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Template Synthesized Membranes for Ion Transport Modulation and Silica-Based Delivery Systems


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1 TEMPLATE SYNTHESIZED MEMBRANES FOR ION TRANSPORT MODULATION AND SI LICA-BASED DELIVERY SYSTEMS By FATIH BUYUKSERIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2007

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2 2007 Fatih Buyukserin

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3 To my parents, my wife Miyase, and my son Faruk Eren Buyukserin.

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4 ACKNOWLEDGMENTS Many individuals have been there to help me during my graduate st udies. I would like to thank Dr. Charles R. Martin for his patience, guidance and support throu ghout my career at the University of Florida. He has been an excellent mentor by providing scien tific discussions about my research and at the same time a great teach er in paper writing and presentation skills. The Martin group members have been very s upportive. I am very grateful to Myungchan Kang, Punit Kohli, Mark Wirtz, Shufang Yu and Dave Mitchell for providing insightful discussions about my experiment s and training me in surface modi fication, analytical detection techniques and instrumentation. Mario Caicedo, Lane Baker, John Wharton, Lacramioara Trofin, Damian Odom, JaiHai Wang and Fan Xu shar ed their experiences and provided helpful suggestions. Miguel Mota spent many hours growing aluminum oxide for my experiments. Heather Hillebrenner was a perfect collaborator a nd a great friend. I want to thank Drs. Weihong Tan and Rick Roge rs for their valued advice on my research and career. I am also grateful to Karen Kelly and Lynda Schneider from the ICBR for their help with SEM and TEM, to Eric Lambers from the MAIC for his help with XPS, and to Colin Medley from the Tan research group fo r his help with confocal microscopy. Finally, my family and friends provided e normous help and moral support during my graduate career. My wife Miyase Buyukserin ha s always been there with her patience and unconditional love. My parents Husniye and Hasan Fehmi Buyukser in, my brothers Mehmet and Mustafa Buyukserin deserve most of the credit for my success as they were constant sources of encouragement and support before and during my gr aduate career. I want to also thank members of the Gainesville Turkish community, especially Fatih Gordu, Erkan Kose, Ahmet Basagalar, Onur Kahya, Zafer Demir, Cem Demiroglu, Kaan Kececi, Nezih Tu rkcu and Ugur Baslanti, for providing a very friendly, cal ming, home-like atmosphere.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF TABLES................................................................................................................. ..........8 LIST OF FIGURES................................................................................................................ .........9 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION AND BACKGROUND...........................................................................13 Introduction................................................................................................................... ..........13 Background..................................................................................................................... ........14 Template Synthesis..........................................................................................................14 Applications in electrochemistry and sensing..........................................................15 Applications in control of ion transport and electromodulation..............................16 Applications with silica and biomolecule nanotubes...............................................17 Track-Etched Polycarbonate Membranes........................................................................19 Electroless plating of polymeric templates..............................................................21 Estimation of nanotube inside diameter...................................................................22 Anodic Alumina Templates.............................................................................................23 Two-step anodization method..................................................................................24 Membrane detachment.............................................................................................26 Sol-Gel Technology.........................................................................................................27 Surface Sol-Gel Method..................................................................................................29 Silane Chemistry.............................................................................................................30 Plasma-Assisted Dry Etching..........................................................................................31 Biomolecule Delivery with Nanoparticles and Viruses..................................................33 Chapter Summaries.............................................................................................................. ...36 2 ELECTROACTIVE NANOTUBES MEMB RANES AND REDOX-GATING...................42 Introduction................................................................................................................... ..........42 Experimental................................................................................................................... ........43 Materials...................................................................................................................... ....43 Electroless Gold Deposition............................................................................................43 Membrane Sample Preparation and Thiol Modification.................................................44 Electrochemical Experiments..........................................................................................45 Transport Experiments....................................................................................................45 Results and Discussion......................................................................................................... ..46 Electrochemistry of the Fc-Thiol.....................................................................................46 Electromodulated Transport Experiments.......................................................................47 Conclusions.................................................................................................................... .........49

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6 3 KINETICS OF FERRICINIUM DECOMP OSITION CONFINED WITHIN GOLD NANOTUBESEFFECT OF THE NANOS CALE ENVIRONMEN T ON KINETICS.......57 Introduction................................................................................................................... ..........57 Experimental................................................................................................................... ........58 Materials...................................................................................................................... ....58 Electroless Gold Deposition............................................................................................58 Membrane Sample Preparation and Thiol Modification.................................................59 Surface Thiol Removal....................................................................................................60 Electrochemical Experiments..........................................................................................60 Results and Discussion......................................................................................................... ..61 Surface Fc-Thiol Removal..............................................................................................61 Electrochemical Decay Studies.......................................................................................62 Conclusion..................................................................................................................... .........64 4 PLASMA-ETCHED NANOPORE POLYMER FILMS AND THEIR USE AS TEMPLATES TO PREPARE NANO TEST TUBES......................73 Introduction................................................................................................................... ..........73 Experimental................................................................................................................... ........74 Materials...................................................................................................................... ....74 Preparation of the Nanopore Alumina-Membrane Masks...............................................75 Preparation of the Nanopore Polymer-Replica Films.....................................................75 Preparation of the S ilica Nano Test Tubes......................................................................76 Results and Discussion......................................................................................................... ..77 Conclusions.................................................................................................................... .........79 5 SILICA NANO TEST TUBES AS DELIVERY DEVICE S; PREPARATION AND BIOCHEMICAL MODIFICATION......................................................................................87 Introduction................................................................................................................... ..........87 Experimental................................................................................................................... ........89 Materials...................................................................................................................... ....89 Preparation of the Nanopore Al umina-Membrane Templates........................................90 Preparation of the S ilica Nano Test Tubes......................................................................90 Silica Nano Test Tube Modification with Fluorophore..................................................92 Antibody Modification....................................................................................................93 Cell Incubation Studies....................................................................................................94 Results and Discussions........................................................................................................ ..95 Defect-Free Silica Nano Test Tube Preparation..............................................................95 Differential Modification.................................................................................................97 Cell Incubation Results....................................................................................................98 Conclusion..................................................................................................................... .......100 6 CONCLUSIONS..................................................................................................................115 LIST OF REFERENCES.............................................................................................................118

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7 BIOGRAPHICAL SKETCH.......................................................................................................129

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8 LIST OF TABLES Table page 2-1 Flux and electromodulated selectivity coefficients ( ) for membranes containing 10-nm and 16-nm diameter nanotubes...............................................................................51 3-1 Fc+ decay constants for different membrane systems and for bulk aqueous solutions of Fc compounds in phosphate solutions at neutral pH.....................................................66

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9 LIST OF FIGURES Figure page 1-1 The chemical structure of polycarbona te and Scanning Elec tron Micrograph (SEM) of the surface of a commercial tr ack-etched polycarbonate membrane............................39 1-2 Top and cross-sectional vi ew of PC membrane before & after the gold plating...............40 1-3 SEM images of the surface of commercially available and home-grown alumina membrane....................................................................................................................... ....41 2-1 Cyclic voltammogram for a Fc-thiol-modi fied Au nanotube membrane and the plot of anodic peak current vs. scan rate from such voltammograms.......................................52 2-2 Effect of electrolyte on the stability of the Fc+/Fc voltammogram...................................53 2-3 Long-term stability of the Fc-thiol layer. .........................................................................54 2-4 Plot of nanomoles of MV2+ transported across a nanotube membrane (nanotube inside diameter = 10 nm) vs. time......................................................................................55 2-5 Moles of electroactive Fc vs. cycle number for a me mbrane containing 16 nm-diameter Au nanotubes...........................................................................................56 3-1 Finding the optimum etching tim e for surface Fc-thiol removal.......................................67 3-2 XPS spectra of the Fc-thiol modified gold membrane after various argon plasma etching periods................................................................................................................ ...68 3-3 Cyclic voltammograms of a Fc-thiol modified membrane before (solid curve) and after (dashed curve) 30 sec of Argon plasma etching........................................................69 3-4 Cyclic voltammograms of four di fferent gold nanotube membranes................................70 3-5 Cyclic voltammograms of m odified gold button electrode...............................................71 3-6 First order kinetic plots for the loss of the Fc+ for gold nanotube membranes with different pore diamaters and for a gold button electrode...................................................72 4-1 Schematic diagrams of the preparati on of closed-end nano test tubes and the preparation of closed-end pores in a substrate material.....................................................80 4-2 SEM images of the nanopore alumina-membrane mask...................................................81 4-3 Cross-sectional SEM of the Al-mas k:Au/Pd-film:polymer-film assembly.......................82 4-4 SEM images of the polymer-film surface and the cross-section of the film after 4 min of O2/Ar plasma etching.....................................................................................................83

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10 4-5 SEM images of the polymer-film after 8 min of O2/Ar plasma etching and the silica nano test tubes synthesized in this template......................................................................84 4-6 SEM images of the polymer-film after 10 min of O2/Ar plasma etching and the silica nano test tubes synthesized in this template......................................................................85 4-7 SEM images of the polymer-film surface and th e cross-section of the film after 12 min of O2/Ar plasma etching........................................................................................86 5-1 Schematic of silica deposition on alum ina surface by the conventional sol-gel and surface sol-gel methods....................................................................................................101 5-2 The structures of the silane s used for surface modifications...........................................102 5-3 Modification of the tube walls with fluorophore.............................................................103 5-4 TEM images of test tube samples obtained from a glass supported alumina template...104 5-5 SEM image of the cross-sect ion of the alumina template................................................105 5-6 TEM and SEM images of the tubes obt ained by the conventional sol-gel method.........106 5-7 Silica deposition with surface sol-ge l method without humidity control........................107 5-8 High resolution TEM image of the silica nano test tube with ~15 nm tube wall thickness...................................................................................................................... .....108 5-9 SEM images of the surface of silica depos ited template after 1 min Ar plasma and after briefly dissolving the alumina template...................................................................109 5-10 SEM and TEM images of silica nano test tubes with different lengths...........................110 5-11 Preparation and differential modifi cation of the silica nano test tubes............................111 5-12 Fluorescence microscopy images of Rhodamine B and Alexa Flour-488 labeled silica nano test tubes........................................................................................................112 5-13 Fluorescence spectra of Rabbit IgG and BS A modified glass slid es after exposure to a solution containing Alexa 488tagged anti-rabbit IgG.................................................113 5-14 Fluorescence images of two different breast carcinoma cell culture samples incubated with Alexa-488 labeled and an tibody-modified silica nano test tubes............114

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11 Abstract of Dissertation Pres ented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy TEMPLATE SYNTHESIZED MEMBRANES FOR ION TRANSPORT MODULATION AND SI LICA-BASED DELIVERY SYSTEMS By Fatih Buyukserin May 2007 Chair: Charles R. Martin Major Department: Chemistry The objective of this research is to prepare membrane platforms for potential applications in ion transport modulation and biomolecule de livery-device fabricati on. Template synthesis approach is used to obtain gold nanotube membra nes and silica nano test tubes that are the two main tools used in this dissertation. Chapter 1 provides an overview of the template synthesis method and its applications. The preparation of the track-etched polycarbonate and anodized aluminum oxide template membranes is pr ovided. Reviews of de position-modification techniques and plasma etching that are us ed in later chapters are then given. Chapter 2 describes an alternative method for electrom odulating ion tran sport through template synthesized Au nanotube membranes. Th is method entails attaching to the nanotubes a molecule that contains a redox-active ferrocene (Fc) substituent. Using these redox-active nanotubes, excess cationic charge can be placed on the membrane by oxidizing Fc to ferricinium (Fc+) by external voltage. It has been found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic permeate species is suppressed relative to when the Fc is in its reduced state. Hence, with these redox-active tubes, th e membrane can be gated between high and low cation-transporting states.

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12 In Chapter 3, the effect of constrained geometry on the decay properties of Fc+ is examined. The Fc+ decay properties of four membrane s with different pore sizes were investigated in an aqueous el ectrolyte and compared to the decay for commercial gold button electrode. After the membrane samples were mo dified with Fc-thiol monolayer, they were exposed to argon plasma that removes Fc-thiol on Au surface films leaving only the Fc-thiol lining the Au nanotube walls. The results suggest that the decay rate increases with increasing pore size and in all cases it is found to obey first order decay kinetics. Chapter 4 describes the fabrication of a uni que nanopore polymer template and its use for silica nano test tube production by sol-gel chemis try. Our objective with these test tubes was to impart multifunctionality through differential modification for de veloping a technology for cell specific biomolecule delivery. A plasma etch method, using a nanopore alumina film as the mask, was used to etch a replica of the alumina pore structure into the surface of a polymer film. The distance that the pores propa gate into the photoresist film is determined by the duration of the etching process. The pores in such plasma-etched nanopore phot oresists films were used as templates to prepare silica nano test t ubes with lengths as small as 380 nm. In Chapter 5, we have compared the prepar ation techniques for si lica nano test tube fabrication from alumina templates and then illu strated the response of br east carcinoma cells to test tubes that have be en biochemically modified. Defect-fre e uniform silica nano test tubes were obtained by the surface sol-gel method. These test tubes were differentially modified with a fluorophore on the inner surface and w ith an antibody (target or control) on the outer surface for the cell incubation studies. The fluor escence data suggest that the t ubes modified with the target antibody attaches much more readily to the cell me mbrane surfaces than the tubes modified with the control antibody. Chapter 6 summarizes the re sults and conclusions of this research.

