio)  for 30 min to generate surface hydroxyl groups; (2) after thorough rinsing with nanopure water, the Si wafer was dried with a nitrogen flow and neat APTES was dropped to cover the entire surface for 10 min; (3) after rinsing with anhydrous ethanol, the APTES treated Si wafer was covered by neat PDMS 5K and heated in oven at 80 oC for 4 hr. The wafer was then rinsed with 2propanol and 1-propanol ( alternately ) in an ultrasonic bath. The
49 PD MS 5K treated Si wafer was blown dry with an Ar flow  A schematic flow chart for the treatment is give n in Figure 3-1. Fabrication of Microengineered Thermoplastic Polymeric Films Both smooth and topographical surfaces were transferred to the three polymers by solution-casting of a polymer/toluene solution (0.15 g polymer per 1 ml solvent) onto the patterned silicon wafers. The resultant topographies contain feature elements with a width of 2 m, a distance ( between the neighboring elements) of 2 m and a varying height of approximately 2 or 3 m The short name of this pattern was Sharklet + 2 SK2 x 2 (for 2 m height) and +3SK2 x 2 (for 3 m height) Pattern fidelity was evaluated with light and scanning electron microscopy (SEM). As the PMMA and PS films were curling after the removal of the solvent thermal press ing was used to ease the unbalanced residual stress. To do this, a PMMA or PS film was s andwiched between two poly ( ethylene terephthalate) (PET) sheets and t wo glass plates wer e preheated in an oven. The desired processing temperature was 88 oC for PMMA, and 97 oSurface Characterization of Polymer Films C for PS. The polymer film (in between the PET sheets) was placed on the preheated glass plate, and covered by another preheated glass plate in the oven. The polymer f ilm was thermal -pressed for approximately 30 ~60 sec under ~2 000 Pa pressure. After cooling, the curling was removed. X -ray photoelectron spectroscopy (XPS) spectra were obtained on a Perkin Elmer PHI5100 ESCA sys X -ray source ( h = 1486.6 eV) at 12 kV and 9 mA in FRR (fixed retardance ratio) mode with analyzer chamber pressure at ~109 torr.
50 Atomic force microscopy (AFM) was used to determine the nano -scale surface roughnes s of the Si wafer and the morphology of the smooth polymer films obtained from solution casting. A Dime n sion 3100 AFM with a Nanoscope V controller (Digital Instruments, CA, USA) was used for all measurements. The probes were silicon nitride cantilevers (V eeco Metrology, CA, USA), with a spring constant of 0.06 N/m. All samples were imaged in air without using liquids. C ontact mode imaging was used to obtain high resolution topographic images of all of the smooth Si wafers and polymer films. The scanned sur faces were 2 x 2 or 5 x 2The w ater contact angle s w ere measured using both dynamic and sessile drop methods with a Ram-Hart goniometer (Netcong, NJ) coupled with DROPimage Advanced software (for image capturing). The d ynamic drop technique was adopted for advancing and recedi ng contact angle measurements. The l iquid used was nanopure n the surfaces via RamHart Auto Pipetting system. Advancing contact angles were initi ally measured with 2~3 a gradual total in increment s and f ive images were taken at one location drops, followed by a gradual intake of the liquid from the same spot, with five images taken at the same location. For each sample, two locations were randomly chosen for measurements with both advancing and receding modes and thus ten images were recorded for each sample. C ont act angles were measured with ImageJ software (public software developed by NIH). at a scan rate of 1 Hz. The s and Six drops were placed at the randomly chosen locations on the test surface. i mmediately after
51 placing th e liquid images were taken in two directions: the spreading direction parallel to the surface feature, and the spreading direction perpendicular to the surface feature. Six images were taken for one test surface. The sessile drop contact angles were also measured with ImageJ software. Surfaces were blown by nitrogen flow at room temperature prior to testing. Results and Discussion PDMS -5K Grafted Si Wafers PDMS 5K was grafted onto Si wafer s according to the treatment scheme in Figure 3 -1. After an oxidativ e piranha wash and thorough rinsing with water the Si wafer ( with a patterned or flat region) was evenly covered by a water layer, which indicated abundant surface hydroxyl groups. When grafting of PDMS -5K was completed, water droplets could not spread on the patterned or flat regions on the Si wafer, providing a rough indication of the PDMS -5K layer on the wafer surface. The surface sil a nization with APTES was the most critical step in the Si wafer treatment because this process determined the surface density of the NH2 end group, which reacts with glycidyl group of the short -chain PDMS (PDMS-5K). Processes using dilute solutions of APTES in water  or in toluene  were employ ed. The PDMS 5K layer s grafted on the Si wafer following the two sila nization treatments, however, were not accept able for demolding. PMMA and PS f ilms had to be peeled off from the mold with the help of dry ice to cool down the Si wafer. Furthermore, the micro topographical features on the obtained PS films were deformed and damaged during demolding, as shown in Figure 3-2. For the PS +2SK2x2 film, the smallest feature (2 m width, 4 m length) was broken (Figure 3-2 A and C), and most features were bent toward one direction (Figure 3-2 D). Although longer immersi on time (18 hr) in 2~12%
52 APTES/toluene solution may give higher surface coverage  the solution treatment was abandoned due to time constraint s A n eat APTES treatment was thus adopted from Suis report  and only took 10 minutes to sil a nize the Si wafer. The result ing PDMS -5K layer on Si wafers was robust : the whole series of polymer samples in this work were cast more than 50 times, and the polymer films from the patterned wafers were peeled off with ease without the aid of dry ice The patterned polymer films showed a high fidelity (see next section) and thus the antisticking treatment protocol was successfully established. Dynamic water contact angle measurement s of both smooth and patterned regions on Si wafers (before and after PDMS 5K grafting) are shown in Table 31. T he advancing (a) and receding ( rFor the flat regions on the Si wafers, the PDMS -5K grafted surface showed no difference i n both ) water contact angles of PDMSe with +2SK2x2 pattern a re also listed as reference s a and r c ompared with a hexamethyldisil a zane (HMDS) treated surface. For the mold regions, which had the recessed Sharklet pattern (2 m depth, denoted as 2SK2x2), the advancing contact angles were the same for the two treating methods, while the receding contact angles were not, with the higher rThe low surface energy methyl groups ( CH for the PDMS 5K treated sur face than the HMDS treated one. 3) on PDMS -5K chains would be in contact with the atmosphere, as are methyl groups in the case of HMDS treated surface T he advancing contact angle is less sensitive to surface roughness and heterogeneity than the receding angle  which explains why the advancing contact angles are the same for both patterned and flat regions with the two treatment methods
53 Contact angle hysteresis, H is defined as the difference of the advancing angle a and receding angle r = (3 -4 ) : The c ontact angle hysteresis can be influenced by many factors, such as the solid surface roughness, the chemical heterogeneity, swelling, penetration of liquid into the solid surface, and reorientation of functional groups [154, 155] The c ontact angle hysteresis was the largest for the PDMS -5K treated Sharklet mold on a Si wafer (5 2o, see Table 31) while it was the least for the smooth PDMSe surface (24oThe XPS survey scans clearly showed that C content (C 1s ~284 eV ) increased and Si content (Si 1s and Si 2p, ~100 eV ) decreased after sila nization with APTES and surface grafting of PDMS 5K on the Si wafers (Figure 33 ). Graf et al  recently developed a protocol for cleaning the Si surface and surface silanization with APTES. Their XPS survey spectra for the bare and APTES-coated Si surfaces showed the same trend as in this study. Si 2p ). Therefore surface roughness together with chemical heterogeneity may explain the difference. 3 peaks were scanned for the Si wafers after each treatment. After the oxidative wash, the chemical states of the Si wafer were mainly Si -(O)4 (~103 eV) and Si(0) (~99 eV) [156 -159] indicating the presence of surface oxidized layer with elemental Si below as shown in Figure 3-4 (A). After the grafting of the short -chain PDMS (Figure 3 4(C)), the surface chemical states shifted to a (C2SiO2) peak (~102 eV)  ; the Si(0) peak still appear ed in the spectra, thoughwith a significant intensity drop (see Figure 3 -4(A)). The spec tra in Figure 34 clearly shows that the Si wafer was covered with short -chain PDMS after the process.
54 Micro -engineered Polymer Films After solvent eva poration and the removal of residual solvent under reduced pressure, t he polymer films with micro topographical features were either easily peeled off (for PDMSe, G1657M, G1650M, and PS/G1650M) or self -peeled (for PMMA and PS) from the PDMS -5K treated sili con wafers. As an example, the whole patterned film (Sharklet AFTM, +2SK2 x 2) replicated onto PMMA i s shown in Figure 3 -5 (A). The fidelity of the microstructure was examined under a light microscope (see Figure 3-5 ( B)). Typica lly twelve (12) microscopic images were taken of random locations from the entire 2.5 x 2.5 cm2SEM images showed the detailed features of the Sharklet pattern replicated to various polymeric substrata. Figure 3 -6 show s the SEM images of the +2SK2x2 pattern on G1657M, PMMA and PS. Images with higher magnifications show s that the etching mar ks, result ing from photolithographic process of Si wafers, were also replicated to the micro -sized features on the PMMA and the PS Sharklet textured surfaces (Figure 37 and 3 -8). However, high magnification images of PDMSe +2SK2x2 micro -features (Figure 39) do not show the nano -sized fine features. The reason may be that PDMSe is a soft material and allows surface shrinkage to minimize surface area after curing. Figure 310 show s the successful replication of the +3SK2x2 pattern onto the G1650M and PS/G1650M blend. The actual feature size was measured from SEM images. For the +2SK2x2 pattern, the measured feature height was 2.1 m of height 2.0 m of width, and 2.0 m of spacing. patterned surface. The number of deformed features was counted and the fidelity was calculated by dividing the number of deformed features by the total number of features in the same area. In this work, all micro patterned samples showed greater than 99 % fidelity.
55 Wettability of the polymer films The type of wetting regime (Wenzel, Cassie -Baxter, or a wicking regime ) can be determined by using a lower ( L C) and upper ( U C = cos (3 -5) ) critical contact angle, according to the following equations [160, 161] : = cos (3 -6) Here, r is the Wenzel roughness factor (i.e., the total surface area divided by the projected plan a r surface area ) and f is the fractional area of the projected surface area that is filled by topographical feature tops. When the contact angle on the smooth surface is higher than the upper critical value, the wetting regime is Cassie -Baxter wetting regime; if the cont act angle is between the lower and upper critical value, it i s in the Wenzel wetting regime; if the contact angle is less than the lower critical value, then it is a wicking regime. The calculated surface parameters are listed in Table 3 -3. According to th e measured water contact angles on the smooth materials (Table 3-5) the wetting regime for the various Sharklet textured surfaces on the selected materials are predicted and listed in Ta ble 34. The calculation predicts that the Sharklet PDMSe, the G1657M, and the PS/G1650M blend are in the Cassie -Baxter wetting regime (air entrapment) while the Sharklet G1650M, the PMMA, and the PS are in the Wenzel regime. The sessile drop water contact angle was measured for all of the smooth and Sharklet textured polymer films that were solution cast (Table 3 3). The contact angle of the smooth PDMSe, PS and PMMA was 113 4o, 89 1o and 76 2o, respectively. The
56 results were in agreement with the literature report of 1 13 1o for PDMSe  90.0 0.5o for PS  and 76 4oFrom Table 35 we can see that the Sharklet textured surfaces on PDMSe, G1657M, G1650M, and PS/G1650M blend can be fitted into the Cassie -Baxter wetting regime. Of interest are the PMMA and PS Sharklet topographies, which show large difference s in the two directions (parallel or normal to the surface feature) None of the model s can predict contact angles in different direct ions on an anisotropic surface. The Cassie -Baxter regime may fit in the perpendicular direction, but not in the parallel direction for the PMMA and the PS Sharklet patterns.  for PMMA. The contact angle mea sured for the smooth G1657M, G1650M, and PS/G1650M blend materials showed that these surfaces were all hydrophobic. Due to the anisotropy of the Sharklet pattern, the shape of the water droplet on the pattern ed surfaces tends to be skewed, and the contact angle measured from different directions should be different. Water droplets sitting on the Sharklet patterned PDMSe, G1657M, G1650M, and PS/G1650M blend are not obviously deformed. However, on PMMA and PS films, elliptical shapes with higher apparent contact angles (in two directions) than on the corresponding smooth films were observed (Figure 312, Table 3 5 ). If the walls and tops of the micro -sized features of the Sharklet textured topogra phies we re perfectly smooth, the measured contact angles should agree with the predicted wetting regimes. However, the solution-cast polymer films replicate well the etching marks on the side walls of all the protruding features. As evidenced in a recent w ork by Nosonovsky  the nano -sized curvatures on the side walls can help pin the liquid -solid contact lines and therefore stabilize the metastable interfaces. In this
57 work, the sid e walls of the topographical features on all of the materials show convex lines. This may explain why the measure water contact angles are usually larger than the predicted values. Surface characteristics of the polymer films The smooth polymer films were scanned by an AFM to compare the nanometer sized features of the various surfaces obtained by solution casting against the PDMS 5K grafted Si wafer (Figure 31 1 ). All 3 -D images of the film surfaces were obtained from 2 x 2 2 regions. The average surface root mean-square roughness ( Rq) values were obtained from analysis of 5 different 2 x 2 2 areas on each sample The Rq = ( ) (3 -7 ) values were calculated from  where ziFrom Table 32 and Figure 31 1 the surfaces of PDMSe and G1657M are smooth (R is the height of a random ly chosen location on the scanned surface, is the mean height of all measured heights, and n is the sample size (i.e., number of height measur e ment s). q < 0.3 nm), the PMMA and PS surfaces are relatively rough (Rq ~1 nm), and the G1650M and PS/G1650M blend surfaces show high roughness (RqConclusion > 1.8 nm) due to the phase separation between the PS block and the ethylene/butadiene block. A simple and effective method was developed to covalently graft short chain Polydimethylsiloxane ( PDMS Mw ~5 kg/mole) onto the surface of a silicon wafer. This process did not require an inert atmosphere and moisture sensitive chemicals.