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13 CHAPTER 1 INTRODUCTION AND BACKGROUND Introduction Nanoscience, the science of small particles of materials, is one of the most important research and development frontiers of modern science.1,2 The systems being studied in nanoscience are measured by nanomet er length scale and a nanometer is one billionth of a meter. Materials of nanoscopic dimensions are of fundamental interest si nce the properties of a material, such as optical, electronic and magnetic etc, can ch ange in this regime of transition between the bulk and molecular scale.3 These new material properties have led to potential technological applications in areas as diverse as mi croelectronics, coati ngs and biotechnology.2 For instance, one such application that is now in use involve s using gold nanoparticles as visual indicators in over-the-counter medical diagnostic kits.4 Nanomaterials can be fabricated through vari ous methods, ranging from chemical methods to lithographic techniques.5,6 The template method, pioneered by the Martin group, is a general approach for preparing nanomater ials that involves the synthesi s or deposition of the desired material within the cylindrical and monodisperse pores of a nanopore membrane or other solid surface.3,6 The applications of template synthesi zed nanomaterials composed of polymers, metals, semiconductors, and carbons have been applied in chemical separation, sensing, catalysis, electrochemistry, biom olecule extraction and delivery.3,4,6-8 Template synthesized gold nanotube membrane s and silica nano test tubes are the main scientific tools used in this research. This chapter provides background information on the preparation and application of thes e tools. An overview of templa te synthesis is given which is followed by past and recent important applicat ions related to the presented research. The preparation of the track-etc hed polycarbonate and anodized aluminum oxide template

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14 membranes is examined. Reviews of electrol ess gold deposition, sol-gel technology, silane chemistry and plasma etching that are used in la ter chapters are then gi ven. Finally, a brief overview of the delivery vehicles used in biomolecule transport is provided. Background Template Synthesis Many methods for the fabrication of nanopart icles have been de veloped, ranging from lithographic techniques to chemical methods.5,6 Our research group has pioneered a general method called template synthesis fo r the preparation of nanoparticles.3,6 This method entails synthesis or deposition of the de sired material within the cylindr ical and monodisperse pores of a nanopore membrane or other solid. We have used nanopore polycarbonate filters, prepared via the track-etch method,9 and nanopore alumina, prepared electrochemically from Al foil,10 as our template materials. A variety of other porous materials such as glass nanochannel arrays, zeolites, and polypeptide tubes can also be used as templates.11-13 Depending on the properties of the synthesized material and the chemistry of the pore wall, hollow nanotubes or solid nanowires can be obtained.6 Probably the most useful feature of the template synthesis is that it is extremely general with regard to the materials that can be prepared For example, we have used this technique to prepare nanotubes and nanowires composed of conductive polymers, metals, semiconductors, carbon, Li+-intercalation materials, and biom olecules such as DNA and protein.6,14,15 Methods used to synthesize such material s within the pores of the template membranes include electroless and electrochemical metal deposition, chemical and electrochemical polymerization, sol-gel deposition, chemical vapor deposition3,6 and layerby-layer deposition.14,15 In addition, template membranes contain cylindrical pores of uni form diameter which yields monodisperse nanocylinders of the desired material with c ontrollable dimensions. Finally, the resultant

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15 nanotubes or nanowires can be assembled into a variety of architectures The nanostructure can remain inside the pores of the template membra ne or they can be freed from the template membrane and collected as an ensemble of free nanoparticles.6 Applications in electrochemistry and sensing One very exciting application of the template synthesis is in the area of electrochemistry.16 The electroless deposition of chemistry allows us to routinely prepare ensembles of gold nanodisk electrodes with diameters as small as 10 nm.17 Long plating times (24 h) results in the deposition of Au nanowires into the pores. These nanoelectrode ensembles (NEE) can be used in ultra trace detection of electroac tive species. The signal-to-background (S/B) ratio at the NEE is orders of magnitude larger than at a macroel ectrode because the double-layer charging currents at the NEE are orders of magnitude lower than th ose at a macroelectrode of equivalent geometric area. This great increase in th e S/B ratio allows detecting ultra trace amounts of electroactive analytes.17 Nanostructured Li+ -intercalation material s that are synthesized by the template method have been used to design novel Li-ion battery electrodes.18 These nanostructured electrodes have improved rate capabilities compared to the thin film electrodes composed of the same material. 19-21 In addition, Sides and Mart in demonstrated that V2O5 nanofibers prepared by sol-gel synthesis in polymer templates show increased low-temperature performances compared to the micrometer-sized V2O5 fibers.22 There has been a significant amount of research in the ar ea of template synthesis of conductive polymers.6 Such nanofibers of conducting polymers have been shown to be more conductive than the bulk material.23,24 A detailed review of this topic can be found elsewhere in the literature.25 Cho et al. recently fabricated well defined nanotube arrays of poly(3,4ethylenedioxythiophene) (PEDOT) that can be used as an extremely fast electrochromic display

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16 (switching time less than 10 ms).26 The thin nature of the temp late synthesized nanotube walls offers a short diffusion distance and re sults in ultrafast switching rates. Finally, there is a great current interest in nanopores that have a conical pore shape and the correspondingly conical nanostructures synthesized via the template method within these pores.27 A number of applications utiliz ing the conical pore geometry ha ve been reported. For example, such conically shaped nanopores can be used as the sensing element for new types of small molecule,28 DNA,29,30 protein,31 and particle32 sensors. Conically shaped gold nanotubes deposited within such pores can also mimic the function of voltage gated ion channels.33 The details of the fabrication of th e pore geometry and the sensing m echanism for such platforms has been recently reviewed by Choi and Martin.8 Applications in control of ion transport and electromodulation Ensembles of Au nanotubes are obtained in the multipore track-etched polycarbonate (PC) templates when the electroless plating is done for shorter times. We discovered that by controlling the Au deposition time, we could prepare Au nanotubes with inside diameters that can be of molecular dimensions.34 We have demonstrated four transport-selectivity paradigms with these Au nanotube membranes (Au-NTM). Fi rst, because the nanot ubes can have inside diameters of molecular dimensions (<1 nm), thes e membranes can be used to cleanly separate small molecules on the basis of molecular size.34 The ability to control the tube diameter has also been used in the separation of a mixture of protein mol ecules with different sizes.35 Second, chemical transport selectivity can be introduced by chemisorbing thiols to the Au nanotube walls.36-38 Third, by using a thiol with both acidic and basic functional groups, ion transport across the Au-NTM can be modulated by controll ing the pH of the contacting solution phases.38 Finally, because the Au nanotubes are electronic ally conductive, excess charge can be placed on the nanotube walls by electrostatic ch arging in an electrolyte solution.39,40 This introduces ion-

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17 transport selectivity as well, and the Au-NTM can be electrom odulated between cation and anion transporting states. Lee and Martin described a unique way for the electromodulation of neutral molecules across Au-NTMs.41 This approach makes use of an anionic surfactant which, when a positive potential is applied to the Au-NTM, partitions into the nanotubes. Because of hydrophobic tail of the surfactant, this re nders the nanotubes interior hydrophobic, and the membrane preferentially extracts and transports hydrophobic molecules.36 The anionic surfactant can then be expelled from the nanotubes by applying a negative potentia l. This provides a route for reversibly electromodulating neutral molecule transport. We have recently been investigating an al ternative method for electromodulating transport in nanotube membranes.42 This method entails attaching to the nanotubes a molecule that contains a redox active ferrocene (Fc) substitu ent. With these redoxactive nanotubes, excess cationic charge can be placed on the memb rane by oxidizing Fc to ferricinium (Fc+) by external voltage. Buyukserin et al. has shown that cation transport through Au-NTMs can be electromodulated by controlling the extent of oxidation of Fcthiol monolayer attached to the Au surface.43 Miller and Martin demonstrated the contro l of surface charge, and thus electroosmotic flow (EOF) in poly (vin ylferrocene) coated ca rbon nanotube membranes.42 Reversible switching between the neutral and polycati onic forms of the redox-active polym er results in changes in the rate and direction of EOF. Applications with silica and biomolecule nanotubes The use of silica nanotubes, whether still embe dded within the template or freed from the template, has been shown in a variety of applications.44-46 The preparation method is generally sol-gel chemistry and the template material is commercial or hom e-made porous alumina membranes. We have shown that silica nanotubes synthesized within the pores of a home-made

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18 alumina template can be used to separa te two enantiomers of a chiral drug.44 An antibody that selectively binds one of the enantiomers of the dr ug was attached to the inner walls of the silica nanotubes. Such membranes selectively transport th e enantiomer that specifically binds to the antibody, relative to the enantiomer that has lower affinity for the antibody.44 Ensembles of silica nanotubes are obtained when such a membrane is dissolved. The nature of template synthesis a llows independent modification of the inner and outer surfaces of silica nanotubes.45,46 For example, silica nanotubes, that ha ve been modified with a fluorophore on the inside and a hydrophobic silane group on th e outside, have been shown to selectively partition into the organic phase in a mixture of aqueous/organic solvents.45 Furthermore, silica nanotubes that have been modifi ed with a certain antibody on both inner and outer tube surfaces can be used to selectively extract the enantiome r that specifically binds to the antibody from a racemic mixture of enantiomers.45 Novel nanostructures called nano test tubes have been recent ly introduced by the Martin group.47-49 Silica nano test tubes are prep ared by sol-gel synthesis of silica in the pores of an alumina template that remains attached to unde rlying aluminum metal. Unlike the previously mentioned nanotubes that are open on both ends, nano test tubes are closed on one end and open on the other. The use of test tubes as potential universal drug delivery vehicles was exploited where these nano test tubes could be filled with payload and then the open end corked with a chemically labile cap.48 For such studies, the tube dimens ions can have an important effect. Buyukserin et al. very recently fabricated a na nopore polymer template that can be used to prepare silica nano test tubes w ith lengths as small as 380 nm.49 Nanotubes composed of biomolecules such as DNA or protein have been fabricated by Hou et al.14,15 Layer-by-layer deposition has been app lied in both cases using a commercial

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19 alumina membrane as the template. Protein na notubes are obtained by a lternately exposing the template to a solution of the desi red protein and then to a soluti on of glutaraldehyde, which acts as crosslinking agent to hold the protein layers together. Biologically acti ve tubes are collected by removing the template and their activity depends on the number of layers deposited.15 The DNA nanotubes, however, have an outer skin of one or more diorganophosphonate/Zr(IV) layers, to provide structural integrity, surrounding an inner core of multiple double-stranded DNA layers held together by hybridization be tween the layers. The DNA components can be released from the nanotube by melting of the DNA duplexes comprising the nanotubes.14 Track-Etched Polycarbonate Membranes The use of nuclear tracks for the production of porous membranes was proposed almost immediately after the discovery of particle track etching in thin sheets of materials.50 Progress in this field was further accomplished through new particle sources, stud ies of new polymeric materials, search for new applications and de velopment of numerous methods of modification.51 There are two basic methods of producing latent tracks in the foils to be transformed into porous membranes.51 The first method is based on the irra diation with fragments from the fission of heavy nuclei such as californium or uranium.9,50 The main advantages of this technique are the relatively low cost, good stability of a particle flux in time, and non-parallel particle flux that enables the production of membranes with high porosity and low percent of overlapping pore channels. The contamination of the tracked foil with the radioactive product is a major limitation of the method which requires cooling of th e material for few months. In addition, angle distributions of pore channels and the range of fission fragments (membrane thickness) are limited.51 The second method involves the use of ion beams from accelerators.9,52-54 Thicker foils with higher pore densities and c ontrollable pore distributions can be obtained with higher energy

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20 non-radioactive ions. Although the cost of irradi ation is higher, the popularity of the ionaccelerator facilities has been increased in the past decade.51 After irradiation, the material is subjected to chemical etching that preferentially removes the latent ion tracks.51 As a result the latent ion track is transformed into a hollow channel. Pore size and pore shape is determined during this chem ical etching stage. The simplest description of pore geometry is based on two parameters: bulk etch rate and track etch ra te. The bulk etch rate depends on the material, on the etchant and on the temperature. The sensitivity of the material, irradiation conditions, post-irradi ation conditions and etching c onditions determines the track etch rate. Cylindrical, conical, funnel-like, and ci gar-like pore shapes can be made by controlling the bulk and the track etching rates.51 Track etched membranes can be prepared from various polymeric materials such as polycarbonate (PC),9 poly (ethylene terephthalate) (PET),51 polypropelene55 and polyimide.53,56 Track membranes are known as precise porous films with a very narrow pore size distribution. The pore diamet er can be from 10 nm to tens of micrometers. The pore density can vary from 1 to 1010 cm-2.51 PC has been used for track membrane production for over thirty years.9 The chemical etching of PC involves the rupture of chem ical bonds on both side s of the carbonate group, leading to the formation of carbonate ions (Figur e 1-1A). PC has a high sensitivity for irradiation which allows producing membranes with a pore diameter as small as ~ 10 nm without UV sensitization stage. When compared to PET, PC has a lower resistance to organic solvents and lower wettability.51 Poly (vinylpyrrolidone) (PVP) coating can be used to render the PC membranes hydrophilic.17 Track-etched PC filtration membranes are commercially available from a number of companies (e.g. Whatman, Os monics). Cylindrical pores are randomly distributed on the membrane surface in these commercial membranes and pore diameters ranging

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21 from 10 nm up to 20 m a nd pore densities between 104 and 109 pores.cm-2 are available (Figure 1-1B). Electroless plating of polymeric templates The Martin group has developed a new class of synthetic membranes that consist of a porous polymeric support that contai ns an ensemble of gold nanotubes.34,36-41 Monodisperse Au nanotubes that span the comple te thickness of the polymeric support can be prepared. The support used in this work is the track-etched polycarbonate filter described above. The gold nanotubes are prepared via electroless deposition of Au onto the pore walls; that is the pores act as templates for the nanotubes (Figure 1-2). Elect roless metal deposition, in general, involves the use of a chemical reducing agent to pl ate a metal from solution onto a surface.57 The key requirement of an electroless deposi tion bath is to arrange the chemis try such that the kinetics of homogeneous electron transfer from the reducing agent to the metal ion is slow. Otherwise, the metal ion would simply be reduced in the bulk so lution. A catalyst that accelerates the rate of metal ion reduction is then applie d to the surface to be coated.17 The electroless deposition method for the prep aration of gold nanotube membranes can be summarized as follows; the template membrane is first sensitized by immersion into a SnCl2 solution which results in deposition of Sn(II) ont o all the membrane surfaces (pore walls and membrane faces). Sn2+ adheres to the membrane because it is precoated with PVP during production to render the membranes hydrophilic. Amine and carboxyl groups of PVP are thought to act as molecular anchors58 that bond the Sn2+ to the membranes surfaces.59 The sensitized membrane is then im mersed into an aqueous basic AgNO3 solution. This causes a surface redox reaction in which the surface-bound Sn(II) is oxidized to Sn(IV) and the Ag+ is reduced to nanoscopic metallic Ag part icles on the membrane surface (Equation 1-1); some silver oxide is also obtained.60

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22 Sn(II)surf + 2Ag(I)aq Sn(IV)surf + 2Ag(0)surf (1-1) The subscripts surf and aq denote species ad sorbed to the membranes surfaces and species dissolved in solution, respectively. The membrane is then immersed into a commercial gold plating solution and a second surf ace redox reaction occurs, to yield nanoscopic Au nanoparticles on the surfaces. Au(I)aq + Ag(0)surf Au(0)surf + Ag(I) aq (1-2) These Au nanoparticles are excellent catalytic sites for the oxidation of formaldehyde and the concurrent reduction of Au. As a result, Au deposition wi ll begin at the pore walls, and Au tubes will be obtained within the pores. In a ddition, the faces of the membrane become coated with thin gold films without bloc king the mouths of the nanotubes.59 The Au nanotubes can have inside diameters of molecular dimensions (<1 nm),34 and inside diameter can be controlled at will.36 Various applications of these membranes are presented in the templa te synthesis section. Estimation of nanot ube inside diameter We use a gas-transport method to determine the effective inside diamet er of the templatesynthesized Au nanotubes.36 Briefly, the tube containing membrane was placed in a gaspermeation cell, and the upper and lower half-cells are evacuated. The upper half-cell will then be pressurized, typically to 20 psi, with He, and the pressure-time transient associated with the leakage of He through the tubes is measured using a pre ssure transducer in the lower half-cell. The pressure-time transient was converted to gas flux ( Q mol.s-1) which is related to the radius of the nanotubes ( r cm) via61,62 Q = (4/3) (2 /MRT)1/2 (nr3 P/l) (1-3) where P is the pressure difference across the membrane (dynes.cm-2), M is the molecular weight of the gas, R is the gas constant (erg K-1 mol-1), n is the number of nanotubes in the membrane sample, l is the membrane thickness (cm) and T is the temperature (K). This equation

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23 is based on the following assumptions: 1) that we know the number of tubes in the sample, 2) that the tubes have a constant diameter down th eir entire length, 3) that the mechanism of gastransport through the membrane is Knudsen diffusion in the nanotubes.59 The presence of cigarshaped pores and bottlenecked tubes causes slight deviations in the first two assumptions. For this reason, the calculated diameter s are sometimes referred to as effective inside diameters. The current plating conditions have shown to d ecrease the formation of these bottlenecked tubes and provide more uniform Au depositions.36 Gas transport through the membranes occurs via three different mechanisms; ordinary (viscous), Knudsen or surface diffusion.63 In addition, a solution-diffu sion model is adopted for describing the transport th rough the non-porous solid-phase Knudsen diffusion occurs when the mean-free path of the gas is much larger than the average pore radius in the membrane. In our case, equation 1-3 is predicated on Knudsen di ffusion in the nanotubes. The validity of this assumption is explored by comparing the diffusion of He/H2 and O2/N2 gas pairs through the Au nanotubes membranes.36 The ratios of the fluxes of the two gases in each pair across membranes of different pore sizes are compared. If the gas transport occurs via Knudsen diffusion, this ratio is the inverse square root of th e molecular weights for the two gases in each pair, and it does not change with changing pore si zes (i.e. plating times). It has been shown that the He/H2 pair perfectly applies the Kn udsen type gas diffusion36 and He gas was used in this work to determine the approximate inside di ameter of Au nanotubes. Anodic Alumina Templates Anodic aluminum oxide (AAO) films formed by the electrochemical oxidation of aluminum have been investigated and used in numerous products for more than 100 years.64-66 In recent years, nanoporous AAO with a hexagonal arrangement of monodisperse nanopores has become a popular template system for the sy nthesis of various functional nanostructures.44,45,67-69

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24 In addition, the use of these well -ordered structures as evapora tion or etching masks yields novel nanometric materials such as nanodots, nanotub es, nanowires, nanowells and nanopores made of metals, metaloxides and semiconductors.70-72 Nanopore arrays with interpore spacing ranging from 50 to 400 nm, pore diameter from 10 to 200 nm, membrane thickness from 0.1 to 200 m, and pore density as high as 1012 pore.cm-2 can be prepared.72-74 Alumina membranes are commercially available as 60 m-thick filtra tion membranes with pores of nominally 20, 100 and 200 nm diameters from Whatma n International, Maidstone, E ngland. Generally the pores of commercial membranes are not uniform in size or shape (Figure 1-3A). Due to these limited and non-uniform membrane parameters, we prepare th e alumina membranes in-house (Figure 1-3B). High purity aluminum metal (99.999%) is used in order to prepare alumina films with highly monodisperse cylindrical pores. This metal is first mechanically polished with sand paper (600 grit) and then electropolished at 15 V in a solution that is 95 wt% H3PO4 and 5 wt % H2SO4 with 20 g/L in CrO3 which prevents pitting. Using smooth electropolished aluminum surfaces is necessary for obtaining ordered hexagonal structures.72 The aluminum is the anode, a Pb plate is the cathode and the voltage is supplied by a variable power supply. The temperature of the electrolyte is kept around 70 C and the polishing is done for peri ods of 5 minutes for at least 2 times on both surfaces for a mirror-like finish. C oncentrated acid solution at high temperature is used for immediate dissolution of alumina.75 Following the electropolishing, the Al foil is subjected to a two step anodization pr ocess developed by Masuda and Fukuda.76 Two-step anodization method Traditionally, the ordered pore arrangements are formed under some specific anodizing conditions after a long anodization time, and as a result, they can only be observed on the bottom part of the films.77 Masuda and Fukuda first showed that straight ordered nanoholes could be formed in a thin membrane of porous alumina by striping away the thick oxides obtained from

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25 the first long anodization and subseque ntly anodizing it for a short time.76 The first long anodization allows sufficient time for self-org anization and homogenization of pore size.75 Once it is removed, an indention or pit is left in the underlying Al substr ate corresponding to each pore. The second anodization at the same voltage and in the same electrolyte results in pore nucleation in these pits that ar e already highly ordered and monodi sperse; thus the alumina film grows as patterned.76 Mechanical imprinting,78 electron-beam79 and focused-ion-beam lithographic methods80 have also been used to create nanosized indentations on the Al surface to precisely control the pore growth process.81 Densely packed ordered hexagonal pore structure, has been repor ted in oxalic, sulfuric and phosphoric acid solutions.76,82-84 We have used 5 wt % aqueous oxalic acid at ~ 1 C under 50 V in both the first and the second a nodization steps. The cathode is a cy lindrical stainless steel tube that supports homogenous ion flow to both surf aces of the aluminum and the solution is vigorously stirred. The solution temp erature is kept between 0 and 4 C for low reaction rates to prevent a runaway reaction a nd to keep Al in contact. The freshly electropolished Al foil is rinsed with purified water and then anodized for ~ 12 h. This first step produces a precursor film which is then dissolved in an aqueous solution that was 0.2 M in CrO3 and 0.4 M in H3PO4 at 80 C. The same conditions were applied to this textured Al substrate fo r different anodization times for the s econd step, and the growth rate we obtained was ~ 12 min anodization per 1 m alumina film thickness. The size of the pores to be grown is dependent on the applied potential and on the type of acid electrol yte used. In general, smaller pores require lower voltages and highl y conductive electrolytes (e.g., sulfuric acid) where as lower conductivity electrolytes (e .g., oxalic acid) are used for larger pores.85 In

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26 addition, immersing the resultan t alumina film in dilute H3PO4 solutions can also be used to tailor the pore dimension as it slowly reacts with alumina film and opens the pore diameter. Membrane detachment After the second-step anodizat ion, the nanopore alumina can be used as a template film while it is still attached to the underlying Al meta l that gives mechanical support to the film. (See Chapter 5.) Generally the alumina is separated from the Al base, however, and further processed into a freestanding membrane of nanopores that is open on the top and bottom and may be used as a base template stencil or mask for fabric ating a variety of highly ordered nanostructures.72 There are three reported ways to separate the alumina film.71,73 Dissolving Al in HgCl2 solution, alumina film separation by voltage reduction and coating an organi c compound layer on the surface of alumina to protect the or iginal morphology from erosive CuCl2-based aluminum removal.86 The first two methods will be discussed here. The simplest way of separating al umina is to dissolve Al in HgCl2. Generally, thin Al foils are most appropriate for dissolv ing, and the solvent does not dama ge alumina. Since there is a nonporous barrier alumina layer closest to the me tal surface, dissolving aluminum results in films that are closed on one end and open on th e other. The resultant film can be further chemically etched to obtain films with pores that are open on both si des. Hazardous Hg is produced during Al dissolution, and one foil is cons umed to prepare one alumina film. The use of progressive reduction in the a nodizing voltage to create a perf oration of the barrier layer and to achieve separation of alumina film fr om Al is described by Furneaux et al.73 When the film reaches the desired thickness, the voltage is reduced to about 70 % of its original value. Since the pore size and the film thickness are dependent on the applied voltage, th e pores at the barrier layer branch to smaller sizes and the barrier layer becomes thinner. After many voltage

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27 reduction cycles, the film/metal composite is immersed into an etchant solution. This quickly dissolves the thin barrier laye r and the alumina is detached. In our case, total reduction pr ocess takes about 1 h, the fina l voltage is 15 V and the etchant is 10 wt % H3PO4. The resultant alumina film has two distinct faces; the barrier side and the solution side. The barrier side has small bran ched pores that can be widened by an acid or base etchant to have uniform pores. In th e Martin group, both commercial and home-grown alumina membranes are extensively used as temp lates and etching masks for the preparation of various functional nanos tructured materials. A detailed discussion is presented under the template synthesis section. Sol-Gel Technology Interest in the sol-gel processi ng of inorganic ceramic and gla ss materials began as early as the mid-1800s with Ebelmanls87 and Graham's88 studies on silica gels. The motivation for solgel processing is primarily the potentially higher purity and homogeneity and the lower processing temperatures associated with sol-ge ls compared with traditional glass melting or ceramic powder methods.89 In addition, the technique can be used to obtain homogeneous multicomponent systems by mixing precursor soluti ons; this allows for easy chemical doping of the materials prepared. Finally, the rheological properties of the sol and the gel can be utilized in processing the material, for example, by dip coa ting of thin films, spinning of fibers, etc.90,91 In sol-gel synthesis a soluble precursor mol ecule is hydrolyzed to form a dispersion of colloidal particles (the sol). Further reaction causes bonds to form between the sol particles resulting in an infinite netw ork of particles (the gel).91 The gel is then typically heated to yield the desired material.92 Organometallic compounds are used as precursor to form the colloids, and in the case of glass, alkoxysilane precur sors such as tetramethoxysilane (TMOS) and tetraethoxysilane (TEOS) are most widely used.93,94 These alkoxysilanes read ily hydrolyze in the

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28 presence of water to form silanols (Equation 1-4). Further polycondensation reactions occur between these silanols with other silanols (Equa tion 1-5, water condensation) and alkoxysilanes (Equation 1-6, alcohol condensation).95,96 R3 Si-O-R + H2O R3 Si-OH +R-OH (1-4) R3 Si-OH + HO-SiR3 R3 Si-O-SiR3 + H2O (1-5) R3 Si-OH + RO-SiR3 R3 Si-O-SiR3 + R-OH (1-6) Simultaneous hydrolysis and polycondensati on of alkoxysilane precu rsors with two or more functional groups form an interconnected 3-D silica gel network. Many factors influence the kinetics of hydrolysis and condensation, and the systems ar e considerably complex as different species are present in the solution.89 In addition, hydrolysis and condensation occur simultaneously. Some important variables are temperature, nature and concentration of electrolyte (acid, base), nature of the solvent and type of alkoxide precursor. Increasing temperature and water amount increa ses the rate of gelation. Acid a nd base catalysts can be used for rapid and complete hydrolysis so either high or low pH extremes will speed the reaction. The nature of solvent influences the reaction rates; for example, 20 times faster rate constants were found in acetonitrile as opposed to formamide.97 Finally, the reaction ra te decreases as the alkoxide group gets longer and bulkier.98 Hypercriticial or ambient conditions are used to convert gel into silica. When the liquid (resultant alcohols or water) is removed as a gas phase from the interconne cted solid gel network under hypercritical conditions (critical-point dryi ng), the network does not collapse and a low density aerogel is produced. If the liquid is re moved at or near ambient pressure by thermal evaporation, shrinkage occurs and the monolith is called a xerogel.89 Materials with various shapes and sizes can be obtaine d through molding or dip-coating of the sol since it is a liquid

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29 form. When a template is immersed in the so l through dip-coating; a gel layer forms at the interface of the template. This layer can be dried and converted silica that replicates the surface topology of the template.95 Template synthesized TiO2, ZnO, WO3, MnO2, Co3O4,V2O5 and SiO2 nanotubes21,91,99,100 can be prepared with the sol-gel method. Surface Sol-Gel Method Precise control over the th ickness and morphology of nanot ubes synthesized with the conventional sol-gel technique can be challenging.101 More reliable control over the quality of planar thin films can be achieved by layer-bylayer deposition techniques, where colloidal particles102,103 or molecular precursors104-106 are successively adsorbed as a layer at a time onto the growing surface. The latter is called surface sol-gel (SSG) me thod and it involves repeats of two-step deposition cycles. In this case, the adsorption of a molecular precursor and the hydrolysis steps (for oxide film growth) are separated by a post-adsorption wash. The washing step desorbs weakly bound molecules that form additional layers.104 The SSG technique ideally can limit each deposition cycle to a single monolayer ; however, in practice, thicker layers have been found for planar oxide SSG films.104,106 Nevertheless, SSG allows very fine control over film thickness because a nanometer or sub-nano meter thick layer is grown in each two-step adsorption/hydrolysis cycle.101 Mallouk and coworkers recently reported the synthesis of silica nanotubes in anodic aluminum oxide membranes using the SSG technique where they have achieved a sub-nanometer control over the tube thickness.101 Furthermore, when coated on metal nanowires, this silica layer can be a high-quality dielectric oxide coating. Fo r this thin silica layer, they have used SiCl4 as the precursor and CCl4 as the solvent/washing solution. See Chapter 5 for more details on SSG based silica nanotubes synthesis. Th e same group has also demonstrated applicability of the first