58 Moreover, the res ult ing antisticking layer had a long life -cycle. This antisticking layer, together with the solution casting process served to replicate high -fidelity (>99%), microengineered topographic patterns onto polymeric substrata with varied mechanical properties and surface energetics. Figure 31. Schematic description of surface treatment of a Si wafer: ( A) Untreated Si wafer (B) Si wafer with surface hydroxyl groups (C ) anchored amine groups on Si surface, (D ) short -chain PDMS covered Si wafer. EtO OEt OEt Si NH2 2Room temp 10 min 80 oC 4 hr 3 O O Si O Me Me Si Me Men((O Si Me Me Me 4 H2SO4:H2O2(30wt%) =1:1 ( vol ) Room temp 30 min1 O O O Si Si Si OH OH O O O Si Si Si O O O O Si Si Si O O Si O NH2 O O O Si Si Si O O Si O NH O OH Si O Me Me Si Me Men((O Me Si Me Me EtO OEt OEt Si NH2EtO OEt OEt Si NH2 2Room temp 10 min 80 oC 4 hr 3 O O Si O Me Me Si Me Men((O Si Me Me Me O O O O Si O Me Me Si Me Men((O Si Me Me Me Si Me Me Me 4 H2SO4:H2O2(30wt%) =1:1 ( vol ) Room temp 30 min1 O O O Si Si Si OH OH O O O Si Si Si OH OH O O O Si Si Si O O O O Si Si Si O O O O Si Si Si O O Si O NH2 O O O Si Si Si O O Si O NH2 O O O Si Si Si O O Si O NH O OH Si O Me Me Si Me Men((O Me Si Me Me O O O Si Si Si O O Si O NH O OH Si O Me Me Si Me Men((O Me Si Me Me NH O OH Si O Me Me Si Me Men((O Me Si Me Me n n (A) (B) (C) (D)
59 Fi gure 32. SEM images of PS film with +2SK2x2 pattern: (A) Top-down view at 1000x magnification, (B) Topdown view at 2000x magnification, (C) taken at 45o tilt to show the protruding features, (D) cross -sectional vi ew
60 Figure 33 XPS survey spectra for Si wafer under various treatments: (A) after H2SO4H2O2 (oxidative) wash, (B) after oxidative wash and APTES treatment, (C) after oxidative wash, APTES treatment, and PDMS 5K grafting. Figure 34 XPS spectra of Si 2p3 peaks for Si under various treatment: (A) after H2SO4H2O2 (oxidative) wash, (B) after oxidative wash and APTES treatment, (C) after oxidative wash, APTES treatment, and PDMS 5K grafting. 0 100 200 300 400 500 600 700 800 Binding energy (eV) N(e) After oxidative wash Silanized Si PDMS-5K grafted Si 97 100 103 106 Binding energy (eV) N (E) A B C 100 97 103 A B C
61 Figure 35 (A) Photo of the replicated PMMA film with Sharklet AFTM (+2SK2 x 2) pattern; (B) Light micrograph of the PMMA Sharklet AFTM (+2SK2 x 2) pattern (top -down view). Figure 36. SEM images of polymers with Sharklet AFTM (+2SK2 x 2) pattern: (A -C) G1657M, (D -F) PMMA, (G -I) PS. G 20 m H 10 m I 5 m A 20 m B 10 m 10 m C 20 m D 10 m E F 10 m G1657M PMMA PS A 20m B
62 Figure 37. SEM images of PMMA +2SK2x2 film, showing the fine structure on the side walls of the features: (A) tilted view, (B) cross -sectional view. Figure 38 SEM images of PS +2SK2x2 film, showing the fine structure of the surface features: (A) Cross -section at 7000x (B) cross -section at 8000x. Figure 39. SEM images of PDMSe +2SK2x2 film, not showing the nano -structure of the surface features: (A) tilted view at 35o (B) cross -section view. 5 m A 5 m B A B 5 m 5 m
63 Figure 310 SEM images of +3SK2x2 polymer film: (A, B) G1650M; (C, D) PS/G1650M. 20 m 20 m 10 m 10 mG1650M PS/G1650M A B C D
64 Figure 31 1 AFM images of various smooth polymer surfaces obtained from solution casting against PDMS 5K treated silicon wafer: (A) PDMSe, (B) Kraton G1657M, (C) Kraton G1650M, (D) PS/G1650M blend, (E) PMMA, (F) PS. Figure 31 2 Photo i mages of an elliptical water droplet sitting on +2SK2x2 PMMA film. (A) top down view of a water droplet sitting on the pattern; (B) side view (direction of water spreading is perpendicular to the surface features); (C) side view (direction of water spreading is parallel to the surface features). (A) (B) (C) (D) (E) (F) A B C
65 Table 3 1 Dynamic water contact angle on the Si wafers Surface feature Contact angle ( o ) H ( o ) a r +2SK2 x 2 PDMSe 160 2 112 3 48 Smooth PDMSe 113 6 89 5 24 HMDS treated Si wafer Sharklet Mold 141 4 98 6 43 flat region 117 2 87 4 30 PDMS 5K grafted Si wafer Sharklet Mold 141 3 89 5 52 flat region 118 2 85 2 33 HMDS: hexamethyldisila zane Table 3 2 R oot mean square roughness of the flat polymer films via solution casting on PDMS 5K treated silicon wafer Material R q nm PDMSe 0.1 1 G1657M 0. 30 G1650M 1.87 PS/G1650M 2.14 PMMA 1.01 PS 1.01 Table 3 3 Calculated surface parameters for microengineered topographies +2SK2x2 +3SK2x2 r f LC ( o ) U C ( o ) r f LC ( o ) U C ( o ) 2.0 0.42 69 111 2.5 0.44 74 106 Table 3 4. Predicted w etting regime for the polymer films with var ied feature height Material +2SK2x2 +3SK2x2 PDMSe Cassie Baxter Cassie Baxter G1657M Cassie Baxter Cassie -Baxter G1650M Wenzel Wenzel /Cassie Baxter PS/G1650M Cassie Baxter Cassie Baxter PMMA Wenzel Wenzel PS Wenzel Wenzel
66 Table 3 5 Sessile drop contact angles on polymer films, with water as testing medium. Material Feature Contact Angle ( o ) Measured Wenzel C B PDMSe Smooth 113 4 +2SK2x2 (parallel) 1 37 5 1 43 138 +2SK2x2 (perpendicular) 1 41 2 +3SK2x2 (parallel) 139 4 1 69 137 +3SK2x2 (perpendicular) 143 1 G1657M Smooth 111 5 +2SK2x2 (parallel) 144 1 1 37 137 +2SK2x2 (perpendicular) 146 2 +3SK2x2 (parallel) 145 2 1 5 4 136 +3SK2x2 (perpendicular) 149 3 G1650M Smooth 103 6 +2SK2x2 (parallel) 135 4 1 17 133 +2SK2x2 (perpendicular) 142 3 +3SK2x2 (parallel) 139 2 1 24 131 +3SK2x2 (perpendicular) 147 1 PS/G1650M Smooth 112 2 +2SK2x2 (parallel) 140 5 1 40 138 +2SK2x2 (perpendicular) 147 2 +3SK2x2 (parallel) 144 5 1 60 137 +3SK2x2 (perpendicular) 152 3 PMMA Smooth 76 2 +2SK2x2 (parallel) 86 1 6 0 119* +2SK2x2 (perpendicular) 120 6 +3SK2x2 (parallel) 86 1 52 117* +3SK2x2 (perpendicular) 133 2 PS Smooth 89 1 +2SK2x2 (parallel) 95 3 88 125* +2SK2x2 (perpendicular) 139 2 C-B estimation for PMMA and PS with Sharklet patterns was based on the air entrapment mode.
67 CHAPTER 4 ULVA ZOOSPORE ATTACHMENT ON THE MICROENGINEERED POLYMERIC FILMS Introduction Biofouling, accumulation of biomass at the interfaces, has been attracting much interest from researchers worldwide Some of the adverse influences of biofouling include the increased fuel cost for ships  accelerated corrosion of marine structure s  and spread of invasive species  With stringent regulations on coating materials applied to marine vessels, the need for cost efficient and environmental -friendly coatings is urgent. Many strategies, in terms of ma terials chemistry, surface energetics, micro -topographical features, and mechanical properties, have been proposed by various research groups to construct antifouling/foul -release (AF/FR) coatings. The study of p olymer based materials are among the most a ctive fields for AF/FR coatings. Among all the efforts on polymer materials, zwitterionic polymers showed promising applications in both antifouling of both protein adsorption and bacteria adhesion[11, 66, 67] Ongoing research with this type of materials also showed excellent resistance to marine organisms  In order to combine the AF property of oligoethylene glycol and the FR property from perfrluoroalkanes, Krishnan et al  synthesized an amphiphilic copolymer. There were oligoethylene glycol and perfluoroalkyl moieties in th e same side chains of their synthesized polymers. In an aqueous environment, the side chains bent with oligoethylene glycol segments sticking toward water and perfluoroalkyl segments bowing toward the coating surface. The obtained surface showed improvem ent against settlements of Ulva and Navicula and higher removal rate of sporelings compared with glass and polydimethylsiloxane
68 (PDMS) surfaces. Linear -chain poly ethylene glycol (PEG) grafted surfaces have long been shown to be effective against protein adsorption and cell adhesion [10, 70, 71] Statz et al  coated Ti surface with PEG chains via conjugation with L 3,4dihydroxyphenylalanine and the result ing surface showed AF/FR against spore and diatom. Other polymeric materials, such as perfluoropolyethers (PFPE) and their PEG blends  and siloxane-polyurathane copolymers  also showed improved AF properties. Surface chemistr y certainly plays a role when a cell determines if it will settle on the surface Ista et al  changed surfac e chemistry (and therefore surface energy) systematically via self assembled monolayers (SAMs) technique and tested the surfaces with marine bacteria and Ulva zoospores. In their work, they prepared two series of mixed SAMs on gold surface: one was alkyl c hains with mixed end groups of COOH and CH3. T he other was alkyl chains with mixed end groups of OH and CH3The concern with all the non-toxic material and surface chemistry work lies in the fact that the surface will eventually be conditioned with organic matter  which will recruit microorganism subsequently. One disadvantage of PEGylated polymer coatings is that PEG can be degraded gradually [9, 75] making it unfavorable for long term applications. Although the adhesion of bacteria complied with the prediction of a thermodynamic model, spore test gave more complicated bioresponses, indicating simple model cannot fully explain the interactions between the somewhat complex microorganisms and the surface. Diatom s were used as test subject on various chemically modified surfaces 
69 Although the adhesion strength between the fouling organisms and the surface energy and mechanical properties of a substratum was investig ated [59, 166] there are few reports on the effect of surface mechanical properties on the settlement/attachment behavior of the marine microorganisms. Recently, Chaudhury et al  varied the elastic modulus of polydimethylsiloxane (PDMS) from 0.2 to 9.4 MPa and found no significant difference on the settlement of Ulva spore. A recent work  suggested that surface mechanical stiffness may influence the adhesion and colonization of bacteria ( Staphylococcus epidermidis and Escherichia coli ), on polyelectrolyte multilayer thin films. Sur face topographical features as a strategy of physical defense have also attracted much attention. One approach, which is based on the biomimetic analogy to the shark scale, has shown effectiveness in antifouling towards many species such as bacteria, spore, and barnacle cyprids [18, 19, 21, 48] Schumachers work  on a nanoforce gradient design didnt show a correlation between the spore settlement and the force gradient. However, the data fitted well with a predicting model in a later work  shown below: log = (4 -1) where A is the spore settlement density on the topographical surface, AS M is the spore settlement density on the smooth surface of the same material, and a is fitting constant, engineered roughness index (ERI) ERIII is defined as = (4 -2)
70 where r is the Wenzel roughness index, n is the number of discrete features on the surface, and f is the fraction of solid in contact with liquid in the Cassie -Baxter model. This prediction model suggests that surface thermodynamic properties are the main controlling factors for microorganisms attachment/settlement onto a surface in the case of PDMSe material. One key issue about this prediction model is the influence of the surface chemistry/energy and subst rate mechanical properties. Therefore, the objective of this study was to test the hypothesis that the Sharklet AFTM pattern is the effective antifouling design in itself no matter on which substrate it is replicated. Thus, thermoplastic polymers with syst ematically varied mechanical and energetic properties were selected to help test the hypothesis. Styeneethylene/butyldiene-styrene triblock copolymer (SEBS) as a thermoplastic elastomer was chosen as a hydrophobic, low modulus (Youngs modulus 6~30 MPa) m aterial. Poly methyl methacrylate (PMMA) was chosen due to its higher modulus (~ 3, 300 MPa) and relatively high surface energy. Another objective of this study was to explore simple technique of fabricating micropatterned polymer films. The Sharklet patte rn was replicated onto five polymeric substrate. The polymers were: poly (dimethyl siloxane ) elastomer (PDMSe) (the standard material on which most topographical studies have been conducted), two types of Kraton SEBS polymers (varied ratio of styrene blocks ), a polystyrene/SEBS blend, and poly(methyl methacrylate) (PMMA). Ulva zoospore settlement assays were performed on the samples.