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30 layer-by layer technique to membrane substrates by preparing uniform an d smooth free-standing semiconductor/polymer nanotubes.107 Silane Chemistry The organofunctional silanes were first introdu ced over 50 years ago as coupling agents for fiberglass and have subsequently proved to be us eful in various fields such as chromatography, catalysis and polymers applications.108-110 Organosilanes form stable covalent bonds with siliceous materials (e.g., silicates, aluminates borates) and various metal oxides. Thus, silanization provides a simple method for tailoring the surface chemistries of such materials. The general formula for an organosilane (RnSiX(4-n)) indicates two classe s of functionality.108 X is a hydrolyzable group typically halogen, alkoxy, acyloxy, or amine. After hydrolysis, a reactive silanol group is formed, which can condense with other silanol groups, for example, those on the surface of siliceous materials. The R group is a nonhydrolyzable organic radical that may possess a functionally that impart s desired characteristics.108 Attachment of proteins, fluorophores, genetic material etc. can be done us ing this R group as reactive handles.44,45 When a monolayer of surface modification is desired, silanes with one hydrolyzable group are used. With a single reactive group, these mol ecules can either bind to the surface or dimerize and the dimers are removed by successive washing steps. Most of the widely used organosilanes have one organic substituent.108,109 There are four steps in the r eaction of these silanes and they are analogous to the steps in sol-gel chemistry. First, hydrolysis of the three labile groups occurs. Condensation of oligomers follows. Th e OH groups of the subs trate then hydrogen bond with the oligomers. Finally, a covalent linkage is formed with the substrate by the loss of water through drying or curing. Water for hydrolysis may come from several sources. Aqueous alcoholic silane solutions that are made acidic wi th acetic acid are common ly used to initiate the

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31 formation of silanols.110 Water can also be present on the s ubstrate surface or it may come from atmosphere. The degree of polymerization of the silanes is determined by the amount of water available and the organic substituent. Th e concentration of the siloxane solution correlates with the thickness of the polysiloxane layer. It has been calculated that deposition from a 0.2% silane solution onto glass could result in eight molecular layers. These multi-layers could be either interconnected through a loose networ k structure, or intermixed, or both, and are in fact formed by most deposition techniques.108 There is a certain amount of re versibility during the formation of covalent bonds to the surface. As water is removed by evacuation for 2 to 6 hours or by heating to 120 for 30 to 90 minutes, bonds may fo rm, break and reform to relieve the internal stress.108 Silanes with four hydrolyzable groups provide a model for substrate reactivity and can be utilized in surface modifications. SiCl4, for example, is commercially important since it can be hydrolyzed in the vapor phase to form amorphous fused silica.108 Organic aprotic solvents can be used for surface treatment of chlorosilanes. Trea tment from dry solvent tends to deposit a more nearly monomolecular layer of silane than can be obtained from water.110 Chlorosilanes react with alcohols to form alkoxysilanes which under go most of the reactions of chlorosilanes. Alkoxysilanes are more convenient reagents than tetr ahalosilanes since they do not generate acid on hydrolysis and are generally less reactive.108 TEOS and TMOS are common reagents used in sol-gel based material synthesis th at have four alkoxy substituents.93,94 Plasma-Assisted Dry Etching The most important subtractive processes enc ountered in miniaturization science are wet and dry etching, focused ion-b eam milling, laser machining, ultrasonic drilling, electrical discharge machining, and traditional precision machining.111 Dry etching involves a family of

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32 methods by which a solid surface is etched in the gas phase, physically by ion bombardment, chemically by a chemical reaction through a reac tive species at the surf ace, or by combined physical and chemical mechanisms. Plasma-assist ed dry etching is categorized according to specific setup as either glow discharge (substrat e and plasma are located in the same vacuum chamber) or ion beam (substrate and plasma are in separate chambers).111 In physical etching, momentum transfer occurs between energetic ions (e.g., Ar+) and the substrate surface. Although the selectivity is poor, directional etching patt erns (anisotropic) are obtained with this method. Some type of chemical reaction takes place in the chemical etching method through which faster and selective etchin g is achieved, but the etched features are isotropic. The most important dry etching technique is the reactive ion etching (RIE).111 It combines physical and chemical etching mech anisms and enables profile control due to synergistic combination of physical sputtering with the chemical ac tivity of reactive species with a high etch rate and high selectivity. A plasma is an area of high energy electric or magnetic field that rapidly dissociates a suitable feed gas to form neutrals, energeti c ions, photons, electrons and highly reactive radicals.111 The simplest plasma reactor consists of opposed parallel-pla te electrodes in a chamber maintainable at low pressures. In ar gon plasma, electrical breakdown of argon gas in this reactor will occur when electrons, accelerate d in the existing electrical field, transfer an amount of kinetic energy greater than the argon ionizat ion potential to the argon neutrals. These energetic collisions generate a positive ion and a second free electron for each successful strike. Both free electrons reenergize, cr eating an avalanche of electrons and ions that results in a gas breakdown emitting a characteristic glow (blue, in the case of argon). In an RF-generated plasma, a radio-frequency voltage applied betw een two electrodes cause s free electrons to

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33 oscillate and collide with gas molecules, leadi ng to a sustainable plasma. Unlike the dc plasma, RF plasma allows etching of dielectrics as well as metals and it sustai ns the plasma at lower potentials.111 There is a wide range of applications for plasma-assisted dry etching from integrated circuit design and micro/nano machining111 to nanobatteries,18 chemical sensors70 and optical lenses.112 In this dissertation we have used physical etching to remove Ferrocene-thiol monolayers from the gold membrane surfaces in Chapter 3, and chemical/physical etching to selectively remove a polymer film to fa bricate silica nanostructures in Chapter 4. Biomolecule Delivery with Nanoparticles and Viruses The use of nanomaterials in biomolecule de livery has been shown to present various advantages such as increased efficacy,113 protection of drugs114 or genetic material115,116 from potential environmental damage and reduced drug toxicity.117 Spherical nanoparticles are almost always used because these shapes are easier to ma ke and can be synthesized from a diverse range of materials, such as liposomes,118,119 polymers,120,121 dendrimers122 and various inorganic compounds.46,115,123 Liposomes are spherical colloidal particles in which the internal aqueous cavity is surrounded by a self-assembled lipid membrane. Due to thei r size, biocom patibility and biodegradability, liposome are very promisi ng systems for biodelivery applications.118 The nature of the liposomes and their features are directly related to th e preparation method, the phospholipid composition and the capability of bi nding other chemical species. Mixtures of egg phosphatydilcholine (PC) are primarily used becau se of their low cost and neutral charge although other neutral phospholipids are al so used, such as sphingomyelin and phosphatidylethanolamine. Although liposomes c ould be formed spontaneously upon hydration of lipids, they do not generally have a thermodyna mically stable structure; so that external

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34 energy, such as sonication, ex trusion or homogenization, is usually required to produce liposomes.124 They have been widely used for both drug delivery120,121,125 and gene transfection118,120,126 after their surface is altered by adding hydrophilic substituents, such as poly(ethylene glycol) (PEG).120 This reduces the liposome uptak e by reticuloendothelial system (RES), thereby prolonging their circulation time.127 The main drawback for the liposome based delivery applications is the stability (either re leasing the biomaterial t oo quickly or entrapping too strongly).121 Polymeric micelles are self-assembling colloi dal aggregates of bl ock copolymers which occur when the concentration reaches the crucial micelle concentration.121 The copolymer involves a hydrophilic and a hydrophobic compone nt where in most cases the hydrophilic component is poly(ethylene oxide).128 There are two principal methods for the preparation of block copolymer micelles, the direct dissoluti on method and the dialys is method. The direct dissolution method simply involve s adding the copolymer to water or buffer solution where as dialysis is used for copolymers with limited water solubility.128,129 In an aqueous environment, the hydrophobic blocks of the copolymer forms the core and the hydrophilic blocks form the corona. These micelles are the most common vehicles for drug delivery130-132 where the lipophilic drug is incorporated in the microenvironment of a hydrophobic micelle core. Another polymer type used for such studies is dendrim ers. Dendrimers are self-assembling synthetic branched polymers with exquisite ly tunable nanoscale dimensions133 and their application in drug delivery134 and targeting135 has been recently investigate d. Their potential for gene delivery has also been examined where increased DNA payloads and decreased cell toxicity were observed with these dendrimer based delivery systems.136,137 Despite various advantages,

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35 polymeric delivery systems can present challe nges for characterization and relatively low payload capacities.121 Viral systems with highly evolved and sp ecialized components are by far the most effective means of DNA delivery, achieving high efficiencies (usually > 90%) for both delivery and expression.126 Most of the recent clinical protocols involving gene therapy use recombinant virus-based vectors for DNA deliv ery. However no definitive evidence has been presented for the clinical effectivene ss of any gene therapy protocol ex cept for a few anecdotal reports of success in individual patients.138 The impotence of current methodology is attributable to the limitations of viral mediated delivery, including toxicity, restricted ta rgeting of specific cell types, limited DNA carrying capacity, production and packaging problems, recombination, and high cost.139,140 These systems are also likely to cause unexpected cytotoxicity and immunogenicity which hamper their routin e use in basic research laboratories.116 For these reasons, nonviral synthetic DNA delivery systems have become increasingly desirable in both basic research laboratories and clinical settings.126 The application of some inorganic nanopart icles for biomolecule delivery has been recently shown; gold and sili ca nanoparticles, for example have been employed in DNA delivery.115,141 Unlike nanoparticles or nanorods, nanot ubes have a unique hollow structure which allows the modification of their inner su rface and filling with specific biomolecules. However, the applications of nanotubes as biomolecule carriers ar e still very rare.116,142 The template method developed in Martin group allows independent modification of inner and outer surfaces of the nanotubes through which multifunctiona l tubes with controllable dimensions can be obtained.46 Multifunctionality is highly required for modern biomedical applications125 and

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36 these differentially modified tubes are potential novel tools for such studies. See Chapter 5 for more details on differentially modifi ed nanotubes and nano test tubes. Chapter Summaries Chapter 2 describes an alternative method for electrom odulating ion tran sport through template synthesized Au nanotube membranes. Th is method entails attaching to the nanotubes a molecule that contains a redox-active ferro cene (Fc) substituen t. Electrochemical characterization of the Fc-thiol modified Au nanotube membranes is first examined. Surface confined cyclic voltammograms were obtained and the stability of these voltammograms was found to depend on the redox state of Fc and th e electrolyte type. Us ing these redox-active nanotubes, excess cationic charge can be placed on the membrane by oxidizing Fc to ferricinium (Fc+) by external voltage. It has been found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic permeate species is suppressed relative to when the Fc is in its reduced state. Hence, with these redox-active tubes, th e membrane can be gated between high and low cation-transporting states. Chapter 3 examines the effect of constrai ned geometry on the decay properties of Fc+. Previous studies have shown that the Fc+ decomposition is a first order decay in bulk aqueous solutions. The Fc+ decay properties of four membrane s with different pore sizes were investigated in an aqueous el ectrolyte and compared to the decay for commercial gold button electrode. After the membrane samples were mo dified with Fc-thiol monolayer, they were exposed to argon plasma that removes Fc-thiol on Au surface films leaving only the Fc-thiol lining the Au nanotube walls. The results suggest that the decay rate in creases with increasing pore size and in all cases it is found to obey firs t order decay kinetics. Furthermore, the decay pattern resembles a surface-like decay as the pore size of the membrane increases. These results

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37 were attributed to the varying hydrophobic characte r of Fc-thiol monolayer and availability of counterions inside the pores as the pore dimensions change. In Chapter 4, the fabrication of a unique na nopore polymer template and its use for silica nano test tube production is described. Our obj ective with these test tubes is to develop a technology for cell specific biomolecule delive ry. A plasma etch method, using a nanopore alumina film as the mask, was used to etch a replica of the alumina pore structure into the surface of a polymer film. The distance that th e pores propagate into the photoresist film is determined by the duration of the etching proce ss. Hence, by controlling the etch time, we effectively control the thickness of the nanopore layer etched into the surface of the photoresist. The pores in such plasma-etched nanopore photoresis ts films were used as templates to prepare silica nano test tubes via sol-gel chemistry. As expected the length of the test tubes is determined by the thickness of the porous part of the photoresist film. Test tubes with lengths of 380 nm were obtained, shorter than any of the nano test tubes previous ly reported where the alumina film was used as the template. Chapter 5 compares the preparation techniques for uniform silica nano te st tube fabrication and then illustrates th e response of breast carcinoma cells to test tubes that have been biochemically modified. Defective test tubes we re obtained with the conventional sol-gel method and it was attributed to the small changes in the viscosity of the gel. La yer-by-layer addition of silica with the surface sol-gel me thod allowed preparation of def ect-free uniform silica nano test tubes. We have differentially modified these test tubes for the cell studies. Before the template was removed, the inner tube surfaces were labele d with a fluorophore. The liberated fluorescent tubes were then modified with a target or a control antibody and then incubated with breast carcinoma cells. The preliminary results suggest that the tubes modified with target antibody

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38 attaches much more readily to the cell membrane surfaces than the tubes modified with control antibody. The results and conclusions of this dissertation are summarized in Chapter 6.

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39 Figure 1-1. A) The chemical structure of polycarbonate. B) Scanni ng Electron Micrograph (SEM) of the surface of a commercial track-etched polycarbonate membrane. C CH3CH3]nO C O O[ A B

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40 Stannous Chloride solutionSilver Nitrate SolutionGold plating Solution Polycarbonate membrane, Top View Polycarbonate membrane, Cross-sectional View Gold nanotube membrane, Cross-sectional View Gold nanotube membrane, Top View Stannous Chloride solutionSilver Nitrate SolutionGold plating Solution Polycarbonate membrane, Top View Polycarbonate membrane, Cross-sectional View Gold nanotube membrane, Cross-sectional View Gold nanotube membrane, Top View Figure 1-2. Top and cross-secti onal view of PC membrane befo re & after the gold plating.