71 Materials and Methods Materials Poly(methyl methacrylate) (PMMA, 350 kg/mole) was purchased from Sigma-Aldrich (St Louis, MO). Styrene ethylene/butyldiene -styrene (SEBS) triblock copolymers, Kraton G1657M (styrene block mass percentage 1 3 %) and Kraton G1650M (styrene block mass percentage 30%), were purchased from Kraton (Houston, TX). Polystyrene (PS, molecular mass 30 kg/mole) was purchased from Polysciences (Warrington, PA). 3 (aminopropyl triethoxysilane) (APTES), and monoglycidyl ether terminated polydimethylsiloxane (PDMS, M W 5 k g/mole, abbreviated as PDMS 5K) were purchased from Sigma Aldrich (St. Louis, MO). Anhydrous etha nol, 1 -propanol, 2propanol, hydrogen peroxide (30 wt%), and concentrated sulfuric acid (95%) were purchased from Fisher Scientific (Pittsburgh, PA). All of the chemicals were used without further p cm) was prepared in the lab with a Barnstead Nanopure DiamondTM lab water system from M illipore (Billerica, MA). Topographical Replication The thermoplastic polymeric films were prepared by a solution-casting method. The polymer toluene solution (0.15 g polymer dissolved in 1 ml toluene) was cast onto a smooth or a pre-patterned silicon wafer. The pattern was transferred to a silicon wafer (2.5x2.5 cm2 in area) by a photolithographic technique [19, 21] To lift the polymer films from the wafer, an antisticking layer (short -chain polydimethylsiloxane, or PDMS 5K) was grafted onto a silicon (Si) wafer in a three-step manner: (1) a Si wa fer was immersed into a freshly mixed concentrated sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) water solution (30 wt%) (1:1 volume ratio)  for 30 min to generate surface hydroxyl groups; (2) after thorough rinsing with nanopure water, the Si wafer
72 was dried with a nitrogen flow. Neat APTES was added dropwise to cover the entire surface for 10 min. (3) After rinsing with anhydrous ethanol, t he APTES treated Si wafer was covered by neat PDMS 5K and heated in an oven at 80 oC for 4 hr. The wafer was then rinsed with 2-propanol. The PDMS -5K treated Si wafer was dried with an Ar flow  Enough of the polymer solution was placed on the wafer dropwise to cover the smooth or patterned area of the wafer. After slow removal of solvent, the desired polymer films were obtained with the size of about 8 cm in length, 3 cm in width and 150 m in thickness. The Sharklet patterned area was located in the middle of the film with a 2.5x2.5 cm2 coverage. Polydimethylsiloxane elastomer (PDMSe) was used as a reference material as described previously by Chung et al  Silastic T2TMThe resultant topographies contain feature elements with a width of 2 m, a distance (between the neighboring elements) of 2 m, and a varied height of approximately 2 .1 or 3 m,. The short name of this pattern was Sharklet +2SK2x 2 (for 2 m height) and +3SK2 x 2 (for 3 m height). Pattern fidelity was evaluated with light and scanning electron microscopy (SEM). (from Dow Corning Corp, Midland, MI) base material and curing agent were mixed at a 10 to 1 mass ratio and cast against the silicon wafer described above. After one day curing at room temperature, the result ing PDMSe films were easily lifted from the silicon wafers. The surface free energy of each sample was determined by a twoliquid method, known as Owens Wendt Kaelble (OWK) approach (geometric mean method) [167, 168] The surface energy of the solid ( S) was the sum of the polar component ( s p) and dispersive component ( s d). In this method, at least a pair of liquids, one polar and one non-polar was needed to determine the surface energy by Equation 4-3:
73 = (4 -3 ) The parameters and which are known, refer to the total surface free energy dispersion and polar components of the probe liquid, respectively Two probe liqui d tests produce two equations from which the and can be calculated. In order to get a more precise surface energies, three probe liquids, water (WT), ethylene glycol (EG) and diiodomethane (DM), which have = 72.8, 48.0 and 50.8 m J /m2, = 21.8, 29.3 and 49.5 m J /m2, and = 51.0 18.9 and 1.3 m J /m2Sample Preparation respectively, were chosen and we took the mean value resulted from the two pair tests, WT -DM and EG -DM, as shown in Table 41 and 4-2 The polymeric films were cut into 1.31.0 inch2Samples were shipped dry to University of Birmingham, UK, and were immersed in seawater for 24 hours prior to the experiment. The samples were not exposed to sonication. Any air visibly trapped in the features was dispersed using a jet of seawater from a pipette. pieces and were then glued to the glass slides by a fast -cure epoxy glue, Araldite 2012, purchased from Huntsman ( The Woodlands, Texas USA). Minimal amount of glue (~0.05 g) was spread onto glass slides followed by attaching of the polymer s amples. The covered area of epoxy glue was larger than the area of the polymer sample. Usually there were two narrow epoxy glue regions exposed to air after attaching the polymer films. The fidelity of each topographical sample was checked under a microsco pe before shipping.
74 Spore Attach ment Assay Spore attachment assay was performed by Dr John Finlay and Dr Maureen Callow at the University of Birmingham, UK. Fertile plants of Ulva linza were collected from Wembury beach, the United Kingdom (5018 N; 402W). Ulva zoospores were released and prepared for attachment experiments as previously described  Samples were transferred to artificial seawater (Tropic Marin) for 1 hour prior to experimentation without exposure to air. Samples were then rapidly transferred to assay dishes to minimize any dewetting of the topographical areas. Ten milliliter of spore suspension (adjusted to 1.5 x 106 ml1The spore concentration was 1.5 x 10 ) were added to each dish and placed in darkness for 45 minutes. The slides were then rinsed and fixed with 2% glutaraldehyde in artificial seawater as described in Callow et al  Spore counts were quantified using a Zeiss epifluorescence microscope attached to a Zeiss Kontron 3000 image analysis s ystem  Thirty images and counts were obtained from each of three replicates at 1 mm intervals along both the vertical (15) and horizontal (15) axes of the slide. 6 spores ml1Statistical Methods ; the settlement time was 45 min, 3 replicates of each sample were tested. Spore density was reported as the mean number of settled spores per mm2Princi pal component analysis was performed with Minitab V15.0 software (Minitab Inc, State College, PA). Many factors, including surface chemistry, surface energy, from 30 counts on each of three replicate slides + / standard error Statistical differences between surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons. Replicate slides (3) of each surface (5) were treated as a nested variable within each surface.
75 mechanical properties, surface topography, tortuosity, and feature dimensions, can affect the atta chment of spores onto a surface. Five parameters that can be quantified for the topographical surfaces were selected as independent variables: the bulk elastic modulus (Youngs modulus E), the surface free energy ( ) the spacing between the two adjacent surface features ( a ), the discrete number of surface features (n ), the Wenzel roughness factor ( r ), and the area fraction of the surface top relative to the projected planar area (f ). The data source came from this study as well as previous work of the Bre nnan research group, shown in Table 42 Results and Discussion Spore A ttachment Affected by the Epoxy Glue The attachment density of Ulva spore on all of the tested surfaces was reported in Figure 41. It was found that the spore settlement density was abnormally high on +3SK2x2 PDMSe surface. As PDMSe w as used as the baseline for the spore attach ment test, the data for other materials we re therefore questionable. It wa s necessary to investigate the reasons so that the spore attachment data could be furth er analy zed After finishing the spore attachment assay, all the tested samples were shipped back to the lab at UF. All the 51 glass slides with polymer samples were scanned under light microscope thoroughly The typical images of each region were shown in Figure 4 2, using +2SK2x2 PS as an example. It was noticed that the spores settled on the epoxy region with extremely high coverage. This high attach ment density on epoxy region can be observed on each glass slide. Spores were also attracted to the cavities between the polymer film and the epoxy layer, as shown in Figure 4 3. These observations demonstrated that the epoxy glue had high attractiveness t o the spores.
76 To test if there was any chemical leaching from the glue, Araldite2012 epoxy glue was spread on two clean glass slides. After one day curing, each slide was immersed in 45 ml D.I. water in a centrifuge tube. A clean glass slide was also soak ed in the same amount of D.I. water to serve as reference. After 38 days of soaking, UV -Vis spectra were taken and shown in Figure 4 4. According to the MSDS provided by Bostik Findley, Araldite 2012 epoxy glue consists two parts: one is epoxy resin contai ning bisphenol A diglycidyl ether polymer (CAS # 25068 38 -6), butanediol diglycidyl ether (CAS # 2425 -79 8) and acrylonitrile butadiene styrene polymer (CAS # 9003569), the other part is a polyamine hardener. The UV spectra showed that the leachant may b e amine/ amide with aromatic groups, having similar structures to N -(3,4dimethoxybenzyl)phthalamic acid compared with a report from the Occupational Safety and Health Administration (OSHA )  The topographical regions of each sample were carefully examined under a microscope. Except for +3SK2x2 PDMSe surface, all other topographical samples kept their fidelity after spore settlement assay (Figure 4 5). Flopped top is prevailing on +3SK2x2 PDMSe surfaces, see Figure 4 6. The retained +3SK2x2 PDMSe sampl e was examined under microscope before and after soaking in D.I. water for 24 hr No flopped top was observed in both cases. The damage of surface features may have occurred during shipping and handling of the samples. Based on the observations, the spore attachment density on +3SK2x2 PDMSe was not considered in the following analysis. Spore Attachment on Various Surfaces It was shown that the chemical attractant from the exposed epoxy regions is strongly influential The instinct assumption was that the influence of the epoxy glue
77 w ould be the same among all the samples. The following observation and statistical analysis were based on this assumption. The attachment density of spores on the smooth surfaces greatly vari ed on the different substrate (Figure 4 1). Spore densities ranged from ~ 300 mm2 on the PDMSe to ~ 1900 mm2 on the PS/G1650 blend. Plotting the spore attachment densities against the square root of the product of elastic modulus and surface energy indicated that spore density increased as elastic modulus increased up to 2 4 0 MPa (PS/G1650M blend )(Figure 4 7 ), but above this the density of settled spores decreased. The correlation was weak, as R2Compared to attachment densities on the smooth surfaces spore attachment was reduced on the + 2SK2x2 patterns on the PDMSe, G1650, PS/G1560 and PMMA. On the G1657 attachment density was slightly higher on the pattern, and on the polystyrene it was much higher (over double). Although the density of settled spores was low on the smooth PDMSe, the reduction of 41% on the patterned area (+2SK2x2) is low compared with previous studies (commonly higher than 70% reduction) Plotted as a function of force gradient spore density increased to a force gradient value of 21 00 nN (PS/G1650M blend) and then fell in a pattern similar to that seen for the elastic modulus of the smooth surfaces (Figure 4 -7). was only about 0.363. In an earlier report, Chaudhury et al.  found no significant difference in spore attachment on PDMSe over a small range of moduli (0.2 ~ 9. 8 MPa) The + 3SK2x2 S harklet reduced spore attachment on the G1657 M G1650 M and the PS/G1650 M blend (Figure 4 -1). In the case of PMMA the attachment density was consider ably greater than on the + 2SK2x2 Sharklet pattern. It is interesting that the
78 small change in height can cause such a difference in attachment density and shows just how sensitive the sensory apparatus of the spores must be. Although, it should be remember imately 15 % of the body length of a spore. A plot of spore attachment density (on the +2SK2x2 and +3SK2x2 patterns) against nano force gradient indicated that there was a possible increase in s pore density with force gradient at (Figure 4 8 ). However, the trend was weak (R2An alternative view of the influence of the leachant from the epoxy glue could be based on the concentration differences of the aromatic amide (s) in the different substrata. The chemical could diffuse into the polymer films. As it contains aromatic rings, it has higher affinity to the substrate containing higher concentration of benzene rings. In this study, G1657M, G1650M, PS/G1650M blend, and PS have higher benzene ring content along the list. Therefore, the concentration of aromatic amide(s) dissolved in the polymer film may be in the following rank: G1657M < G1650M < PS/G1650M blend < PS. Th e data in Figure 4-1 exhibited the higher spore attachment density following the trend. The film thickness will determine the concentration gradient of the dissolved amin/ amide. The abnormally high attachment densities on the patterned sample of +2SK2x2 PS, may be caused by the variations in the thickness of the polymer films compared with the smooth PS film The solubility of the aromatic amides may be smaller in PDMSe and PMMA compared with PS -containing polymers, but they could still disturb the attachment behavior of the spores. = 0.38) and might again reflect other parameters rather than force gradient per se.
79 If, in the worst case of scenario, the leachant from the epoxy glue is overwhelmingly strong to disturb the sensory functions of the spores, the spores would show random attach ment behavior on all the samples, i.e., there would b e no statistical difference among all of the smooth and the Sharklet textured surfaces for all of the materials However, the attachment density data did show difference among different material groups. Therefore, it could be concluded that material proper ties may have influence on the spore attachment process. Site Selection by Spores Images of the spores on the patterns are shown in Figure 4-9 and 410. On both types of pattern, on all substrates the spores selected the wider channels which separated the diamond patterns to settle in. They were especially prevalent at either end of the diamond where the smallest features were placed (probably the widest part of the channel). The channels between the individual bars of the pattern were narrower and proved less favorable for attachment Correlation between the Surface Parameters and Ulva Spore Attachment The three surface parameters ( n, r, f ) were used to construct the concept engineered roughness index ( ERIII) in previous works and excellent predictive correlation was obtained for the Ulva spore attachment on PDMSe material  The p rincipal component analysis (PCA) method was first applied to the three parameters to determine if any of the three parameters contribute more to the total variance. The data set was shown in Table 4 3 PCA analysis on the three param eters shows that the first component has an Eigenvalue of 2.4, explains 80.4% of the total variance in the group. The eigenvalues of the other two components are all less than 1 (0.359 and 0.230 respectively) and should not be considered. For the first com ponent, the loading of each
80 variable is 0.560 ( n ), 0.590 ( r ), and 0.581 ( f ), respectively. The results show that the three surface parameters contribute equally to the total variance and should all be considered in the correlation with the response. Howeve r, the plot of the Ln(A/ASM) against ERIII values gave weak correlation (R2When three more surface parameters ( E and a ) were introduced, p rincipal component analysis (PCA) method was used to reduce the surface parameters (listed in Table 4 3 ) into two groups of components. As seen in Table 4-4 the two components can explain a total of 7 2. 9 % of the varia nce of the quantified surface parameters which describ e the surface energy, mechanical property, surface topography (roughness) and tortuosity. In the first component, the three parameters n r, and f showed the highest loadings (higher than or close to 0.5). In the second component, E and are the main contributors with loadings (absolute values) higher than 0.6. = 0.24, Figure 4 -11). The grouping o f the surface parameters inspired new correlation of spore attachment vs. surface properties. Brady  showed that the relative adhesion of fouling organisms is proportional to the square root of the product of elastic modulus and surface energy. Callow et al.  demonstrated that when a spore is in touch with a surface, it secrets a small amount of adhesive to test if this is a suitable spot for it to settle. It suggests that the mechanical strength of the initial test adhesion is among the factors that determine the likelihood of th e spore to settle on the surface. Considering the second component in PCA analysis, it is reasonable to introduce new parameters into the spore attachment model. When all the data from this study and previous works by Wilson  Schumacher [19, 20] and Long  were included, the reduction of spore attachment density on a surface is expressed as follows (shown in Figure 412 ),
81 = 10.7 (R 2As observ ed in the data set from Wilsons work  the spacings between the neighboring features were all no less than 5 m, which is about the diameter of the spores. Spacing of 5 m channeled PDMSe surface greatly increased spore attachment  Late r surface designs all considered spacings less than this critical size. Therefore, when the data from Wilsons work were not included, the predictive correlation is (also in Figure 4 -13 ) = 0.49) (4 -3) = 11.6 (R 2In Figure 413 there are still data points higher than point zero when ERI = 0.68) (4 -4 ) IIIt should be noted that PCA analysis was to find the correlations among the independent variables and to simplify the multi -dimensional correlation to 2or 3 dimensional correlation. The PCA method used in this section could help group the surface parameters for correlation wit h the spore attachment data As the spore attachment was greatly influenced by the epoxy glue in this study, the data obtained and the correlation results should be interpreted with caution. Nevertheless, the values are small. It is possible that other factors such as surface chemistry play significant role in some cases. In a bacteria -surface interaction study  the surface energy term was considered as a sum of Lifsthitz van der Waals (LW) and acidbase (AB) interactions; and AB term was further subdivided into electron-donor and electron acceptor contributions. These divisions may be more accurately describing the contributions from the variation of surface chemistry to the surface energy term for the different materials.
82 statistical analysis gave a potential direction for the predictive model in the future. A new spore attachment study on the samples without using epoxy glue will be performed and should clarify the above concerns. Conclusions Sample analysis showed that the epoxy glue used for adhering the polymeric f ilms onto the glass slides could leach out strong attractant (aromatic amide) to the Ulva spores. Therefore, the spore attachment data in this study should be interpreted with caution. New batch of polymeric films was prepared and the spore attachment test will be performed with future studies to eliminate the concerns about the leachant Observations showed that regardless of which material the patterns were made from, the majority of spores settled in the channels between the diamonds which were larger than the channels between the individual elements. The data would suggest that either G1650 or PS/G1650 would probably be the best of the more rigid materials from which to mak e a S harklet patterned coating. The principal component analysis (PCA method ) s howed that the two groups of surface parameters one composed of n r and f and the other composed of E and were important in interpreting the relationships among the surface parameters. Since the analyses were performed for the surface parameters, the grouping of the surface parameters provided potential direction for the correlation of the spore attachment and the surface properties in the future.
83 Figure 41. The attachment densities of Ulva spores on sharklet patterns Each point is the mean from 90 counts on 3 replicate slides. Bars show 95% confidence limits.
84 Figure 42. The schematic drawing of +2SK2x2 PS film attached to a glass slide. Microscopic images were taken from each region of the sampleattached glass slide : (a) glass region, (b) Araldite2012 epoxy region, (c) smooth polymer region, (d) topographical region. Scale bars were all equal to 20 m. c d a b c d b a
85 Figure 43. Spores can be observed in the cavity between the PS film and the underneath epoxy glue. Figure 44. UV -Vis spectra of water extracts from Araldite layer and glass slide stored in D.I water in centrifuge tubes.
86 Figure 4 5. Some representative micro images of topographical features on various substrates. +3SK2x2 G1657M +3SK2x2 G1650M +3SK2x2 PS/G1650M +3SK2x2 PMMA 20 m 20 m 20 m 20 m
87 Figure 46 Micrograph of +3SK2x2 PDMSe surface after spore attachment assay. Flopped tops of the features were prevailing on the whole sample. Figure 47 The attachment densities of Ulva spores on smooth surfaces vs. natural logarithm value of the square root of the product of elastic modulus and surface energy of the corresponding material Each point is the mean from 90 counts on 3 replicate slides. 20 m y = 203x 598 R = 0.363 0 400 800 1200 1600 2000 4 6 8 10Spore attachment (no/mm2)Ln(E 1/2
88 Figure 48 The attachment densities of Ulva spores on the S harklet patterns plotted as a function of nano force gradient Each point is the mean from 90 counts on 3 replicate slides.
89 +2SK2x2 +3SK2x2 PDMSe G1657M G1650M PS/G1650M PMMA Figures 4 9 Images of spore settled on the S harklet patterns (fixed sample). Image that exist between the diamonds units of the pattern.
90 Figures 4 10. Image of spores settled on the +2SK2x2 PS patterns (fixed sample). Figure 411. Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII The data sets used were from this study, Schumacher [19, 2 0] and Long  ln(A/ASM) = 0.040ERIIIR = 0.24 2 1.5 1 0.5 0 0.5 1 1.5 0 5 10 15 20 25Ln(A/ASM)ERIII
91 Figure 412 Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII divided by the square r oot of ( E ). The spore attachment data were from this study and previous works of Wilson  Schumacher [19, 20] and Long  Notice that in Wilsons work the spacing s between the neighboring features were no less than 5 m while all others the spacings were about 2 m. Figure 413 Correlation of the spore attachment density on the topographical relative to the smooth surface vs. ERIII divided by the square root of (E ). The spore attachment data were from this study and previous works of Schumacher [19, 20] and Long  The spacings between the neighboring features were al l about 2 m, which was less than the diameter of the spore (~5 m). ln(A/ASM) = 10.7ERIII/(E )1/2R = 0.49 2 1.5 1 0.5 0 0.5 1 1.5 0 0.05 0.1 0.15Ln(A/ASM)ERIII/(E )1/2 ln(A/ASM) = 11.76RIII/(E )1/2R = 0.68 2 1.5 1 0.5 0 0.5 1 1.5 0 0.05 0.1 0.15Ln(A/ASM)ERIII/(E )1/2
92 Table 4 1. Static contact angle measurement on various smooth surfaces with different probe liquid. Material Water (WT) Diiodomethane ( DM ) Ethylene glycol ( EG ) PMMA 75.9 1.2o 36.0 1.1o 51.6 0.8o G1657M 111.8 0.8o 54.2 1.5o 82.3 1.2o G1650M 103.0 3.1o 42.1 1.9o 72.0 1.8o PS/G1650M 108.6 1.4o 44.4 1.2o 68.6 1.3o Table 4 2 The mechanical property and surface free energy of the selected materials. Material E MPa mJ/m 2 Literature report Measured PDMSe 1.4  22  21.5  Kraton G1657M 6.3 35.0 Kraton G1650M 34 40.8 PS/G1650M blend 2.4 10 2 40.7 PMMA 3.3 10 3  41. 2  42.4 PS 3.1 10 3 40. 2 
93 Table 4 3 Surface parameters in the current and pr evious spore attachment studies Material Surface E, MPa mJ/m 2 a m n r f PDMSe SM 1.4 21.5 0 0 1 0 +2SK2x2 1.4 21.5 2.0 4 2.05 0.417 G1657M SM 6.3 35 0 0 1 0 +2SK2x2 6.3 35 2.0 4 2.05 0.417 +3SK2x2 6.3 35 1.9 4 2.51 0.438 G1650M SM 34 40.8 0 0 1 0 +2SK2x2 34 40.8 2.0 4 2.05 0.417 +3SK2x2 34 40.8 1.9 4 2.51 0.438 PS/G1650M SM 240 40.7 0 0 1 0 +2SK2x2 240 40.7 2.0 4 2.05 0.417 +3SK2x2 240 40.7 1.9 4 2.51 0.438 PMMA SM 3300 42.4 0 0 1 0 +2SK2x2 3300 42.4 2.0 4 2.05 0.417 +3SK2x2 3300 42.4 1.9 4 2.51 0.438 PS SM 3100 40.2 0 0 1 0 +2SK2x2 3100 40.2 2.0 4 2.05 0.417 PDMSe SM 1.4 21.5 0 0 1 0 (Schumacher) triangle pillar 1.4 21.5 2.0 2 2.23 0.37 ridge 1.4 21.5 2.0 1 2.5 0.5 pillar 1.4 21.5 2.0 1 2.36 0.23 SK 1.4 21.5 2.0 4 2.51 0.42 SM 1.4 21.5 0 0 1 0 GR0 1.4 21.5 2.0 1 2.5 0.333 GR1 1.4 21.5 2.0 2 2.78 0.444 GR2 1.4 21.5 2.0 2 2.5 0.4 GR3 1.4 21.5 2.0 2 2.5 0.417 GR4 1.4 21.5 2.0 2 2.5 0.4 GR5 1.4 21.5 2.0 1 2.5 0.428 SK 1.4 21.5 2.0 4 2.51 0.42 PDMSe SM 1.4 21.5 0 0 1 0 (Long) SK(+1.8) 1.4 21.5 2.0 4 1.9 0.38 SK( 2.0) 1.4 21.5 2.0 4 2 0.6 SM 1.4 21.5 0 0 1 0 SK(2.7) 1.4 21.5 2.0 1 2.4 0.38 SK(2.7) 1.4 21.5 2.0 2 2.4 0.43 SK(2.6) 1.4 21.5 2,0 3 2.3 0.46 SK(2.9) 1.4 21.5 2.0 4 2.5 0.48 SK(2.6) 1.4 21.5 2.0 5 2.3 0.49
94 Table 4 3 Continued Material Surface E, MPa 2 a m n r f PDMSe (untreated) sm 1.4 21.5 0 0 1 0 channel(5x5) 1.4 21.5 5 1 2 0.5 (Wilson) channel(5x10) 1.4 21.5 10 1 1.67 0.333 channel(5x20) 1.4 21.5 20 1 1.5 0.2 pillar (5x5) 1.4 21.5 5 1 2 0.25 pillar (5x10) 1.4 21.5 10 1 1.44 0.111 pillar (5x20) 1.4 21.5 20 1 1.16 0.04 channel (1.5x5) 1.4 21.5 5 1 1.3 0.5 channel (1.5x10) 1.4 21.5 10 1 1.2 0.333 channel (1.5x20) 1.4 21.5 20 1 1.12 0.2 pillar (1.5x5) 1.4 21.5 5 1 1.3 0.25 pillar (1.5x10) 1.4 21.5 10 1 1.13 0.111 pillar (1.5x20) 1.4 21.5 20 1 1.05 0.04 (Si oil 50 cst, 5%) sm 1.4 22.8 0 0 1 0 channel(5x5) 1.4 22.8 5 1 2 0.5 channel(5x10) 1.4 22.8 10 1 1.67 0.333 channel(5x20) 1.4 22.8 20 1 1.5 0.2 pillar (5x5) 1.4 22.8 5 1 2 0.25 pillar (5x10) 1.4 22.8 10 1 1.44 0.111 pillar (5x20) 1.4 22.8 20 1 1.16 0.04 channel (1.5x5) 1.4 22.8 5 1 1.3 0.5 channel (1.5x10) 1.4 22.8 10 1 1.2 0.333 channel (1.5x20) 1.4 22.8 20 1 1.12 0.2 pillar (1.5x5) 1.4 22.8 5 1 1.3 0.25 pillar (1.5x10) 1.4 22.8 10 1 1.13 0.111 pillar (1.5x20) 1.4 22.8 20 1 1.05 0.04 (Si oil 50 cst, 20%) sm 1.4 22.5 0 0 1 0 channel(5x5) 1.4 22.5 5 1 2 0.5 channel(5x10) 1.4 22.5 10 1 1.67 0.333 channel(5x20) 1.4 22.5 20 1 1.5 0.2 pillar (5x5) 1.4 22.5 5 1 2 0.25 pillar (5x10) 1.4 22.5 10 1 1.44 0.111 pillar (5x20) 1.4 22.5 20 1 1.16 0.04 channel (1.5x5) 1.4 22.5 5 1 1.3 0.5 channel (1.5x10) 1.4 22.5 10 1 1.2 0.333 channel (1.5x20) 1.4 22.5 20 1 1.12 0.2 pillar (1.5x5) 1.4 22.5 5 1 1.3 0.25 pillar (1.5x10) 1.4 22.5 10 1 1.13 0.111 pillar (1.5x20) 1.4 22.5 20 1 1.05 0.04
95 Table 4 3 Continued Material Surface E, MPa 2 a m n r f (Si oil 5000 cst, 5%) S m 1.4 23.3 0 0 1 0 channel(5x5) 1.4 23.3 5 1 2 0.5 0 channel(5x10) 1.4 23.3 10 1 1.67 0.33 channel(5x20) 1.4 23.3 20 1 1.5 0.2 0 pillar (5x5) 1.4 23.3 5 1 2 0.25 pillar (5x10) 1.4 23.3 10 1 1.44 0.11 pillar (5x20) 1.4 23.3 20 1 1.16 0.04 0 channel (1.5x5) 1.4 23.3 5 1 1.3 0.5 0 channel (1.5x10) 1.4 23.3 10 1 1.2 0.33 channel (1.5x20) 1.4 23.3 20 1 1.12 0.2 0 pillar (1.5x5) 1.4 23.3 5 1 1.3 0.25 pillar (1.5x10) 1.4 23.3 10 1 1.13 0.11 pillar (1.5x20) 1.4 23.3 20 1 1.05 0.04 0 Table 4 4 Loadings for the two components generated from principal component analysis using the variables from the five variables data set (Table 43 ). Surface parameter Component 1 Component 2 Elastic modulus ( E ) 0. 212 0.6 33 Surface free energy ( ) 0.2 69 0.6 19 Spacing between the adjacent features ( a ) 0.340 0.145 Number of discrete features ( n ) 0.5 11 0.090 Wenzel roughness factor ( r ) 0.5 20 0.272 Area fraction of solid in contact with liquid ( f ) 0. 485 0.336 Percent of total variance explained 45.4 % 27 .5%