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41 Figure 1-3. SEM images of the surface of anodized aluminum oxide (alumina) membranes. A) Commercially available alumina membra ne. B) Home-grown alumina membrane. A B

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42 CHAPTER 2 ELECTROACTIVE NANOTUBES MEMB RANES AND REDOX-GATING Introduction We have developed a new class of syntheti c membranes that contains monodisperse Au nanotubes with inside diameters that can be of molecular dimensions (<1 nm).34,36-41 The Au nanotubes span the complete thickness of the me mbrane and can act as conduits for molecule and ion transport between solutions placed on either side of the membrane. We have demonstrated four transport-sele ctivity paradigms with these Au nanotube membranes. First, because the nanotubes can have inside diameter s of molecular dimensions (<1 nm), these membranes can be used to cleanly separate sm all molecules on the basis of molecular size.34 Second, chemical transport selectivity can be introduced by chemisorbing thiols to the Au nanotube walls.36-38 Third, by using a thiol with both acidic and basic functional groups, ion transport across the Au nanotube membrane can be modulated by controlling the pH of the contacting solution phases.38 Finally, because the Au nanot ubes are electronically conductive, excess charge can be placed on the nanotube wall s by electrostatic charging in an electrolyte solution.39-41 This introduces ion-transport selectivity as well, and the Au nanotube membranes can be electromodulated between cati on and anion transporting states. We have recently been investigating an al ternative method for electromodulating transport in nanotube membranes.42 This method entails attaching to the nanotubes a molecule that contains a redox-active ferrocene (Fc) substitu ent. With these redox-active nanotubes, excess cationic charge can be placed on the membrane by using the potential applied to the membrane to driving the fo llowing redox reaction:42,143-145 Fc Fc+ + e(2-1)

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43 We have found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic permeate species is suppressed relative to when th e Fc is in its reduced state. While similar results have been achieved using membra nes composed of re dox-active conductive polymers,146-148 this paradigm for gating ion transport has not been demonstr ated for redox-active nanotube membranes. We describe the results of such redox-modulated transport experiments here. Experimental Materials Polycarbonate filtration membranes (6 m-thick, 30 nmand 50 nm-diameter pores, 6x108 pores cm-2) were obtained from Osmonics Inc. Commercial gold-pl ating solution (Oromerse SO Part B) was obtained fr om Technic Inc. Na2SO3, NaHCO3, NH4OH, HNO3, KCl, methanol and formaldehyde were obtained from Fish er and used as received. SnCl2, methyl viologen dichloride hydrate, and 1,5-na phthalene disulfonic acid disodi um salt hydrate were used as received from Aldrich, as were KClO4, AgNO3 and triflouoroacetic acid from Acros Organics, ethanol (absolute) from Aape r, and 11-ferrocenyl-1-undecaneth iol from Dojindo Chemicals. Purified water was obtained by passing house-di stilled water through a Millipore, Milli-Q system. Electroless Gold Deposition The electroless deposition or plating method de scribed previously was used to deposit gold nanotubes within the pores of th e nanopore polycarbonate membranes.59 In general terms, this entails depositing gold along the pore walls so that each pore becomes lined with a gold nanotube. Briefly, the template membrane was first immersed into methanol for five minutes and then immersed for 45 min into a solution that was 0.025 M in SnCl2 and 0.07 M in trifluoroacetic acid. This yields th e Sn-sensitized form of the membrane.17 The membrane was

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44 then immersed into an aqueous ammoniacal AgNO3 solution (0.029 M Ag+) for 7.5 minutes and then immersed in methanol for 5 minutes. The gold plating bath was prepared by mixing 0.5 ml of the commercial gold-plating solution with 20 mL of an aqueous solution that was 0.127 M in Na2SO3, 0.625 M in formaldehyde, and 0.025 M in NaHCO3. The bath pH was lowered to 10 by drop wise addition of 1 M H2SO4 prior to immersion of the membrane. During electroless deposition, the temperature of the bath was maintained at 4 oC. Membranes were placed in the gold-plating bath for different periods of time to obtain nanotubes of different inside diameters.36,149 The inside diameter of the nanotub e was determined using the gas-flux measurement described previously.36 Membrane Sample Preparation and Thiol Modification The electroless-plating method yields the Au nanotubes lining the pore walls as well as thin Au films covering both faces of the membrane.17 The Au films do not block the mouths of the nanotubes at the membrane faces and can be us ed to make electrical contact to all of the nanotubes in parallel.39 This was accomplished by applying a copper tape with a conductive adhesive (3M, #1181) to the outer edge of one Au surface film.17 The membrane sample was prepared by sandw iching the nanotube membrane between two pieces of electrically insulating plastic tape (3M Scotch brand no. 375). Each piece of tape had a 0.2 cm2-area hole punched through it, and the holes were aligned on either side of the membrane. This insulating tape also covered the conductive tape used to make el ectrical contact to the membrane. The end of the copper tape protruding from the membrane sample was used as the electrode lead for electrochemical experiments in which the membrane sample was the working electrode. Details of this elect rode fabrication method can be f ound elsewhere in the literature.17 The Au surface films and Au nanotube walls we re modified with the thiol 11-ferrocenyl-1undecanethiol, here after called Fc-thiol. This was accomplished by mounting the assembled

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45 membrane sample between the two halves of a U-tube permeation cell34,36,39 and filling both half-cells with a 2 mM solution of Fc-thiol di ssolved in ethanol. The membrane sample was exposed to this solution for 20 h, fo llowed by thorough washing with ethanol. For some membranes, the Fc-thiol on the Au surface films was removed by brief (30 sec) exposure to a mild Ar plasma. A Samco model RI E-1C reactive-ion etch system was used. The plasma conditions were as follows: 13.56 MHz, 50 W, 10 Pa Ar pressure, Ar flow rate =12 sccm. Electrochemical Experiments Electrochemical experiments were done with the membrane sample mounted in the Utube cell. Electrolyte solution was added to both half-cells, a nd the Au nanotube membrane was made the working electrode in a conventional thre e-electrode experiment. The counter electrode was a Pt wire and the reference was an Ag/AgC l electrode with 3 M NaCl. In the transport experiments one half-cell solution, the feed half -cell, contained the permeating species and the other half-cell received the perm eating species. The reference and counter electrodes were placed in the feed half-cell. A Solartron SI 1287 electrochemical interface module (Solartron Analytical, Hampshire, England) connected to a PC running CorrView and CorrWare software (Scribner Asc. Inc., NC) was used. Transport Experiments The same U-tube cell was used for the transport experiments. The permeating specie investigated was the di cation methylviologen (MV2+). The feed half-ce ll was charged with 20 mL of a 20 mM aqueous MV2+ solution, and the receiver half-cell was charged with 20 mL of purified water. The flux of MV2+ from the feed half-cell, th rough the membrane and into the receiver half-cell was obtaine d by continuously measuring the UV absorbance (at 260 nm) of the receiver half-cell solution. A flow-through Agilen t 8458 spectrophotometer was used.34,39,150

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46 The data were processed as plots of moles MV2+ transported vs. time. Straight line plots were obtained, and the flux of the permeati ng ion was calculated from the slope. Results and Discussion Electrochemistry of the Fc-Thiol Figure 2-1A shows a cyclic voltammogram for a Fc-thiol-modified Au nanotube membrane (nanotube inside diameter = 8 nm). The redox waves associated with the oxidation of the Fc to Fc+ and the re-reduction back to Fc are clearly seen.145,151-153 Figure 2-1B shows that the anodic peak current is linearly related to scan rate as would be expected for a surfaceconfined voltammogram.154 It is of interest to note, however, that there are in essence two different Au surfaces in these membranes The Au on the inside walls of th e nanotubes running through the membrane and the Au surface films on both faces of the membrane. If the number of moles of Fc obtained from the area under the anodic wave is divided by the tota l Au area (tube walls plus surface films), a coverage by Fc of 1.0x10-9 moles.cm-2 is obtained. This is about a factor of two larger than the value calculated from the footprint of the Fc molecule on an atomically flat Au surface.145,153 The higher value obtained experimentally here simply reflects the surface roughness of our electrolessly deposited gold. Figure 2-2 shows the effect of electr olyte on the stability of the Fc/Fc+ redox couple. When KCl was used, the voltammogram current d ecayed continuously with scan number (Figure 2-2A). As has been discussed previously153, this is due to nucl eophilic attack of Clon the Fe(III) center of Fc+. As shown by the analogous set of 30 cyclic voltammograms in Figure 22B, the redox chemistry is much more stable in 0.1 M KClO4.153 This is because ClO4 is a poorer nucleophile than Cl-. For long-term use, however, it is best to store the Fc-thiol-modified

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47 membrane in its reduced (Fc) state. If this is done in the KClO4 solution, Fc-thiol electrochemistry can be observed, unchanged, for periods of at least one week (Figure 2-3). Figure 2-2 also shows that the ox idation of Fc-thiol proceeds at more negative potentials in KClO4 than in KCl. Such effects have been observed previously for ferrocene-modified electrodes and have been attributed to the different extents to which the anions of the electrolyte form ion-pairs with Fc+.151,152 Fc+ is a lipophilic cation, present in a lipophilic monolayer film, and therefore ion pairs preferenti ally with the more lipophilic ClO4 -. This ion-pair interaction makes the oxidation thermodynamically easier in ClO4 vs. Cl-. The shift in the position of the Fc-thiol voltammetric wave with time in KCl (F igure 2-2A) has also been observed previously, although no explanation was offered.153 We suggest that as decomposition of the lipophilic cyclopentadienly ring occurs (wit h increasing scan number in KC l, Figure 2-2A) the monolayer film becomes less lipophili c, and this allows Clto have greater ion-pairing access to the remaining intact Fc+ groups. Electromodulated Transport Experiments A solution of the cationic permeating species MV2+ was placed on one side of the Fc-thiolmodified membrane, and the quantity of this sp ecies transported through the nanotubes and into the receiver solution on the opposit e side was measured as a func tion of time (Figure 2-4). During the time interval from 0 to ~1700 sec, a po tential of 0.7 V was applied to the membrane. At this potential the ferrocene is present as oxidized Fc+, yielding excess positive charge on the nanotube walls and membrane faces. This charge causes MV2+ to be electrostatically repelled from the membrane, yielding the low-flux state for MV2+ transport. Complete exclusion of MV2+ is not observed because at the 20 mM salt (MVCl2) concentration used in this experiment, the electrical double layer on the walls of the 10 nm-diameter nanotube does not completely fill the total nanotube volume. As we have discussed in detail previously,39,40 this means that there

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48 is a region in the center of the nanotube where MV2+ is not excluded, and transport occurs in the region. At 1800 sec a potential of 0 V was applied to the membrane At this potential the ferrocene on the nanotube walls, and membrane faces, is present as neutral Fc. Because there is now no excess positive charge on the membrane, MV2+ is not repelled, and a higher MV2+ flux (relative to the short time data) is obtained (dat a points for line 2, Figure 2-4). The slopes of the straight-line segments in Figur e 2-4 provide the fluxes for MV2+ across the nanotube membrane. We define an electromodulation-transport cycle as a period when 0.7 V was applied (low flux state) followed by a period when 0 V was applied (hi gh flux state). This allows us to define an electromodulated-transport selectivity coefficient ( ) as the flux during the high-flux state (0 V) divided by the flux during the low-flux state (0.7 V). The larger the value of the greater is the electromodulated cati on-gating effect. Table 2-1 shows flux and values for various cycle numbers for membranes with 10 and 16 nm-diameter Au nanotubes. Considering the flux data first, we see as would be expected, that the fluxes in the membrane with the larger-d iameter nanotubes is higher. However, the selectivity for the membrane containing these larger diameter nanotubes is lower. Again, this is due to the fact that the el ectrical double layer that is responsible for repelling MV2+ fills a smaller fraction of the total nanotube volume for the larger diameter nanotube.39,40 The electromodulated selectivity coefficient, decreases with increasing cycle number (Table 2-1). Part of this decay in selectivity is due to the fact that the magnitude of the flux in the low-flux (Fc+) state increases with each successive cy cle. To understand the origins of this effect we obtained a cyclic voltammogram afte r each cycle, and from the area under the anodic wave obtained the moles of electroactive Fc rema ining in the membrane (Figure 2-5). We see

PAGE 49

49 that there is a steady drop in amount of electroactive Fc with cy cle number. While this may at first seem to contradict the data in Figure 23, the key difference is that in Figure 2-3 the ferrocene was left in the neutral Fc state between cycles, and in Figure 2-5 the Fc was held in the charged Fc+ for long periods (Figure 2-4) during each cycle. Because it is the Fc+ state that is susceptible to nucleophilic attack,155,156 electroactivity decays much more quickly in Figure 2-5 than in Figure 2-3. This steady drop in electroactive Fc in the membrane with cycle number (Figure 2-5) explains why the selectivity decays with cycle nu mber (Table 2-1). This is because it is the positively charged Fc+ groups that repel MV2+, and since the quantity of Fc+ decreases with cycle number, the selectivity decreases with cycle numbe r. The other factor causing the selectivity to decay with cycle number is that the magnitude of the flux in the high flux state decreases with cycle number (Table 2-1). This suggests that me mbrane fouling occurs. One possible source of membrane fouling is that the decomposition produc ts that result from nu cleophilic attack on the Fc+ causes partial occlusion of the nanotubes. Conclusions We have shown that cation transport through Au nanotube membranes can be electromodulated by controlling the extent of oxidation of a Fc-thiol attached to the Au surfaces. We have defined an electromodulation sele ctivity coefficient for cation transport, As would be expected, higher values are obtained for membranes containing smaller inside-diameter nanotubes. For the 10 nm-diameter nanotubes a maximum value of = 9.4 was obtained. It is possible to make smaller diameter nanotubes,34 and it would be of interest to see if correspondingly higher se lectivity coefficients could be obtained. Unfortunately, the electromodulated selectivity decreases with membra ne use because when the Fc is present in the Fc+ state it is susceptible to nuc leophilic attack and decompositi on. It is well-known that

PAGE 50

50 decamethyl-ferrocence is less susceptible to this degradation pathway,157,158 and for this reason would be a better choice for the nanot ube-bound electromodulating agent.

PAGE 51

51 Table 2-1. Flux and electromodulat ed selectivity coefficients ( ) for membranes containing 10-nm and 16-nm diameter nanotubes. Nanotube Diamater (nm) Cycle Number Low Flux nmole min-1cm2High Flux nmole min-1cm210 1 1.2 11 9.4 10 2 1.5 11 7.3 10 3 2.0 10 5.1 16 1 6.4 38 5.9 16 2 7.3 38 5.2 16 3 7.8 34 4.3 16 4 8.3 32 3.8 16 5 9.5 28 2.9

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52 0.00.20.40.60.8 -40 -20 0 20 40 Potential / V vs. Ag/AgClI / AA 0.00.20.40.60.8 -40 -20 0 20 40 Potential / V vs. Ag/AgClI / AA 0 10 20 30 40 50 60 020406080100120 Scan Rate/ mV s-1Anodic Peak Current/ AB 0 10 20 30 40 50 60 020406080100120 Scan Rate/ mV s-1Anodic Peak Current/ AB Figure 2-1. A) Cyclic voltammogram for a Fc-thiol-modified Au nanotube membrane with nanotube inside diameter = 8 nm. Scan rate = 70 mV s-1. B) Anodic peak current from such voltammograms vs. scan rate. The elec trolyte in both half-cells was 0.1 M KCl.