96 CHAPTER 5 BIOFILM INHIBITION ON SURFACE MICROENGINEERED POLYMERIC FILMS Introduction Invasive medical devices from catheters to artificial heart valves are being increasingly employed to improve health and life quality. However, inserted or implanted medical devices have a substantial risk of infections. Deaths due to hospital acquired infections was estimated to be about 100,000 cases in the US alone in the year 2002  Staphylococcus aureus (S. aureus ), a common bacterium that exists on the skin and in the nose, is among the major source s of healthcare associated infections  Although treated with various antibiotic agents, S .aureus is evolving to be resistant to many of them. Now more than 63% of the isolated strains from hospitalized patients are methicillin-resistant S. aureus (MRSA)  B acterial biofilms are often responsible for the chronic infections among patients  To avoid bacterial adhesion to biomedical devices, two basic strategies have been proposed and used for biomaterials: bioadhesion -free coatings, and incorporated antimicrobial agents  As the effectiveness of the antibiotic treatment is increasingly challenged by MRSA, alternative methods are needed which take advantage of the surface characteristics on the inhibition of biofilm formation. A biomimetic surface microtopographical design, Sharklet AFTM (Figure 5 -1), has been shown to be effective against the settlement of various marine species [17 19] Recent work showed qualitatively that this pattern could delay t he formation of a S. aureus biofilm  The quantitative characterization of biofilm is difficult and still under intensive investigation. Recently, new dyes were found to distinguish the matrix and the
97 bacterial cells buried in it  Still, quantifying the cell counts in an aged biofilm is difficult One objective of this work was to test the hypothesis that the surface microengineered topographical features alone can inhibit the formation of the bacterial biofilms. To test this we fabricated microengineered poly mer films (all in Sharklet pattern) with systematically varied mechanical and energetic properties and t he micro engineered surfaces were challenged with S. aureus inocula for 7 days. Another objective of this study was to explore possible mechanism for t he inhibition of the biofilm formation on the microengineered surfaces. Therefore new clinical treatment strategies for medical device associated infections could be inferred from this work. The 7day cultured polymer samples were challenged with a high d ose of antibiotic culture medium for extended period of times and the bioactivity of the bacterial microcolonies was examined with a relatively simple and accurate means called BioTimer assay. The microbial colonies/biofilms formed on the surfaces of thes e polymeric samples were evaluated by the BioTimer method reported by Pantanella et al.  This method takes advantage of the metabolic process of Staphylococcus : the bacteria can fully digest glucose in the culture medium convert ing it to CO2 and water in the presence of air. The dissolved CO2 will gradually lower the pH of the culture medium in an enclosed culture well and thus a pH -sensitive reagent serves as an indicator for t he level of metabolic activity of the bacteria. The time requir ed for the pH sensitive color switch of the culture medium can be correlated to the initial bacterial concentration. The bioactivity in terms of metabolic behavior was statistically analyzed between microcolonies formed on the control (smooth surface) and the Sharklet textured surface
98 Materials and Methods Materials Poly(methyl methacrylate) (PMMA MW 350 k g/mole) was purchased from SigmaAldrich (St Louis, MO). Styrene ethylene -butyldiene -styrene (SEBS) triblock copolymers, Kraton G1657M (styrene block mass percentage 10%) and Kraton G1650M (styrene block mass percentage 30%), were purchased from Kraton (Houston, TX). Polystyrene (PS, MW 30 kg/mole) was purchased from Polysciences (Warrington, PA). 3 -(aminopropyl triethoxysilane) (APTES), and monoglycidyl ether terminated polydimethylsiloxane (PDMS, Mn 5000 g/mole, abbreviated as PDMS -5K) were purchased from Sigma Aldrich (St. Louis, MO). Anhydrous ethanol, 1 -propanol, 2propanol, hydrogen peroxide (30 wt%), and concentrated sulfuric acid (95%) were purchased from Fisher Scientific (Pittsburgh, PA). All chemicals were used without further p cm) was prep ared in the lab with Barnstead Nanopure Diamond TM lab water system from M illipore (Billerica, MA). Tryptic soy broth (TSB) and Mueller Hinton broth (MH) were purchased from BD Biosciences (Franklin Lakes, NJ), and D -(+) -glucose and phenol red were obtain ed from Sigma -Aldrich (St Louis, MO). Cetylpyridinium chloride (CPC) was purchased from Acros (Thermal Fisher Scientific, Rockford, IL). These materials for the bioassays were used as received. Fabrication of Microengineered Polymeric Films The thermoplastic polymeric films were prepared by a solution-casting method. The polymer toluene solution (0.15 g polymer dissolved in 1 ml toluene) was cast onto a smooth or a pre-patterned silicon wafer. The pattern was transferred to a silicon wafer vi a a photolithographic technique [19, 21] To lift the polymer films from the wafer, an
99 antisticking layer (short -chain polydimethylsiloxane, or PDMS 5K) was grafted onto the Si wafer in a three-step manner: (1) a silicon (Si) wafer was immersed in a freshly mixed concentrated sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) water solution (30 wt%) (1:1 volume ratio)  for 30 min to generate surface hydroxyl groups; (2) after thorough rinsing with nanopure water, the Si wafer was dried with nitrogen flow and neat APTES was dropwise added to cover the whole surface for 10 min; (3) after rinsing with anhydrous ethanol, the APTES treated Si wafer was covered by neat PDMS -5K and heated in an oven at 80 oC for 4 hr. The wafer was then rinsed with 2propanol. The PDMS 5K treated Si wafer was dried with an Ar flow  Eno ugh polymer solution was placed on the wafer dropwise to cover the smooth or patterned area of the wafer. After slow removal of the solvent, polymer films with the desired thickness of about 150 m were obtained. Polydimethylsiloxane elastomer (PDMSe) was used as a reference material as described previously by Chung et al.  Silastic T2TMThe resultant topographies contain feature elements with a width of 2.1 m, a distance (between the neighboring elements) of 1.9 m, and a height of approximately 3.0 m. The short name of this pattern was +3SK2 x 2 (for 3 m height). Pattern fidelity was evaluated with light and scanning electron microscopy (SEM). The pattern is shown in Figure 5 -1. (from Dow Corning Corp Midland, MI) base material and curing agent were mixed at a 10: 1 mass ratio and cast against the silicon wafer described a bove. After curing at room temperature for one day the result ing PDMSe films were easily lifted from the silicon wafers.
100 The surface free energy of each Kraton samples was determined by a two-liquid method  Nanopure water ( with resistivity higher than 18.0 M cm) and methylene iodide were used as test liquid. Sample Preparation All of the smooth and patterned polymeric films were cut into approximately 3x3 cm2For the biofilm formation assays involving only the PDMSe samples, the samples were adhered to the Petri dish es with the help of a 70% ethanol/water solution. After removing the ethanol/water solution in a biological hood, the PDMSe samples were attached to the Petri dishes Standard ethylene oxide gas sterilization was employed. squares and placed in 8-cm Petri dishes as shown in Figure 5-2. For the biofilm formation assays performed on various materials (PDMSe, SEBS, PS/SEBS blend, and PMMA), the polymeric films were secured to the Petri dishes with a petroleum based grease Lubriseal (Thomas Scientific, Swedesboro, NJ) The samples were then sterilized in a dessicator with a mixture of commercial bleach and dry acetic acid (1:2 volume ratio) at room temperature for 30 min. A Petri dish with a layer of the grease was also sterilized and served as a positive control. This sealant was found not to be non-toxic to S. aureus Bio film Formation Assay S. aureus (ATCC 35556) was subcultured in a tryptic soy broth (TSB) growth medium and grown at 37 C overnight with shaking. Optical absorbance measurements w ere correlated with the colony forming units (CFUs) to obtain the bacteria gr owth curve. The bacterial concentration was determined by spectrophotometry by interpolating CFUs per milliliter from the linear optical density -CFUs regression. The samples were statically immersed in a 107 CFUs/ml bacterial suspension and kept in a
101 5% CO2After thorough rinsing of the samples, an antibiotic agent oxacillin was used to treat the samples which had been cultured with S. aureus f or 7 days. Oxacillin was dissolved in a sterilized TSB medium at a concentration of 1 g/l. This concentration is considerably higher than the minimum inhibit ory concentration (MIC) for S. aureus to kill any bacterial cells that are not well protected by t heir extracellular polymeric substance (EPS). The three Petri dishes were handled in the following manner: (1) a randomly picked Petri dish was not treated with the antibiotic medium and the samples were punched for the BioTimer assay described in the nex t section; (2) 20 ml of the antibiotic medium was placed into the remaining two Petri dishes; the two dishes were put into the CO incubator for 7 days. T he dishes were daily put on a rocker for 1 min at 40 rpm and the medium was then replaced to allow for continued bacterial growth. When the 7day culture is completed, the d ishes were rinsed three times by a 20 ml Tween 80 (5 ppm) -PBS (phosphate buffered saline) solution, followed by three rinses with 20 ml sterilized, distilled water each time. 2Except for the BioTimer assay, the samples were fixed with 10 mM CPC water solution and two disks punched out from each samples were thoroughly scanned under SEM. Typical images were recorded for ea ch punchouts. (5%) incubator, cultured for 12 hr and 24 hr, respectively; then the samples were rinsed with sterilized water three times af ter cultur ing and were then ready for the BioTimer assay. BioTimer Assay The BioTimer medium (BTM) was prepared according to the following recipe  : Mueller Hinton broth (21 g), glucose (10 g), and phenol red (25 mg) were dissolved in
102 1000 ml distilled water. The medium was sterilized at 121 C for 15 min. The pH value was the determined to be 7.1, and t he final medium was red and transparent A calibration curve can be obtained by corr elating t he initial concentration of the bacteria with the time required for the pH sensitive color switch of the BTM A volume of 0.1 ml of S. aureus / TSB overnight cultures w as injected into 0.9 ml BTA in a 48well plate. Then serial ten-fold dilutions in a 0.9 ml BTM were performed. A volume of 220 l of the mixed culture medium was drawn from each of the wells and the plate counting (for CFUs) was performed immediately. The 48 well plate, which had 680 l of S. aureus -BTM remaining in each well, was put into an incubator at 37 oC. The color of the inoculated BioTimer assay was checked at regular time intervals. For each dilution, the time required for the color switch of the BTM was recorded and plotted versus the log10Statistical Methods of CFUs. The mean number of cell counts on each of four punched coupuns + / standard deviation was reported Statistical differences between surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons  Minitab ( version 15, Minitab Inc, State College, PA) was used for this purpose. Results Characterization of Bacterial Colonies on the PDMSe Samples The calibration curve for the planktonic CFUs counts and time used for color switch shows (Figure 5 3) a good correlation (R2 ~0.97). The time required for the medium to switch color was correlated to the initial bacterial concentration (in CFUs/ml). A smaller color switch time corresponds to a higher initial concentration of planktonic
103 bacterial cells (in CFUs) The calibration curve was used to estimate the planktonic equivalent CFUs counts for the bacteria attached to or colonized on the surfaces of t he polymer samples after a 7 -day culture. As all culture medi a w ere prepared specifically for a series of experiments, the BioTimer calibration was also performed each time to account for the slight variation in the amount of the phenol red used in the Bio Timer medium. The BioTimer assay results for the antibiotic treatment are shown in Figure 54. The results showed that without the antibiotic treatment, the time for a color switch (red to yellow) w as longer on smooth samples than on the Sharklet pattern on the PDMSe material. The difference was statistically significant (P<0.05). This observation means that there were more metabolically active S. aureus cells on the Sharklet textured surface than on the smooth surface. A further examination by SEM imaging showed that multi layered colonies were observed all over the smooth PDMSe surfaces ( Figure 55 (A) and (B)). However, on the surfaces of the Sharklet pattern, only singlelayer to three-layer small colonies dwelling in between the protruding features were prominent (Figure 56 ( A) and ( B)). In some regions of the Sharklet surfaces, there were small multi layered colonies observed ( Figure 56 (C )), which were usually smaller in size than those on the smooth surfaces and covered less than 5% of the whole punched disk (8 -mm diameter) by a rough estimation. After the 12 hr antibiotic treatment, the concentration of the active bacteria on the Sharklet surface dropped more than that on the smooth surface. Compared with the untreated samples, the planktonic equivalent CFUs counts on the Sharklet surface showed about a two-log reduction while less than a one log reduction occurred on the
104 smooth surface. Furthermore, after the antibiotic treatment, the planktonic equivalent CFUs counts on the Sharklet surface were s ignificantly less than those on smooth surface ( 72% reduction, p<0.01) as determined by the BioTimer assay. After a 24 hr antibiotic treatment, the BioTimer assay gave the same planktonic equivalent CFUs count estimates for smooth and Sharklet surfaces (~ 2 000 CFUs/ml). T he SEM imaging ( Figure 56 ) shows that although there were not many continuous bacterial colonies formed on the Sharklet pattern, some material covered the spacing between adjacent features. Th is material may come from the deposition of t he culture medium or the secreted/metabolic products from the bacterial cells dwelling in the spac e between features. Therefore, the PDMSe feature, together with the thick organic capsule material, may have protected some of the bacterial cells on the surface of Sharklet pattern. Bacterial Biofilm Formation on Various Substrates As shown in Table 5 1, a series of polymeric substrates with systematically varied mechanical properties and surface energies were selected to determine the characteristics of biofilm formation on the smooth and Sharklet -patterned surfaces. The BioTimer assay results for the smoo th and Sharklet surfaces ( Figure 5-7 ) showed that it took less time for the BTM to change color on the Sharklet samples than on the smooth samples for the same material (P<0.05), except for Kraton G1650M surfaces where they were statistically not distingui shable. Th u s the numbers of metabolically active bacteria on the surface of the Sharklet textured surface are more than those on the corresponding smooth surfaces. Tukey test was performed for the group of the smooth samples and the Sharklet textured sampl es. For the color switch time, there was no difference among the smooth surfaces and among the Sharklet textured surfaces
105 Typical images obtained from SEM imaging ( Figure 58 ) showed the opposite trend compared with the BioTimer assay results Biofilms we re observed on the smooth surfaces (except G1657M), while there were no biofilm s observed on the Sharklet patterns. As the secreted materials from the biofilm covered the bacterial cells and the bioactivity of the biofilms cells is less than the planktonic cells, finding the opposite result from the BioTimer assay is understandable. Discussion Development of Bacterial Microcolonies on the Surfaces The metabolic activities are different for the bacterial cells buried in the microcolonies. The work on Escherichia coli (E. coli) and Pseudomonas putida (P. putida) by Sternberg et al  demonstrated that the metabolic activity of bacterial cells in the centers of the largest biofilm microcolonies is less than that of the cells at the outer layers. While investigating the metabol ic activity in the biofilms of Staphylococcus epidermidis (S. epidermidis ) and S. aureus t he Stewart group  found a depthdependence within the biofilm microcolonies ; they categorized the physiological states of the bacterial cells into at least four groups: aerobically growing, fermentatively growing, dormant and dead. Since they found about 10% of the total population was dead cells in the report  and the dead cells are consider ed a constituent of the biofilm, we decided to analyze two types of cells in the microcolonies on a surface: bioactive and dormant cells after a 7day culture. After antibiotic challenge, we consider ed dead cells also Based on the SEM imaging and BioTimer assay results on the smooth and Sharklet patterned PDMSe surfaces, one possible scheme was proposed to illustrate the time -dependent development of bacterial microcolonies on the two surfaces, see
106 Figure 59 On both the smooth and Sharklet patterned surfaces, planktonic cells attach on the surface and begin to secret EPS to enclose themselves ( Figure 59 both images (A) and (B), steps (a) and (b)), followed by the increase of the microcolonies and the beginning of differentiation of cell phenoty pe ( Figure 5 -9 both images (A) and (B), step (c)). With time the microcolonies on the smooth surface increase in size without any limitation ( Figure 59 (A), step (d)), therefore the quorum sensing (QS) system is triggered and the cells/cell clusters are then dispersed into the culture medium ( Figure 5 -9 (A), step (e)). This is in agreement with the work by Yarwood et al  However on the Sharklet surface, the microcolonies are separated by the micro-sized features (Figure 59 (B), step (c)). Physically, the microcolonies are disrupted. To develop larger microcolonies, bacterial cell s need to either grow from the microcolonies or deposit from the culture medium to fill the spacings between the protruding features. This becomes a rate limiting step. By the time mature biofilms are dominant on the smooth substrate, there are only a few continuous microcolonies distributed in a few regions on the Sharklet patterned surface ( Figure 5-9 (B), step (d)). Since the culture medium is changed every day, the entir e process occurs all the time. Therefore, we can observe all forms of cell aggregat es : from individual cells to microcolonies on the Sharklet surface and from individual cells to mature biofilms on the smooth surface (as shown in Figure 510 (A) after 7 -day TSB culture). From one report  i n a thick S. aureus biofilm, which was cultured in a capillary tube under continuous flow of culture medium with access to air, the bioactive cel l layer marked by an active expression of green fluorescent proteins showed an average ~40 m thickness out of the total ~170 m thick biofilm Oxygen concentration
107 was determined to reach as deep as ~50 m in the 2 day old biofilm in the report  In another work  cel ls become more densely packed in a S. aureus biofilm as it aged in a 2day culture. In our study, the inoculated samples were kept in a 5% CO2Many antibiotic agents showed penetration and killing to some degree on the cells in the form of biofilms for S. epidermidis [191, 192] atmosphere in an incubator. The large and densely packed biofilms c ould be observed everywhere on the smooth PDMSe surface. However, on the Sharklet textured surface, the pr ominent colony patterns are the discontinuous, several layered (commonly less than 3 layers as compared by the 3m feature height) microcolonies. With aging, the biofilm cells seemed to be less bioactive and in a dormant or dead state  Theref ore we speculate (supported from the BioTimer assay results ) that most of the cells dwelling on the Sharklet textured surface are exposed to the culture medium and in a bioactive state. This indicates (as was observed) the time required for the color switc h for the BioTimer assay would be less for the Sharklet textured surface than that for the smooth samples. and S. aureus [190, 193] Fux et al.  examined the oxacillin resistance of the detached aggregates from S. aureus bio films formed in a flow mode. They found that large clumps were far more resistant to oxacillin challenge compared with the mechanically dispersed clusters detached from the same biofilms  Therefore, oxacillin treatment is expected to kill more cells in small micro colonies on a surface than large ones. After 12 -hr of oxacillin treatment, it is postulated that most cells in small clusters separated by the protruding features on the Sharklet surface are killed (although some dormant cells residing in the center of the microcolonies were protec ted ). Similarly, the EPS protected the cells in the inner portion
108 of the microcolonies and mature biofilms on the smooth surface. Upon removal of the antibiotic medium and submerging into the BTM the cells in the center regions become nuclei of regrowth ( Figure 5-10 (B)). As there may be more large microcolonies on the smooth surface than on the Sharklet as shown from SEM imaging the BioTimer assay results show a longer time required for the color switch for the Sharklet samples than the smooth ones. When treated by 1000 g/ml oxacillin for 24 hr, most cells on the surfaces are killed wi th less than 1% of the total population on both the smooth and the Sharklet textured surfaces surviv ing In a recent review, Lewis  summarized works on persister bacterial cells, which are described not as antibiotic -resistant mutants, but tolerant to antibiotics by maintaining a dormant an d nondividing state. Upon removal of the antibiotic treatment, the persister cells can revive in a non-toxic culture medium and will still be killed by antibiotics. Singh et al.  provided direct evidence that persister cells exist among S. aur eus cells in both planktonic and biofilm forms. It is postulated that the persister cells that are protected by the thick EPS in the microcolonies on the smooth and Sharklet textured surfaces survive the harsh treatment. This hypothesis could explain why i t takes a long time for the dormant cells to regrow in the fresh BT M after 24 hr antibiotic treatment and why the CFUs counts are not statistically different for the two types of surfaces. Surface Properties on the Development of Bacterial Microcolonies The influence of surface properties on the adhesion of bacterial species has been investigated extensively. In a recent paper, Lichter et al.  reported the positive correlation of surface stiffness on the adhesion of various bacterial species. Surface
109 chemical composition and surface charge [124, 126, 197] also play important role s in the inhibition of adhesion and development of bacter ial cells on surfaces. In our study, five materials with systematically varied mechanical and energetic properties were tested. For the smooth surfaces, the SEM images show differences i n the morphologies of the bacterial microcolonies/biofilms ( Figure 5 -7 ), which might suggest that there is an effect on the development of microcolonies from the mechanical and/or nano-sized topographical properties of the substrata. The BioTimer assay results, however, do not show a statistical difference in the CFUs counts for the smooth samples. This can be interpreted to mean that the numbers of the active cells on the surfaces are statistically the same. For the Sharklet patterned surfaces, the SEM images show that most microcolonies are found between the features. Thus the physical obstacles appear to play the major role in separating the clusters of the bacterial cells in the case of Sharklet patterned surfaces. This observation provides the basis for proposing that the application of the microengineered surface feature s disrupt the formation of bacterial biofilms. In contact with culture medium, all the surfaces can be conditioned with organic matter, therefore disguising the chemical/energetic cues of the substrate. Based on the results and the above discussion, it appears that S. aureus cells tend to form thin-layered, discontinuous microcolonies on the Sharklet textured surface in a 7-day in vitro culture. About 98% of the bioa ctive cells dwelling on the Sharklet textured surface can be killed by the antibiotic agent oxacillin in a 12hr treat ment. This t gives a new possible treatment method for biofilm associated infections, i.e., the physical disruption of the microcolonies c ombined with antimicrobial treatment.
110 Conclusion The experimental protocol for a biofilm formation assay with S. aureus as test bacterium was established. Based on the BioTimer assay results, there was no difference i n the colony forming units attached to the smooth polymer films with varied mechanical and energetic properties and t he same results h e ld for the Sharklet AFTM patterns. However, the BioTimer assay results showed that ther e were more metabolic activity from the bacteria formed on the Sharklet patterns than on the smooth surfaces. SEM imaging showed that biofilms were formed on the smooth surfaces while no or little biofilm formed on the Sharklet AFTM patterns. After a highdose antibiotic treatment for 12 hr, the measured metabolic rate from t he Sharklet surfaces was less than that from the smooth surfaces. In terms of planktonic equivalent CFUs, there was about a 2 -log reduction of the population of bioactive cells on the Sharklet textured surface while less than a 1 -log reduction of bacterial population was achieved on the smooth surface, as determined by the BioTimer assay. Therefore the physical obstruction of the micro -sized features on the surface may inhibit the development of microcolonies. The bacterial cells attached to the Sharklet te xtured surface may mostly keep their bioactive state and may be easy to kill with antibiotics due to less protection from thick cell layers and EPS compared with the large biofilms formed on the smooth surface. New treatment strateg ies for the medical dev ices can be inferred from this work, i.e. the combination of micro -textured surfaces with antibiotic treatment, taking advantage of the inhibition of biofilm formation and eas ily kill ed bioactive cells on the microengineered surfaces.
111 Figure 5 1. SEM images of Kraton G1650M with +3SK2x2 pattern: (A) top-down view, (B) 40o tilted view to show the protruding features. Figure 5 2. Layout of sampling plan for biofilm formation assay. The two polymeric films (3x3 cm2 ) were adhered to the bottom of one polystyrene Petri dish. After 7day culture with S. aureus/TSB, the surfaces were rinsed and then four 8 -mm punches were obtained for the BioTimer assay to estimate the colony forming units (CFUs) on the surfaces. Two punches were used for SEM imaging.
112 Figure 5 3. The correlation of the CFU counts in the BioTimer assays and the time required for color switch (from red to yellow). The inset pictures show the color of the BioTimer medium before (red) and after (yellow) planktonic S. aureus culture. y = 3E+07e 0.011x R = 0.9721 1.00E+00 1.00E+01 1.00E+02 1.00E+03 1.00E+04 1.00E+05 1.00E+06 1.00E+07 0 300 600 900 1200 1500 Time, minCFUs
113 Figure 5 4. Planktonic -quivalent CFUs counts on PDMSe samples after 7 -day culture followed by an antibiotic treatment. The numbers on top of the columns are the mean planktonic equivalent CFUs counts from 4 BioTimer assays in a triplicate set of experiment s for each surface 0 1 2 3 4 5 6 7 non treated oxacillin 12 hr oxacillin 24 hrLog(CFUs) Smooth +3SK2x23.5E+5 1.2E+6 6.4E+4 1.8E+4 2.5E+3 2.2E+3
114 Figure 55. SEM images of the smooth surface after 7-day S. aureus culture: (A) Topdown view, (B) 40o tilted view. A B 50 m 1 0 m
115 A B Figure 5 6. SEM images of the Sharklet patterned surface after 7 day S. aureus culture: (A) Top down view, (B) 40 o tilted view, arrows clearly showing EPS covered cell clusters, (C) bacterial colonies that were sporadically observed on Sharklet surface. 50 m 10 0 m 1 0 m C
116 Figure 5 7 T he t ime required for color switch on smooth and Sharklet surfaces after 7day culture. Star (*) denotes statistically different data pair (P<0.05). *
117 Figure 5 8 SEM images of the surfaces of smooth and Sharklet polymer films after 7day S. aureus /TSB culture. Scale bars all equal to 20 m.