PAGE 53

53 0.00.20.40.60.8 -15 -10 -5 0 5 10 15 AI/ A 0.00.20.40.60.8 -15 -10 -5 0 5 10 15 AI/ A 0.00.20.40.60.8 -15 -10 -5 0 5 10 15 B Potential / V vs. Ag/AgClI / A Figure 2-2. Effect of electro lyte on the stability of the Fc+/Fc voltammogram. The potential was swept continuously throu gh the voltammetric waves for 30 scans at 20 mV s-1. The membrane contained nanotubes with inside di ameter of 26 nm. A) Electrolyte was 0.1 M KCl. The arrow points in the direc tion of increasing scan number (scan 1 to scan 30). B) Electrolyte was 0.1 M KClO4.

PAGE 54

54 0.00.20.40.60.8 -20 -10 0 10 20 I / APotential / V vs. Ag/AgCl Figure 2-3. Investigation of th e long term stability of the Fc-thiol layer. The Au nanotube membrane sample (nanotube inside diamet er = 10 nm) was mount ed in the U-tube cell with 0.1 M KClO4 in both half-cells, and volta mmograms were obtained after 2 days (solid black curve), 4 da ys (solid gray curve), and 6 days (dashed black curve) of storage unpotentiostated in th e reduced (Fc) state. The ha lf-cell solutions were not degassed and the U-tube cell was not protected from light.

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55 0 20 40 60 80 100 120 140 160 180 200 020004000600080001000012000 Time/ secNanomoles transported1 2 3 5 4 6 0 20 40 60 80 100 120 140 160 180 200 020004000600080001000012000 Time/ secNanomoles transported1 2 3 5 4 6 Figure 2-4. Plot of nanomoles of MV2+ transported across a nanotube membrane (nanotube inside diameter = 10 nm) vs. time. Data point s for lines 1, 3 and 5 were obtained with a potential of 0.7 V vs. Ag/AgCl applied to the membrane. Data points for lines 2, 4 and 6 were obtained with a potential of 0 V vs. Ag/AgCl applied to the membrane. The slopes of these straight lines ar e used calculate to the flux of MV2+. The feed solution was 20 mM in MV2+.

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56 0 0.2 0.4 0.6 0.8 012345 Cycle numberNanomoles of electroactive Fc 0 0.2 0.4 0.6 0.8 012345 Cycle numberNanomoles of electroactive Fc Figure 2-5. Moles of electroactive Fc vs. cycle number fo r a membrane containing 16 nm-diameter Au nanotubes.

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57 CHAPTER 3 KINETICS OF FERRICINIUM DECOMP OSITION CONFINED WITHIN GOLD NANOTUBESEFFECT OF THE NANOS CALE ENVIRONMENT ON KINETICS Introduction We have been investigating a general method for preparing nanomaterials called template synthesis.3,4,6 This method entails synthesis of the desi red material within the cylindrical and monodisperse pores of a nanopore membrane or othe r solid. Using this method, a new class of synthetic membrane was developed that cont ain monodisperse Au nanotubes with inside diameters that can be of molecular dimensions (<1 nm).34,36-39,41 The Au nanotubes span the complete thickness of the membrane and can ac t as conduits for molecule and ion transport between solutions placed on either side of th e membrane. We have been using these gold nanotube membranes to investigate how pore si ze, charge and chemistry affect transport selectivity in membranes. Of particular releva nce to the work reported here, ion and chemical transport selectivity can be su ccessfully introduced and modulated by chemisorbing thiols to the Au nanotube walls.36,38,41 We have recently reported an alternative me thod for electromodulating ion transport in Au nanotube membranes. This method entails chemis orbing to the Au nanotubes an alkyl thiol that contains a redox-active ferrocene (Fc) substituen t. With this membrane system the charge density on the nanotube walls can be electromod ulated Faradaically by using the potential applied to the Au nanotube membrane to contro l the position of equilibrium for the following redox reaction:42,143-145 Fc Fc+ + e(3-1) We have found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic permeate species is suppressed relative to when th e Fc is in its reduced state. However, the flux difference between these states is lost with memb rane use because when the Fc is present in the

PAGE 58

58 Fc+ state, it is susceptible to nuc leophilic attack and decomposition.153 The extent of Fc+ decomposition is directly related to the strength of the nucleophile155 and it is a fi rst order decay in aqueous solutions.159 In this chapter, we report the resu lts of nanotube pore size affect on Fc+ decomposition. For this purpose, it was necessary to remove the Fc-thiol on Au surface films leaving only the Fc-thiol lining the Au nanotube walls. This was accomplished by briefly (30 sec) exposing both faces of the membrane to an argon plasma (mild conditions). The behavior of four membranes with different pore sizes were investigated and co mpared to the decay in commercial gold button electrode. The results suggest that the decay rate increases with increasing pore size and in all cases it is found to obey first order decay kinetic s. Furthermore, the decay pattern resembles a surface-like decay as the pore size of the membrane increases. Experimental Materials Polycarbonate filtration membranes (30 nm -, 50 nm-, 200 nmand 600 nmdiameter pores) were obtained from Osmoni cs Inc. Commercial gold-plating solution (Oromerse SO Part B) was obtained from Technic Inc. Na2SO3, NaHCO3, NH4OH, HNO3, methanol and formaldehyde were obtained from Fish er and used as received. SnCl2 was used as received from Aldrich, as were KClO4, AgNO3 and triflouoroacetic acid fr om Acros Organics, ethanol (absolute) from Aaper, and 11-fe rrocenyl-1-undecanethiol from Dojindo Chemicals. Purified water was obtained by passing house-distilled wa ter through a Millipore, Milli-Q system. Electroless Gold Deposition The electroless deposition or plating method de scribed previously was used to deposit gold nanotubes within the pores of th e nanopore polycarbonate membranes.59 In general terms, this entails depositing gold along the pore walls so that each pore becomes lined with a gold

PAGE 59

59 nanotube. Briefly, the template membrane was first immersed into methanol for five minutes and then immersed for 45 min into a solution that was 0.025 M in SnCl2 and 0.07 M in trifluoroacetic acid. This yields the Sn-sensitized form of the membrane.17 The membrane was then immersed into an aqueous ammoniacal AgNO3 solution (0.029 M Ag+) for 7.5 minutes and then immersed in methanol for 5 minutes. The gold plating bath was prepared by mixing 0.5 ml of the commercial gold-plating solution with 20 mL of an aqueous solution that was 0.127 M in Na2SO3, 0.625 M in formaldehyde, and 0.025 M in NaHCO3. The bath pH was lowered to 10 by drop wise addition of 1 M H2SO4 prior to immersion of the membrane. During electroless deposition, the temperature of the bath was maintained at 4 C. Membranes were placed in the gold-plating bath for different periods of time to obtain nanotubes of different inside diameters.36,149 The inside diameter of the nanotube was determined using the gas-flux measurement described previously36 where the pore diameter was < 50 nm. For bigger pores, elec tron micrographs of the pores obtained via Hitachi S4000 FESEM were used to calculate the pore diameter Gold nanotube membranes with pore diameters 10 2.0, 28 2.6, 65 7.5, and 284 20 nm were used in this work. Membrane Sample Preparation and Thiol Modification The electroless-plating method yields the Au nanotubes lining the pore walls as well as thin Au films covering both faces of the membrane.17 The Au films do not block the mouths of the nanotubes at the membrane faces and can be us ed to make electrical contact to all of the nanotubes in parallel.39 This was accomplished by applyi ng a copper tape with a conductive adhesive (3M, #1181) to the outer edge of one Au surface film.17 The membrane sample was prepared by sandw iching the nanotube membrane between two pieces of electrically insulating plastic tape (3M Scotch brand no. 375). Each piece of tape had a 0.2 cm2-area hole punched through it, and the holes were aligned on either side of the membrane.

PAGE 60

60 This insulating tape also covered the conductive tape used to make el ectrical contact to the membrane. The end of the copper tape protruding from the membrane sample was used as the electrode lead for electrochemical experiments in which the membrane sample was the working electrode. Details of this elect rode fabrication method are descri bed elsewhere in the literature.17 The Au surface films and Au nanotube walls we re modified with the thiol 11-ferrocenyl-1undecanethiol, here after called Fc-thiol. This was accomplished by mounting the assembled membrane sample between the two halves of a U-tube permeation cell34,36,39 and filling both half-cells with a 2 mM solution of Fc-thiol di ssolved in ethanol. The membrane sample was exposed to this solution for 20 h, followed by thorough washing with ethanol. A commercial gold button electrode (Bioanalytic al Systems, Inc. IN) was modified under the same conditions after being polished with alumina nanoparticles. Surface Thiol Removal The Fc-thiol modified gold nanotube membra ne sample was placed into the vacuum chamber of a reactive-ion etchi ng system (Samco model RIE-1C). The plasma conditions were 13.56 MHz, 50 W, 10 Pa Ar pressure, Ar flow rate =12 sccm. In order to confirm the removal of Fc monolayer from the membrane surface, we have used a Kratos Analytical Surface Analyzer XSAM 800 with a Mg source that is normal to the sample surface. This instrument was used to detect the surface Fe 2p3/2 peak for membranes before and after Ar plasma etching for different etching times. Electrochemical Experiments After the plasma etching, the membrane was washed with ethanol and water and then subjected to electrochemical e xperiments. Electrochemical e xperiments were done with the membrane sample mounted in the U-tube cell. 0.1 M KClO4 electrolyte solution was added to both half-cells, equilibrated for 1-2 days and bubbled with Argon for 30 minutes before the

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61 experiment. Argon was also purged into th e system throughout the experiment. The Au nanotube membrane was made the working el ectrode in a conventi onal three-electrode experiment where the counter electrode was a Pt wire and the reference was an Ag/AgCl electrode with 3 M NaCl. A Solartron SI 1287 electrochemical inte rface module (Solartron Analytical, Hampshire, England) connected to a PC running CorrView and CorrWare software (Scribner Asc. Inc., NC) was used. In order to observe and ca lculate the decay in the Fc+, the membrane sample was held at 0.7 Volts for ~ 6 hours during which cyclic vol tammograms (CVs) were taken periodically. The cathodic half cycles of these CVs were then used to calculate the amount of redox-active Fc for each CV. The same conditions were also applie d to Fc-thiol modified gold button electrode which was not exposed to any plasma treatment. Results and Discussion Surface Fc-Thiol Removal In order to study the effect of pore size on Fc+ decay, we needed a technique to remove all Au surface Fc but do not destroy the Fc-thiol lini ng the Au nanotube walls. We have first used O2 plasma conditions, but it removed nonspecifically all Fc-thiol from the gold membrane even at short times under mild conditions. Ar plasma etching, however, proved to be useful to selectively remove the surface Fc-thiol monolay er. Figure 3-1 shows the cyclic voltammograms of freshly modified membranes before and after the Ar plasma treatment with different etching times. In order to find the minimum etching tim e that is necessary to remove Au-surface Fcthiols, we have used membranes that have pores filled with Au. Since these membranes can not have any Fc-thiol inside the por es, a successful plasma removal s hould show no sign of Fc in the voltammogram. This is achieved at 30 seconds (Figure 3-1B) and furthe r proved by XPS studies (Figure 3-2, Curve C). The Fe 2p3/2 peak at 711 ev disappears even after 5 seconds (Figure 3-2,

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62 Curve B) although the voltammogram (Figure 3-1A) still shows some trace which indicates the greater sensitivity of the CV method. When these conditions were applied to a memb rane with open pores, the plasma removes surface Fc monolayer and leaves the Fc monolay er inside the Au nanotube walls. Figure 3-3 shows the voltammograms of a membrane before a nd after plasma treatment. This membrane has pores with 20 nm inside pore diameter. In this case the amount of re dox-active Fc is decreased by 40 %, which is equivalent to the relative amounts of Au surface-film vs. Au nanotube-wall surface area (assuming cylindrical po res of 10 nm radius). Voltamm ograms like Figure 3-3 (solid line) were also used to calculate the surface coverage of ferrocene. The coverage for all membrane systems were ~ 2 times the predicted packing limitation of 4.5 x 10-10 mol/cm2,145,160 which is due to the rough surface structur es of electroless plated gold membranes.161 Electrochemical Decay Studies Figure 3-4 shows cyclic voltammograms of f our membranes with different inside pore diameters that are subjected to 0.7 Volts for ~ 6 hours. The same conditions were also applied to a Fc-thiol modified commercial gold button electr ode to compare the Fc decomposition for a flat surface with no pores (Figure 3-5). The spiky peak s observed in Figure 3-5 suggest that there are strong attractive interacti ons in this environment.162 Examination of CVs in Figures 3-4 and 3-5 indicates that the bigger the pore size the faster the decay and the more it resembles a flatsurface-like behavior. For pore sizes 65 nm, there is clearly a negative shift with increasing time which is most pronounced for R = 10 nm (Figure 3-4A). We suspect that the mild hydrophobicity of Fc-thiol is responsible for this observation. We and others163 have obtained contact angles ( ) < 80 for Fc terminated alkane th iol monolayers on gold surfaces where as SAMs formed by long-chain alkane thiols have values of ~ 115.164

PAGE 63

63 This shift in the CVs to more negative potentials as it decays indicates that the environment around the Fc groups becomes more hydrophilic with increasing scan number. This has been observed before, and indicates that with prolonged scanning the Fc/Fc+ groups in the monolayer film become more acces sible to water and counterions.165 The hydrophobicity is most pronounced with the smallest pore be cause the volume of the Fc-thiol that is filling the pore has the biggest ratio in the R =10 nm case. As the por e size gets bigger this ratio gets smaller. There is no clear shift where R = 284 nm (Figure 3-4D ). In this case the hydr ophobic contribution is minimal and the Fc groups are already accessible to water and counterions as there is no clear shift just similar to the flat surface gold electrode. In order to compare the decay constants, se mi logarithmic plots of normalized cathodic charge against time166 were examined (Figure 3-6). Linear plots were obtained for each system, obeying the first order decay kine tics that is previously obser ved in aqueous solutions for ferricinium.159,166 Studies in aqueous solution have shown that ferricinium cations (Fc+) decompose through an exchange of cyclopentadienyl anions (Cp-) with another nucleophile. (e.g., OH-, Cl-, NO3 -)153,155,156 The rate of exchange increases with the donor strength of the nucleophile. The decomposition of Fc+ can be summarized as follows:155 FeCp2 + + n L FeLn 3+ + 2 Cp(3-2) assuming that in a primary step ligand exchange around the Fe (III) ion occu rs. In this reaction L can be a solvent molecule, a neutral nucle ophilic agent or a monovalent anion. The Cpcan then reduce undissociated FeCp2 + to FeCp2 in a follow-up reaction and Cp radicals form. Fc+ decomposition is observed in electr olytes containing perchlorate anion.153,163 It is found that increasing the pH increases the extent of decomposition substan tially which is due to the increased concentration of hydroxide ion.153 In the current system, both ClO4 and OHcan

PAGE 64

64 initiate the Fc+ decomposition although th e latter has a much smaller concentration ([ClO4 -] = 0.1 M and [OH-] = 2.0 x 10-6 M). Table 3-1 shows the increase in decay constants with increasing pore size. This constant approaches to that of a flat gold surface for R = 285 nm. As mentioned above, the increasing accessibility of water and counterions to Fc groups with increasing pore size should be a factor in such an observation. More importantly, the tendencies of ClO4 vs. OHtowards an alkane-like environment are different. Extraction of ionpairing complexes of perchlorat e into organic phases is a well defined technique to detect trace amount s of perchlorate in aqueous samples.167-169 In this case, perchlorate being a weak lipophi lic nucleophile is the dominant anion inside the alkane-like environment of the small pores which results in sl ower decay rates. As the pore gets larger and more hydrophilic, OH(strong nucleophile) partitioni ng into that pore increas es and thus the rate constant gets bigger. Other potential nucleophile in th is system is water, but its donor strength is insufficient for Fc+ decomposition.155,170 It is also interesting to not e that the decay constants of ferrocene and 1,1dimethyl ferrocene molecules in bulk aqueous phosphate buffer has similar values166 as the Fc monolayers studied in this work (Table 3-1). Conclusion Recently, we have shown the affect of Fc+ decomposition on electromodulating ion transport through gold nanotube membranes.43 In this paper, we have elucidated the nanotube pore size affect on Fc+ decomposition. Fc-thiol monolayers on Au surface film were successfully removed by briefly exposing both surfaces of the membrane to argon plasma. The decomposition of Fc+ inside the Au nanotube walls were th en studied for four membranes with different pore sizes and compared wi th a flat surface electrode. Th e results suggest that the decay rate increases with increasing pore size and in all cases it is found to obey first order decay

PAGE 65

65 kinetics. Furthermore, the decay pattern resemb les a surface-like decay as the pore size of the membrane increases. We suspect that limited accessibility of the counterions inside the small pores and their different tendencies towards a lipophilic environm ent are responsible for the slower decay rate. This is due to the constrained geometry of these small pores and the more pronounced hydrophobic character of Fcthiol monolayers. As th e pore size gets bigger, both of these affects are lost and the membrane behaves just like a fl at-surface electrode. The negative shift in the voltammograms was also more pronounced for sm aller and more hydrophobic pores. This shift in the CVs to more negative pote ntials as it decays indicates th at the environment around the Fc groups becomes more hydrophilic w ith increasing scan number.

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66 Table 3-1. Fc+ decay constants for different membrane systems and for bulk aqueous solutions of Fc compounds in phosphate solutions at neutral pH.166 Case Studied Decay Constant (sec-1) R = 10 nm 0.7 x 10-5 R = 28 nm 0.9 x 10-5 R = 65 nm 1.4 x 10-5 R = 284 nm 1.9 x 10-5 Gold Button Electrode 2.1 x 10-5 Ferrocene 1.4 x 10-5 1,1-dimethyl ferrocene 0.6 x 10-5

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67 0.00.20.40.60.8 -6 -4 -2 0 2 4 6 A 0.00.20.40.60.8 -6 -4 -2 0 2 4 6 Potential (V vs. Ag/ AgCl)B Figure 3-1. Finding the optimum etching tim e for surface Fc-thiol removal. Cyclic voltammograms of Fc thiol modified gold nanotube membranes before (solid curves) and after (dashed curves) Argon plasma etching. The electrol yte is 0.1 M KClO4 and the membranes have pores fill ed with gold. Increasing the Argon etching time from A) 5 sec to B) 30 sec removes all Surface-Fc.

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68 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 700705710715720725 Binding Energy (ev)Intensity (counts)A B C 140000 150000 160000 170000 180000 190000 200000 210000 220000 230000 700705710715720725 Binding Energy (ev)Intensity (counts)A B C Figure 3-2. XPS spectra of the Fc -thiol modified gold membrane afte r A) 0 sec, B) 5 sec and C) 30 sec of Argon plasma etching. The Fe 2p3/2 peak is detected at 711 eV and it disappears even after 5 second etching. A Kratos XSAM surface analyzer with a Mg source normal to the membrane surface has be en used. The gold membrane is plated overnight to fill the pores with gold completely.

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69 0.00.20.40.60.8 -10 -5 0 5 10 Potential (V vs. Ag/ AgCl) Figure 3-3. Cyclic voltammograms of a Fc-thiol modified membra ne before (solid curve) and after (dashed curve) 30 sec of Argon plasma etching. The membrane has pores with 20 nm inside pore diameter. The dashed curve corresponds to Fc-thiol monolayer lining only inside the nanotube walls.

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70 0.00.20.40.60.8 -30 -20 -10 0 10 20 30 CI (A)Potential (V vs. Ag/AgCl)0.00.20.40.60.8 -15 -10 -5 0 5 10 15 AI(A)0.00.20.40.60.8 -15 -10 -5 0 5 10 15 BI (A)0.00.20.40.60.8 -30 -20 -10 0 10 20 DI (A)Potential (V vs. Ag/AgCl) Figure 3-4. Cyclic voltammogr ams of four different gold nanotube membranes with pore diameters A) R = 10 nm, B) R = 28 nm, C) R = 65 nm, and D) R = 284 nm. Scans were recorded sequent ially after holding Eapp at 0.7 V for 0 min (black), 35 min (red), 105 min (blue), 175 min (green), 245 min (vio let) and 315 min (orange). Scan rate is 20 mV/sec.

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71 0.00.20.40.60.8 -300 -200 -100 0 100 200 300 400 I (nA)Potential (V vs. Ag/AgCl) Figure 3-5. Cyclic voltammogr ams of modified gold button el ectrode. Scans were recorded sequentially after holding Eapp at 0.7 V for 0 min (bl ack), 35 min (red), 105 min (blue), 175 min (green), 245 min (violet) and 315 min (orange). Scan rate is 20 mV/sec.

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72 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 050100150200250300350Time (min)-ln (Q/Q 0)cathodicC B A D E 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 050100150200250300350Time (min)-ln (Q/Q 0)cathodicC B A D E Figure 3-6. First order kineti c plots for the loss of the Fc+ for A) R = 10 nm, B) R = 28 nm, C) R = 65 nm, D) R = 284 nm and E) gold button electrode.

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73 CHAPTER 4 PLASMA-ETCHED NANOPORE POLYMER FILMS AND THEIR USE AS TEMPLATES TO PREPARE NANO TEST TUBES Introduction We recently introduced a new class of tubul ar nanostructures called nano test tubes.47,48 Unlike conventional nanotubes, which are open at both ends, nano test tube s are open on one end and closed on the other. They are made by the template-synthesis method, in which the pores in a nanopore material are used as templates to prepare nanotubes.3,4,6 The key to obtaining nano test tubes is using a template in which the pores are closed on one end (F igure 4-1A). When the tube-forming material is deposited within such pores, both the por e walls and the closed pore end get coated with this material, and closed-end test tubes are obtained. Th e outside diameter of these nano test tubes is determined by the pore di ameter of the template, and the length of the tubes is determined by the template thickness.47 Nanopore alumina films, prepared by el ectrochemical oxidation of Al metal,76,171have pores that are closed on one end, provided th e alumina is not removed from the underlying Al surface.18 In our prior work, we used such alumina films as template s to prepare silica nano test tubes.47 There is, however, a limitation with regard to the dimensions of th e nano test tubes that can be obtained with these nanopore alumina templa tes. Specifically, it is difficult to obtain short (<500 nm long) test tubes. This is because such short nano test tubes require ultra-thin alumina templates, which means that very brie f anodization times must be used. However at very short times, anodization of aluminum shows irregular growth patterns and the resulting alumina film does not have a regular pore structure.71 Our motivation for making smaller nano test tubes comes from our interest in investigating uptake of such tubes by living cells with the ultimate goal of us ing these tubes as drugor DNAdelivery vehicles. We believe that for such appli cations it would be adva ntageous to have tubes

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74 that are small in length relative to the dimensions of the cell. B ecause of this limitation with the alumina templates, we have been investigati ng methods for preparing thinner nanopore templates so that shorter nano test tubes might be obtained. One such method builds on Masudas concept of using a nanopore alumina membra ne as a plasma etch mask.172,173 This technology entails removing a nanopore alumina film from the underlying Al surface so that the pores are open at both faces of the resulting alumina membrane. The free-standing alumina membrane is then placed on a substrate, and a plasma is used to etch a replica of the alumina pore structure into the surface of the substrate (Figure 4-1B). We ha ve used this method to prepare nanopore carbon anodes for battery applications18 and nanowell glass surfaces for applications in analytical chemistry.70,174 We have recently modified this mask/etch technology so that it can be used to produce pores in an underlying polymer ( photoresist) film, as opposed to the harder materials (glass,70,174 diamond,172,173 graphite18) etched previously. Furthermore, we have shown that with this modified mask/etch method the distance that the po res propagate into the photoresist film can be controlled by varying the etch time. Hence, by c ontrolling the etch time, we effectively control the thickness of the nanopore layer etched into the surface of the photoresist. We have used such plasma-etched nanopore photoresist films as templa tes to prepare silica nano test tubes. As expected the length of the test tubes is determined by the th ickness of the porous photoresist layer, and test tubes with lengths of 380 nm were obtained, shor ter than any test tubes obtained using an alumina template.47 We report preliminary results of these investigations here. Experimental Materials Aluminum foil (99.99%) was obtained from Al fa Aesar, and micros cope premium finest glass slides from Fisher. PMGI SF 15, a pol ydimethylglutarimide-based positive photoresist,

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75 was purchased from MicroChem Corp. Ethanol (absolute, Aaper), tetraethyl orthosilicate (Aldrich), HCl (Fisher), and 1165 Microposit Remover (a 1-methyl -2-pyrolidinone-based system for dissolving the PMGI photoresist, Shipley) we re used as received. Purified water was obtained by passing house-disti lled water through a Millipore, Milli-Q system. Preparation of the Nanopore Alumina-Membrane Masks The nanopore alumina membranes were prepared in house using the well-known two-step electrochemical anodization method.18 Briefly, after annealing a nd polishing the aluminum foil, a nanopore alumina film was formed across the Al surface by anodization. This film was then dissolved in acidic CrO3, and a second anodized alumina film was formed. This film was removed from the underlying Al surf ace using the voltage-reduction method.175 The resulting free-standing nanopore alumina membrane has tw o faces the one that was exposed to the solution, and the one that was adjacent to the Al substrate, during anodization. These faces are not identical,18 and we delineate them, here, as the solu tion-side and the Al-side faces. The pore diameter, as determined from scanning electron microscopic (SEM) images of the solution-side face (Figure 4-2A), was 79 nm. The alumina memb rane thickness was ~1.5 m (Figure 4-2B). SEMs were obtained using a Hitachi S4000 FE-SEM. Prior to imaging, the surface of the SEM sample was sputtered with a thin Au/Pd film us ing a Desk II Cold Sputter instrument (Denton Vacuum, LLC). Preparation of the Nanopore Polymer-Replica Films Glass microscope slides (2 cm x 2 cm) were washed with copious amounts of ethanol and blown dry with nitrogen. A Model 6700 spincoat er (Speedline Technologie s, IN) was used to coat one surface of the slide with the PMGI SF 15 photoresist; ~2 ml of the photoresist were dispensed, the terminal spin speed was 10,000 rpm, and the spin time was 45 sec. The resulting polymer film (~4 thick) was cured in air at 190 C for 15 minutes.