118 Figure 5 9 Schematic illustration of bacterial microcolonies which developed on the smooth (A) and microengineered (B) surface with time: (a) individual bacterial cells attach reversibly to the surface; (b) the cells anchor to the surface irreversibly by secreting extracellular polymeric substances (EPS), and the cells lo se their motility; (c) early development of discrete bacterial colonies with start of differentiation of enclosed cells; (d) cells in the microcolonies differentiate into two main types: one is more active and dwells in the outer layer in contact with the culture medium and another is hibernating in the center and bottom layer ; (e) single cells or cell clusters disperse from the mature biofilm to start a new cycle. Active cell Dormant cell a b c d e Active cell Dormant cell a b c d (A) (B)
119 Figure 5 10 Schematic illustration of the microcolonies formed on the topographical and smooth surfaces and the states of the cells after antibiotic treatment. (A) Microcolonies are well established on the surfaces after 7-day culture. (B) When treated by oxacillin for 12 hr most cells in small microcolonies and in the outer layers of biofilms were killed. (C) After a longer period of antibiotic treatment, most cells attached to the surface of the smooth and the microengineered pattern are killed, while some dormant ( persister) cells in the center and bottom of the microcolonies are protected by the EPS and/or the surface features. (A) (B) (C)
120 Table 5 1. Physical properties of the selected materials. Materials Youngs modulus, MPa Surface free energy, mJ/m 2 PDMSe 1.4 a 21.5 c Kraton G1657M 6.3 35.0 Kraton G1650M 34 40.8 PS/G1650M blend 2.4 10 2 40.7 PMMA 3.3 10 3 b 42.4 a From previous work  b From reference  c From reference 
121 CHAPTER 6 NEW UNDERCUT SURFACE FEATURE AND BIORESPONSE Introduction Biofouling the undesired attachment and accumulation of biological matter on surfaces imposes huge economic losses on societ y For example, it is estimated that the US Navy spends an extra amount of over one billion dollars per year on fuel costs due to the increased drag resulting from biofouling  Due to biotoxicity and accumulation along the food chain previously employed organo-tin based coatings were banned and phased out by the year 2008. Other biocides containing metal oxides (such as copper) also showed hazardo us effect s on marine organisms. Thus environment al friendly coatings are needed for antifouling applications. F or some time, it has been known that surface topography play a significant role in how surfaces are wet by liquids. Wenzel  examined the effects of added surface area arising from surface features upon wetting. Micro -surface features can also inhibit wetting by f orming vapor liquid interfaces .This phenomenon was examined by Cassie and Baxter  On smooth surfaces, wettability has a profound influence upon Ulva zoospore attachment and removal  Surface topographical features can significantly change the apparent water contact angle for a material. However, there are no report s on fouling studies for a compound surface which has stable liquidvapor solid interfaces. It may be attributed to the instability of the air pockets in the underwater environment for most of the surface microstructures.
122 With under -cut surface micro -features, obtaining stable liquidvapor -solid interfaces is possible Theref ore, the surface wetting regime may be changed on a surface with undercut micro-patterns [199, 200] Furthermore, stable air pockets will hide some of the surface in contact with the liquid causing microorganisms to have a reduced chance of touching the surface. These facts inspired the design of under -cut surface features on thermoplastic materials. Theory The concept of the engineering roughness index (ERI), based upon surf ace wetting, was first proposed by Schumacher et al  in a model for the bioresponse of marine spore s toward micro engineered surfaces. To account for the effect of various distinct surface features o n the designed micro engineered surfaces, th e calculation of ERI was modified into the following form by Long et al.  = (6 -1) where r is the Wenzel roughness factor defined by the total surface area divided by the projected planar surface area  n is the number of the distinct surface features, and f is the fraction al area of the solid surface in contact with the liquid in the Cassie -Baxter (C -B) model  The basis for th e predic tive model was built upon the bioresponse assays performed on polydimethylsiloxane elastomer (PDMSe) material. To apply this model to various other materials with different surface chemistry (and thus surface energy and wettability) and nano-sized topograp hies we proposed to use the water contact angle to estimate r or f depending on whether the wetting regime is in the Wenzel, C -B, or
123 wicking regime. It can be determined by a lower ( L C) and upper ( U C = cos (6 -2 ) ) critical contact angle, according to the following equations [160, 161] : = cos (6 -3 ) When the contact angle on the smooth surface is larg er than the upper critical angle, the wetting regime is Cassie -Baxter ; if the contact angle is between the lower and upper critical angl e s the surface is in the Wenzel wetting regime; if the contact angle is less than the lower critical angle, the surface is in the wicking regime. After determining the wetting regime for a topographical surface, the terms in Equation (61) can be estimated by the following eq uations: (6 -4 ) = Air entrapment (6 -5 ) = Liquid wicking where, cos cos* r is the apparent contact angle and is the intrinsic contact angle (liquid in contact with a flat surface). Therefore, if the wetting regime is in the Wenzel state, then Equation 64 is used to calculate the roughness index value, and f is calculated by the feature dimensions; if the wetting regime is in the C -B state, then the upper line in Equation 6 5 is used to estimate the value of f and the Wenzel roughness ratio is calculated from the topographical dimensions. The aim of this approach is to estimate
124 the actual area fraction (relative to the planar surface area) in contact with the liquid. It should be noted that there are constraint s for r (r and f (0 f <1). The engineered roughness index value calculated from the water contact angle data is denoted as ERIII *Chapter 3 showed that the Sharklet textured PDMSe surfaces were all in the C -B regime. ERI in this work. II vavlue can be calculated using equations 6 1 and 6 5 (the air pocket situation) Good correlation can be obtained for spore attachment density change relative to smooth surface vs. ERIII = 0 .058 ( 2= 0 82 ) (6 -6 ) (see Figure 61): In Equation 6 -6, A denotes the spore attachment density on the topographical surfaces and ASMThe correlation between Ln( A/A i s the spore attachment density on the smooth surface. SM) and ERIII = 0 .071 (2= 0 88 ) (6 -7) which was based on the measured dimensions of the topographical features in Longs work  is cited belo w for comparison : Materials and Methods Materials The polymer films used in this work were all +3SK2x2 films made from Kraton G1650M, PS/G1650M blend, and PMMA. The preparation method was described in detail in Chapter 3.
125 Process of Making Undercut Micro -features on Polymer Films To produce undercut micro -features pressure and heat were applied to deform the tops of the micro-features, as illustrated in Figure 6-2. The softening and melting ranges of s ome common polymers a re listed in Table 61. Specifically, the prepatterned polymer film was placed on a flat substrate. A hot plate was then driven in contact with the polymer film to exert sufficient pressure for a specified amount of time. The top of the micro features w as deformed ; Figure 63 show s some examples of the flattened surface structures. The experimental setup i s shown in Figure 6 4. Two stacks of glass slides (two pieces in each stack) were put on a flat glass plate as spacer s The polymer film was placed on one glass slide and placed in between the two stacks. A PDMS 5K grafted silicon wafer was then put on top of the polymer film and the spacer. The preheated glass beaker with silicone oil ( total weight 205 g) was placed on the silicon wa fer for 30 sec to deform the top of the micro-features without damaging the entir e surface structure. The app lied pressure was about 2200 Pa on the polymer film. The silicone oil in the container was preheated to (1) 160 oC for G1650M, (2) 170 oC for PS/G1 650M, and (3) 200 oSurface Characterization of Polymer Films C for PMMA The w ater contact angle was measured usi n g the sessile drop method with a Ram -Hart goniometer (Netcong, NJ) coupled with DROPimage Advanced software (for image capturing). Nanopure w istivity) droplets were placed o n the surfaces via a Ram Hart Auto Pipetting system. On each sample, six 5-l droplets were randomly placed; the images of each droplet were taken in two directions: parallel and perpendicular to the surface features. In this manner, twelve images were recorded
126 for each sample and contact angles were measured with ImageJ software (public software developed by NIH). For each sample, t he contact angles were reported in two directions. Sample surfaces were imaged using scanning electron microscopy (SEM). The samples were sputter coated with Au/Pd under Ar (~45 mTorr) at 38 mA for 1 min. A Jeol 6400 SEM was used to take the images. The operation al conditions were: (1) working distance 15 mm, (2) accelerating voltage 5 kV, (3) beam current 3~6x1010Statistical Methods mA. Statistical differences between surfaces were evaluated using a nested analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons Results and Discussion Polymer Films with Undercut Micro-Topographical Features The three polymer materials (G1650M, PS/G1650M, and PMMA) with surface feature +3SK2x2 (Sharklet AFTM pattern, feature height ~3 m, feature width ~2 m, and spacing between two neighboring features ~2 m) were t herm al pressed. Each treated sample was observed with a light microscope to ensure there were no obvious defects after the treatment. The SEM images were taken for all three materials with Sharklet AFTM pattern after therm al pressing (Figures 65, 6-6, and 6 7). The images showed a wider feature width at the top than the other part of the same feature from the cross-sectional SEM images. The feature width and spacing on each polymer ic material we re measured and are listed in Table 6-2 The dimensions of the features were measured using more than 5 features in one SEM picture. Usually more than 4 pictures were used for a dimension measurement. The standard deviation was small (<0.05 nm), and therefore was not included in Table 6 -2 The measured dimensions show e d that the
127 tops we re flattened The features on G1650M and PS/G1650M after therm al pressing showed an upside -down wedge shap e. Features on PMMA after thermal pressing (Figure 67) more closely resembled the cross-sectional view shown in Figure 64. Estimation of Fouling Reduction on the Undercut Surface s The undercut Sharklet pattern was formed on the three test materials (G1650m, PS/G1650M blend, and PMMA) after therm al pressing and the spacing between the tops of the neighbori ng features wa s reduced (Table 6-2). Therefore, the area fraction of the tops that can be wet by the liquid relative to the projected planar surface area increased, thus decreasing the heterogeneity of the surface pattern. Sessile drop water contact angles we re measured in two directions: parallel to and perpendicular to the features based on the water expansion direction. After the undercut shape is formed on the Sharkle t pattern for the same material the difference between the water sessile drop contact angles measured in the two directions (parallel and perpendicular) decreased significantly for all three materials. It should be noted that for G1650M and PS/G1650M blend, the undercut +3SK2x2 topographies showed no heterogeneity in terms of the water sess ile drop contact angle data (Table 62). As analyzed in Chapter 3, the normal and undercut +3SK2x2 Sharklet textured surfaces on the G1650M and PS/G1650M blend may all fall into the Cassie -Baxter wetting regime. Based on the measured sizes of the topographical features for the normal and undercut patterns, the ERIII values can be calculated from Equation 6 -1, and are shown in Table 62. The relative reduction of spore attachment density was then estimated, based on Longs work  using Equation 6-7 The ERIII values increase for the undercut topographical patterns relative to the normal features for all three materials As s hown in Table 6 2, t he estimated reduction in the spore attachment density on the two
128 types of Sharklet textured surface s relative to the corresponding smooth surface s was slightly greater on the undercut surface for G1650M and PS/G1650M blend, and was mo re improved on the undercut surface for PMMA, when compared with the normal Sharklet surface. The wetting regime was determined by the lower ( L C) and upper ( U C) critical contact angle calculated with Equation 6-2 and 63, based on the geometry of the surface topographical features. As shown in Table 6 3, normal and undercut Sharklet textured surfaces on G1650M and PS/G1650M blend fall in the Cassie-Baxter regime. For PMMA, the two types of topographical surfaces were both in the Wenzel regime. The sessile drop water contact angle for PMMA with the two types of topographical patterns are all higher than that of the smooth surface of PMMA. This phenomenon may be caus ed by the nano -sized convex surfaces on the side walls of the protruding features on the Sharklet PMMA film. For the undercut +3SK2x2 PMMA surface, the water contact angle in perpendicular direction is less than that for the normal +3SK2x2 PMMA film (mean value 125o vs. 133o). Zheng  demonstrated that contact angle is reversely proportional to the area fraction of the solid in contact with a liquid ( f ) for C B wetting regime. The decrease in contact angle for the undercut PMMA surface (relative to the normal Sharklet surface) shows that the wetting regime should be in C -B state, although the calculated lower an d upper critical contact angles predicted it to be the Wenzels wetting regime. Because the thermodynamically favored wetting regime is the Wenzel state for the two Sharklet patterns on PMMA, the high apparent water contact angles are certainly transient s tate s and susceptible to disturbance s such as shaking and ultrasonic treatments.
129 The ERIII value s can also be evaluated with E quations 6 -1 6 4, 6 -5 and 6 -6. The contact angle data in the parallel direction were used to estimate the ERIII values and percent reduction (see Table 6 4 ). The estimated percent reduction based on water contact angle measurement and ERIII *Underwater Stability of the Undercut Surface s show s higher resistance against spore attachment on the undercut Sharklet patterns than on the normal Sharklet pattern replicated on G1650M and PS/G1650M There is no difference i n the estimated percent reduction of spore attachment density for normal and undercut Sharklet textured PMMA film s When a surface with nano/microengineered topographi cal features is immersed in water, an issue of concern is whether the surface can hold the composite liquid vapor solid contact lines underwater. The ability to keep the composite contact interface intact can help minimize the contact area between the fouling species and the surface i n water, therefore deferring or preventing the biofouling process  Marmur  used thermodynamic reasoning to show the feasibility of the underwater superhydrophobicity. On the assumption of no liquid penetration through the tops of the features, he found that the following condition must be satisfied, > + 1 + (6 -8) where r is the surface roughness ratio, rmin is the minimum required value of the surface roughness ratio, fo is the area fraction of the projected solid top that is wet by the liquid without penetration through the protruding features, and is the intrinsic contact angle (Youngs contact angle on the smooth material). As r >1, Equation 6 8 constrains to be greater than /2  mean ing the starting material must be hydrophobic. PMMA is a
130 hydrophilic material and therefore it will not show stable underwater superhydroph obicity. The hydrostatic stability can be expressed as the critical pressure that the composite surface can withstand before water penetrates into the vicinities among the surface topographical features. T he equilibrium of the composite interface requires [200, 201] ( ) = cos ( + ) (6 -9) where pc is the critical penetration pressure, Ac is the planar area of a unit cell of the periodic surface feature, A is the area of the tops of the features in the unit cell is the surface free energy of the liquid (for water = 72.8 mJ/m2 = ( ) (6 -10) ), L is the perimeter of the top of the surface feature, is the intrinsic contact angle (Youngs contact angle), and is the g eometric angle of the surface feature (see illustrations in Figure 6-8 ) The critical pressure can be evaluated by T heoretically the deepest position (h ) on the surface that w ill maintain the composite interface underwater will be = (6 -11) Here, is the density of the liquid (the density of water is 1000 kg/m3) and g is the gravitational acceleration (9.8 m/s2The surface roughness ratio for the untreated and therm al pressed +3SK2x2 surfaces (G1650M, PS/G1650M blend and PMMA) are all about 2.5. I n the case of the Sharklet pattern, the unit cell is shown in Figure 6 8. The surface pattern is anisotropic ).
131 and t herefore the perimeter of the top of the features is calculated for the features in the unit cell and the average critical pressure is estimated based on the geometry of the unit cell. As shown in Table 6-5 rminThe work by Tuteja et al [199, 200] showed that under -cut surface micro features could facilitate stable water vapo r-solid interfaces. Undercut surface nano or micro top ographical features with proper chemical surface modification on the polymeric materials may therefore help maintain stable underwater superhydrophobicity and improve long-term performance for antifouling applications. values for the two microengineered surfaces on G1650M are 2.9 and 2.8, respectively. Therefo re, the two surfaces cannot hold the water air -solid interfaces underwater, even though they have a relatively high penetration pressure (15 and 29 kPa, respectively). PS/G1650M blend will maintain a stable composite interface underwater, withstanding water pressure as high as 24 and 39 kPa for the normal Sharklet textured surface and the undercut +3SK2x2 surface, respectively. The rmodynamically the untreated and therm al pressed +3SK2x2 PMMA surface cannot hold the composite interface underwater However, w ith the undercut topographical features the surface can be covered with a 12 -cm height of water layer and still keep the composite interface (see Table 65 ). Conclusion In this work, it was demonstrated that a thermal pressing technique can be carefully employed to obtain flattened-top (under -cut) microstructures on the polymeric substrate. The feature dimensions clearly showed the flattened tops of the Sharklet AFTM patterns. The w ater contact angle s showed less heterogeneity on this surface compared with the untreated topographical surfaces The increased area fraction of the feature top s uggest s a more stable water air -solid composite interface.
132 The result ing surfaces are expected to show high er biofouling resistance t o marine microorganisms.
133 Figure 61. Correlation of spore attachment density and ERIII Spore attachment data and water contact angle measurements are adapted from the work of Schumacher et al.  and Long et al  respectively. Figure 62. Schematic illustration of processing method for fabricating undercut surface topographies. = 0 058 2 = 0 82 ERIII
134 Figure 63. Some examples of cross -sectional view (A) and topdown view (B -D) of the undercut surface topography. Solid (i.e. polymer) Side view Top down view 1 Top down view 2 Top down view 3 (A) (B) (D) (C)
135 Figure 64. Experimental setup for processing polymer films to obtain undercut surface topographical features Silicon wafer Heat sink Polymer film Glass slide
136 Figure 65. SEM images of Kraton G1650M undercut +3SK2x2 film: (A) topdown view at 1000x magnification; (B) topdown view at 3000x magnification; (C) Cross sectional view; (D) cross-sectional view 50 m 50 m 10 m 10 m 10 m 10 m 5 m 5 m A B C D
137 Figure 66. SEM images of the PS/G1650M undercut Sharklet +3SK2X2 film: (A) top down view at 1000x magnification; (B) topdown view at 3000x magnification; (C) 40o tilted view 10 m 10 m 50 m 50 m 10 m 10 m A B C
138 Figure 67. SEM images of the under -cut PMMA Sharklet +3SK2X2 film, (A) top -down view, (B) cross -sectional view. A B
139 (A) (B) Figure 68. (A) a unit cell of the surface features of the Sharklet pattern (topdown view), the blue area represents the tops of the surface features and the area in the thick black lines is the planar area of the unit cell ( Ac ). (B) Schematic illustration of the underc ut surface features can increase the critical pressure exerted on the top of the features  Table 6 1 Softening and melting ranges of some common thermoplastic polymers Polymer Softening range, o C Melting range, o C Polyethylene (PE) 65 110 95 135 Polypropylene (PP) 120 130 140 170 Polymethylmethacrylate (PMMA) 105 125 220 240 Polystyrene (PS) 85 100 190 260 Polyvinyl chloride (PVC) 65 150 100 260 Polycarbonate (PC) 84 -140 230280 Polyethylene terephthalate (PET) 120 150 240 260 Table 6 2. Measured geometry of the Sharklet surface features and the correlation of Ulva spore attachment reduction. Material Feature Feature size, m ERI I I % reduction* Width Height Spacing G1650M +3SK2X2 2.1 3.0 1.9 17.9 72 Under cut +3SK2X2 2.3 2.9 1.6 19.4 7 5 PS/G1650M +3SK2X2 2.1 3.0 1.9 17.9 72 Under cut +3SK2X2 2.3 2.9 1.6 19.4 7 5 PMMA +3SK2X2 2.0 3.0 2.0 17.7 72 Under -cut +3SK2X2 2.2 3.0 1.8 21. 6 79 % reduction is relative to the smooth surface of the same material predicted by Equation 67.
140 Table 6 3. Sessile drop water contact angle (CA) measurement and the wetting regime for the topographical surfaces Material Feature CA ( o ) LC ( o ) UC ( o ) Regime G1650M Smooth 103 6 +3SK2x2 parallel 139 2 74 106 C B perpendicular 147 1 Under -cut +3SK2x2 parallel 147 3 76 104 C B perpendicular 149 2 PS/G1650M Smooth 112 2 +3SK2x2 parallel 144 5 74 106 C B perpendicular 152 3 Under -cut +3SK2x2 parallel 146 3 76 104 C B perpendicular 147 2 PMMA Smooth 76 2 +3SK2x2 parallel 86 1 74 106 Wenzel perpendicular 133 2 Under cut +3SK2x2 parallel 84 4 75 105 Wenzel perpendicular 125 6 Parallel: indicating the spreading direction of water droplet i s in parallel to the longitudinal direction of the surface features Perpendicular: indicating the spreading direction of water droplet i s in perpendicular to the longitudinal direction of the surface features. Table 6 4. Estimation of the ERIII Material values and %Reduction of the attachment density of spores based on the correlation in this study. Feature r f ERI II % Reduction G1650M +3SK2X2 2.5 0.32 32 84 Under cut +3SK2X2 2.5 0.21 48 94 PS/G1650M +3SK2X2 2.5 0.30 33 85 Under cut +3SK2X2 2.5 0.27 37 88 PMMA +3SK2X2 2.5 0.86 12 49 Under cut +3SK2X2 2.5 0.89 11 48 %Reduction is estimated by Equation 66. Table 6 5 Estimated penetration pressure for the various surface topographies. Material Feature ( o ) f o r min p c k Pa h m G1650M +3SK2X2 90 0.44 2.9 15 1.5 Under cut +3SK2X2 80 0.49 2.8 29 3.0 PS/G1650M +3SK2X2 90 0.44 1.9 24 2.5 Under cut +3SK2X2 80 0.49 1.8 39 4.0 PMMA +3SK2X2 90 0.43 Under cut +3SK2X2 75 0.46 1.2 0.12
141 CHAPTER 7 CONCLUSIONS AND FUTURE WORK Conclusions In this work, the fabrication techniques of microengineered topographical polymer films were explored. The fabricated polymeric substrates were tested against adhesion of marine microorganism s and bacteria. New surfaces with designed propertie s (chemical, mechanical, topography) could be inspired from these bioresponse results. Fabrication of Micro-engineered Polymeric Films A s olution casting method was chosen as the fabrication process under current laboratory conditions. A difficult y of th is process was the demolding of the fabricated polymer films without damaging the micro -sized features. A simple wet process was developed to covalently graft a release agent onto the surface of a silicon mold. A fabrication process for polymer films was dev eloped so that micro engineered topographical features could be replicated onto various polymeric materials. Quality control of the result ing polymer films was simplified as the fidelity (> 99%) of the microfeatures and integrity of the whole film was ensured. Marine Antifouling Assay The polymeric films created with specified feature dimensions, mechanical properties, surface energies and chemical compositions were tested with an Ulva linza zoospore assay. All the polymeric films were attached to the glass slides by an epoxy glue, which was found to be strongly attractive to the spores. The data analyses and interpretation were based on the assumption that the influence of the epoxy glue on the spores was th e same among all the samples. The Sharklet AFTM pattern was effective against spore attachment with some exceptions. The p rincipal component analysis
142 (PCA) revealed that two groups of surface parameters may be most important New correlations w ere obtained between the reduction of spore attachment and the surface parameters including the engineered roughness index, mechanical property and surface energy. PCA analysis provided potential direction of the improvement on the surface parameters for spore attachm ent studies although the spore attachment assay results need to be interpreted with caution. Bacteria Biofilm Assay The experimental protocol for the biofilm formation assay with S. aureus as test bacterium was established. Based on BioTimer results, th ere was no difference i n the colony forming units attached on the smooth polymer films with various mechanical properties and t he same result was found for the Sharklet AFTMNew Undercut Surface Features patterns. However, BioTimer assay showed that there were more metabolically act iv e cells from the bacterial mi crocolonies that formed on the Sharklet patterns than on the smooth surfaces. SEM imaging showed that biofilms formed on the smooth surfaces while no biofilm s form ed on the Sharklet patterns. After a highdose antibiotic treatm ent for 12 hr, less metabolic activities were determined by the BioTimer assay on the Sharklet textured surfaces than on the smooth surface s indicating that more cells on the Sharklet surfaces may be killed than on the smooth. Therefore a formation model was proposed for the development of bacterial microcolonies on the smooth and the micro engineered surfaces. N ew treatment strateg ies for the surface att ached bacterial microcolonies were also proposed based on this study. Pro per processing conditions to fabricate the under -cut micro features on various thermoplastic materials were explored. The feature dimensions clearly reveal ed the
143 flattened tops of the Sharklet AFTMFuture Work patterns. The dimensions of the undercut features are expe cted to stabilize the water vapor -solid interface, supporting higher stability of underwater superhydrophobicity for PS/G1650M blend. The resulting undercut surfaces are predicted to show a higher biofouling resistance against marine microorganisms based on predictions from two models. Marine Bioresponse Assays on the Polymer ic Films A new batch of micro engineered polymeric films was prepared without using any extra adhesives. Th e glass slides were enclosed with the test polymer by a thermal pressing method, e.g. G1657M was used to enclose the glass slides if the test engineered films were fabricated from G1657M. The free -standing micro engineered films were then attached to the enclosed glass slides with the help of its toluene solution. After removing the residue solvent, the samples were checked and sent to the University of Birmingham (UK) for spore attachment assay on June 18, 2010. The samples are currently undergoing tests and characterizations. We expect to obtain the results in August 2010. The undercut surface features on polymer films were successfully processed, and the dimensions showed higher stability for resistance of water penetration when underwater. As discuss ed in Chapter 6, even small changes in the contact angle could induce large variations in the engineering roughness index and t hus the bioresponse is believed to change accordingly. This hypothesis therefore needs to be tested using the newly fabricated un d e rcut features.
144 In vivo Study of the Efficacy of the Micro-engineered Polymer Films Based on the protocol established for the biofilm formation assay, biofilm/microbial colonies can be formed and recovered from the polymeric substrates. To further investiga te the efficacy of micro -topographical surface structrues against biofilm formation, we propose to implant the 8mm punches from the smooth and Sharklet AFTMTo evaluate the effectiveness of the proposed treatment strategy, a procedure for preparing an animal model could be: (1) the paired samples (the smooth and topographical surfaces) are c ultured to develop bacterial microcolonies on the surface in a 3day to 7 day culture period; (2) 8mm discs are punched out from the bacteriacovered samples and implanted into the bodies of the test animals (either rat or rabbit). One group of animals ar e implanted with the smooth samples and another group of animals are implanted with the topographical smaples. (3) the two groups of animals are subdivided into four groups, and are treated with or without antibiotics. Two more groups of animals implanted with just sterilized samples w ill be used as controls (see Table 7 1 ). after a 7 day S. aureus /TSB culture period The paired samples (smooth and topography) will be imp lanted subcutaneously. The animal conditions, including body temperature, body mass, activity and wound healing, will be monitored during the test period. After a specified period of time, the samples will be retriev ed from the implanted sites and the resi dual bacteria on the samples will be measured with a BioTimer assay.
145 Table 7 1. In vivo test groups to evaluate the effectiveness of the proposed treatment strategy. Groups 1, 2 and 3 consist of the same amount of animals. group 1 ( S. aureus covered) 2 ( S. aureus covered) 3 (sterilized surfaces) Smooth Sharklet Smooth Sharklet Smooth Sharklet Treatment No No antibiotics antibiotics No No Observation Body mass, temperature, activity, mortality, healing rate, bacterial cell counts after retrieving of the samples
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163 BIOGRAPHICAL SKETCH Liwen Jin (Kevin) son of Zhongjiang Jin and Yun Zhou, was born in Yili, Xinjiang, China. He was raised there until thirteen years old, when he moved with his family to Bole city, Xinjiang. Liwen finished his high school study from the Forth High School of Bole City and p assed the entrance examination of college. He was then admitted to Petroleum University (East China), majoring in Applied Chemistry. During the four year college life, not only did he receive scholarship every semester, but he also took part in various cam pus activities including physical exercises, Englishlearning group, ball -room dancing, and research. After graduating from the university, Liwen has been working for several organizations, mainly focusing on research and consulting in the area of chemical engineering. He found that he was eager to absorb more knowledge in this area. In 2001, he was admitted to the Department of Chemical Engineering at Clemson University, SC, USA. He finished his masters degree over there and worked for a local company as a research intern. In 2006, Liwen joined Dr Baney research group in the Department of Materials Science and Engineering at the University of Florida. Under the guidance of Dr Baney and Dr Brennan, he has been working on antifouling of both marine microorg anisms and bacteria using micro engineered polymeric materials. Liwen will receive his PhD degree in August 2010.