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76 As per our prior work,18,70 the general strategy was to place the nanopore aluminamembrane mask onto the surface of the polymer film, and use a plasma-etch method to burn a replica of the alumina pore structure into the polymer surface (Figure 4-1B). However, we discovered that when the alumina mask was placed directly on top of the polymer film, a replica of the alumina pore structure coul d not be obtained; instead, larg e diameter (~500 nm) pits were burned into the surface of the polymer film. In order to obtain a faithful replica, it proved necessary to sputter-coat the pol ymer film with a thin metal film, and then place the nanopore alumina-membrane mask on this metal film (Figur e 4-3). Three different metals Au, Ag, and Au/Pd were investigated, with the best results obtained with Au/Pd. The Au/Pd films were sputtered using the Desk II Cold Sputter instrument, with 45 mA sputtering current, 75 mTorr Ar pressure, and 60 sec sputtering time The film thickness was ~30 nm. The alumina-membrane mask was placed on top of the Au/Pd-coated polymer film with the solution-side face of the membrane facing down The masked substrate was placed into the vacuum chamber of a reactive-ion etching system (Samco model RIE-1C) and subjected to two plasma-etch treatments. The first was a 2-minute Ar-plasma etch (physical etch,111). The plasma conditions were 13.56 MHz, 140 W, 10 Pa Ar pr essure, Ar flow rate =12 sccm. The second etch was a chemical etch111,176using an O2/Arplasma. The plasma conditions were 13.56 MHz, 140 W, 10 Pa O2 pressure, O2 flow rate = 10 sccm, 10 Pa Ar pressure, Ar flow rate = 12 sccm. Preparation of the Silica Nano Test Tubes A key objective of this work was to show th at the pores in these nanopore polymer-replica films could be used as templates to prepare na no test tubes. To dem onstrate this, a sol-gel method described previously47 was used to deposit silica nano test tubes within the pores of the polymer-replica films. Briefly, a 50/5/1 (by vo lume) mixture of ethanol, tetraethyl orthosilicate

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77 and 1M HCl was prepared and allowed to hydrolyze for 30 min. The nanopore polymer-replica film was immersed into this sol (PMGI SF 15 is insoluble in ethanol) with sonication for 30 sec and then kept under vacuum in a desiccator for 5 more minutes. The sol-impregnated film was dried in air, and then oven cured for ~5 h at 100 C, to yield silica nano test tubes47 within the pores of the polymer-replica film. To liberate the nano test tubes, the na nopore polymer-replica film was dissolved by overnight immersion in the 1165 Microposit Remover solution. The liberated test tubes were collected by filtration and ri nsed with copious amounts of the remover and ethanol. Transmission electron microscopy (TEM) samples were prepared by re-suspending the liberated nano test tubes in ethanol and immersing a TEM grid into this suspension. TEM images were obtained with a Hitachi H-7000 microscope. Results and Discussion As noted above, it proved necessary to coat th e surface of the polymer film with a thin Au/Pd layer prior to applying the alumina etch mask and plasma etching. The aluminamask:Au/Pd:polymer-film assembly (Figure 4-3) was then first etched with an Ar plasma (physical etch).111 This brief Ar-plasma etch removes th e portions of the Au/P d film beneath the pores in the nanopore alumina mask. Put another way, the Ar plasma creates a replica of the alumina pore structure in the Au/Pd film, and thus exposes the portions of the polymer film in the regions beneath the alumina pores. The asse mbly (Figure 4-3) was then subjected to an O2/Ar plasma (chemical etch)111 to remove the exposed portions of the polymer film beneath the alumina pores; i.e., the O2/Ar plasma is responsible for replica ting the pore structure of the mask in the polymer film. Figure 4-4 shows surface and cross-sectional imag es of the polymer film after four minutes of etching with the O2/Ar-plasma. Some reproduction of th e pore structure of the alumina-mask

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78 can be seen in the surface image (Figure 4-4A ), but the pores propagate only a very small distance into the polymer film (Figure 4-4B). Analogous images after 8 minutes of etching with the O2/Ar-plasma show that the pore structure has been faithfully reproduced in the surface of the polymer film (Figure 4-5A), and that the pores obtained propagate, with uniform diameter, ~380 nm into the upper surface of the film (Figure 4-5B). The pore diameter is 81 nm identical to the diameter of the pores in the alumina mask. When the pores in this polymer film were used as templates to prepare silica nano te st tubes, tubes with diameters of 83 nm and lengths of 380 nm were obtained (Fi gure 4-5C). As would be expected,47 not only are the diameters equivalent to the pore diameter, but th e length is equivalent to the thickness of the porous part of the polymer film. By controlling the O2/Ar-plasma etch time; the distance that the pores propagate into the upper surface of the polymer film can be varied. For example, a film that was etched for 10 min had 85 nm diameter pores (Figure 4-6A) that pr opagated ~1 m into the polymer film (Figure 4-6B). Correspondingly, the nano test tubes synthe sized within the pores of this film were ~100 nm in diameter and 1000 nm in length (Figur e 4-6C and D). In th is case obtaining an accurate value for the tube diameter is problema tic, because as can be seen in Figure 4-6B, the pore is wider at the mouth than at the bottom. As a result the outside diameter of the tubes is likewise larger at the mouth (Figures 4-6C and D). Note that the metal film is still present on top of the polymer film (Figure 4-6B). When longer etch times (e.g., 12 min) were used, much larger scale damage is produced in the polymer film, and faithful reproduction of the pores in the alumina mask is no longer achieved (Figure 4-7). This is because for such long etch times the metal film on the surface of polymer film is damaged and partly removed and, as a result, the pores merge at the polymer

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79 film surface (Figure 4-7A). This damage could be detected with the naked eye, as the faint black color of the Au/Pd coating could no longer be observed. Hence, ag ain, we see the essential role played by the metal film in producing a faithful replica of the alumina mask in the underlying polymer. Conclusions We have extended the alumina-mask, plasma-etch c oncept to a new substrate material a photoresist polymer film. In so doing we created a new type of nanopore polymer template for use in template synthesis of nanomaterials. An appealing feature of this new template is that the distance that the pores propagate into the su rface of the polymer film can be controlled by varying the plasma etch time. This allows fo r corresponding control over the lengths of the nano test tubes prepared by template synthesis within the pores. Via this route, we have successfully prepared silica nano test tubes that were over 100 nm shorter than the shortest tubes prepared in an alumina-film template.47 It is also of interest to note that this general procedure can be thought of as a relatively high throughput nanotube synthe sis technology. This is because there are ~1010 pores per cm2 of template area; so for example, with 10 cm2 of template, we can make 1011 nano test tubes. Another appealing feature of these new polymer-f ilm templates is that they can be used for both aqueous-based (including both acidic and basic solution) and organic-based (including most aliphatic alcohols, ketones and ethers) template synthesis. Nevertheless, these films can be dissolved, when needed, in the photoresist re mover solution to liberate the nano test tubes synthesized within the pores. We are currently further explor ing the plasma-etch process in attempts to make even thinner nanopore polymer replica films.

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80 Porous material with closed-end pores Deposit tubeforming material Nano test tubes ANonporous material to be etched Plasma etch and remove alumina Closed-end pores Alumina etch mask B Porous material with closed-end pores Deposit tubeforming material Nano test tubes ANonporous material to be etched Plasma etch and remove alumina Closed-end pores Alumina etch mask Porous material with closed-end pores Deposit tubeforming material Nano test tubes ANonporous material to be etched Plasma etch and remove alumina Closed-end pores Alumina etch mask Alumina etch mask B Figure 4-1. Schematic diagrams of A) the concep t of using a template with closed-end pores to prepare correspondingly closed -end nano test tubes, and B) the alumina-mask plasma-etch method to prepare closed-end por es in an underlying substrate material.

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81 Figure 4-2. SEM images of the nanopore alum ina-membrane mask; A) Top view; B) crosssectional view. A B

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82 Figure 4-3. Cross sectional SEM of the Al-mask:Au/Pd-film:polymer-film assembly. Alumina mask Au/Pd film Polymer Film Glass Support

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83 Figure 4-4. SEM images of A) the polymer-film surface and B) the cros s-section of the film after 4 min of O2/Ar plasma etching. B A

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84 Figure 4-5. SEM images of A) the polymer-film surface and B) the crosssection of the film after 8 min of O2/Ar plasma etching. C) SEM images of silica nano test tubes synthesized in the pores of this polymer film. A B

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85 Figure 4-6. SEM images of A) the polymer-film surface and B) the cros s-section of the film after 10 min of O2/Ar plasma etching. C) SEM and D) TEM images of silica nano test tubes synthesized in the por es of this polymer film. A C B D 200 nm

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86 Figure 4-7. SEM images of A) the polymer-film surf ace and B) the cross-section of the film after 12 min of O2/Ar plasma etching. A B

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87 CHAPTER 5 SILICA NANO TEST TUBES AS DELIVERY DEVICE S; PREPARATION AND BIOCHEMICAL MODIFICATION Introduction The application of nanomaterials such as nanoparticles, nanotubes, nanorods, and nanowires in biological systems has attracted great interest in the fields of materials science and biochemistry.2,177 Because of their dimensions, which make them suitable for application in biological systems, the potential of nanomaterials for biodetection,178-181 bioseperation,45 and biomolecule delivery118,120,121,125,126,142 has been explored.116 In particular, the use of nanomaterials in biomolecule delivery has been shown to present variou s advantages such as increased efficacy,113 protection of drugs114 or genetic material115,116 from potential environmental damage and reduced drug toxicity.117 Spherical nanoparticle s are almost always used because these shapes are easier to make a nd can be synthesized from a diverse range of materials, such as liposomes,118,119 polymers,120,121 dendrimers122 and various inorganic compounds.46,115,123 Unlike nanospheres, nanotubes have unique hollow structures however their use as biomolecule carrier s are still very rare.116,142,182 We have pioneered a technology, called templa te synthesis, for preparing monodisperse nanotubes of nearly any size and co mposed of nearly any material.3,183,184 These nanotubes have a number of attributes that make them potential candidates for biom olecule delivery applications. First, nanotubes have larger inner diameters th an nanoparticles which al low nanotubes to carry a correspondingly larger payload. In addition, the template method allows independent modification of the distinct i nner and outer surfaces of the tubes. Multifunctional delivery vehicles can be obtained by this differential m odification scheme. Such delivery tools attracted great interest in biomedical applications, for example, multifunctional nanomaterials are

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88 considered to be ideal units for the cancer-specific therapeutic and imaging agents.125 Finally, the tubes can be synthesized from various materi als and their dimensions are easily controlled.45 We have shown the application of differe ntially modified silica nanotubes as smart nanophase extractors for enantiomeric drug molecules.45 Chen and colleagues demonstrated the preparation of fluorescent si lica tubes for gene delivery.116 After the attachment of quantum dots, the tubes were loaded with gr een fluorescent protein (GFP) pl asmid and incubated with monkey kidney COS-7 cells. The loaded tubes are shown to be non-toxic to th e cells, they initiate approximately 10-20 % of the cells to express G FP and they also act as physical shields to protect the genetic material form enzymatic degr adation. The tubes, however, lack differential modification and capping as they are necessary for targeted delivery46,47 and the tube size is controlled by physical polishing which is inappropria te for obtaining tubes with lengths < 1 m. Novel nanostructures called nano test tubes have been recent ly introduced by the Martin group.47,48 Silica nano test tubes are pr epared by sol-gel synthesis of silica in the pores of alumina template that remains attached to unde rlying aluminum metal. Unlike the previously mentioned nanotubes that are open on both ends, nano test tubes are closed on one end and open on the other. The use of test tubes as potential universal drug delivery vehicles was exploited where these nano test tubes could be filled with a payload and then the open end corked with a chemically labile cap.48 We have developed a capping strate gy that involves the Schiffs base reaction to form imine linkages between the te st tubes and the aldehyde -modified polystyrene corks.48 Lee and coworkers have described a sele ctive partial functionalization method using controlled gold nanoparticle diffusion in nanotub es and prepared Au-capped silica nano test tubes by seed-mediated gold-growth.185 The same group has also introduced magnetic nano test tubes that has a layer of Fe3O4 prepared by dip-coating.186

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89 In our earlier work, we have used the conven tional sol-gel method to obtain silica nano test tubes in the pores of alumina template.46,47 Although the procedure is easy, it can be challenging to control the thic kness and morphology.101 This chapter compares the preparation techniques for silica nano test tube fabrication using the conventional and surface sol-gel methods and illustrates the subsequent differential tube modi fication strategy for their use in cell incubation studies. Defective test tubes were obtained w ith the conventional sol-gel method and it was attributed to the small changes in the viscosity of the gel. Layer-by-laye r addition of silica with the surface sol-gel method allowed the preparation of defect-free uniform silica nano test tubes. We have differentially modified th ese test tubes using silane and Schiff-base chemistry to impart biochemical functionality for the cell studies. Befo re the template was removed, the inner tube surface was labeled with a fluorophore. The liberate d fluorescent-tubes were then modified with a target or a control antibody and then incubated with br east carcinoma cells. The preliminary results suggest that the tubes modified with the ta rget antibody attaches much more readily to the cell membrane surfaces than the tubes m odified with the control antibody. Experimental Materials Aluminum foil (99.99%) was obtained from Alfa Aesar. Microscope premium finest glass slides, methanol, chromium tr ioxide, oxalic acid, NaOH, H3PO4, H2SO4 and HCl were obtained from Fischer and used as received. Tetraethyl orthosilicate(TEOS), silicon tetrachloride, carbon tetrachloride, 3-(amino-propyl)triethoxysilane(APT S), Rhodamine B Isothiocyanate, sodium cyanoborohydride, IgG from Rabbit serum, and Al bumin Bovine Serum were used as received from Sigma-Aldrich as were ethanol (absolut e) from Aaper, N,NDimethylformamide from Acros, Alexa 488 carboxylic acid-succinimidyl es ter and Alexa Flour 488 labeled goat antirabbit IgG from Invitrogen, and 3-(trimethoxysil yl)propyl aldehyde from UCT Chemicals. IGF-

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90 IR and IGF-IR rabbit polyclonal antibodies were obtai ned from Santa Cruz Biotechnology, Inc. Purified water was obtained by passing hous e-distilled water through a Millipore, Milli-Q system. Preparation of the Nanopore Alumina-Membrane Templates The nanopore alumina membranes were prepared in house using the well-known two-step electrochemical anodization method.18,171 Briefly, after annealing and polishing the aluminum foil, a nanopore alumina film was formed across the Al surface by anodization. This film was then dissolved in acidic CrO3, and a second anodized alumina f ilm was formed using oxalic acid electrolyte. This yields the desired ordered nanopore alumina film on both surfaces of the aluminum film. Unlike the work described in th e previous chapter, the alumina film is not detached from the aluminum so the template remains attached to the underlying Al metal. It is also important to note that in the first work we reported the preparation of silica nano test tubes; we have attached a glass substr ate to one surface of Al with e poxy for stability reasons, which yielded alumina growth only on one side of Al metal.47 Preparation of the Silica Nano Test Tubes Two different sol-gel methods were used to de posit silica nano test tubes within the pores of the nanopore alumina template (Figure 5-1). In the convent ional sol-gel method:47,48 a 50/5/1 (by volume) mixture of ethanol, tetraethyl orthosilicate and 1M HC l was prepared and allowed to hydrolyze for 30 min. The alumina template was immersed into this sol with sonication for 30 sec and then kept under vacuum in a desiccator for 5 more minutes. The sol-impregnated template was dried in air, and then oven cured fo r ~5 h at 100 C, to yiel d silica nano test tubes47,48 within the pores of the nanopore alumina template. The surface film was removed by wiping the membrane surface with a laboratory tissue soaked in EtOH.

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91 In the surface sol-gel method;101 two-step deposition cycles, in which the adsorption of a molecular precursor (SiCl4) and the hydrolysis steps are sepa rated by a post-adsorption wash. An alumina template was immersed in SiCl4 solution in CCl4 (85 mol-%) for 2 min and quickly soaked in a CCl4 beaker. The template was then washed with CCl4 and immersed in a second CCl4 beaker for 15 min to remove unbound SiCl4 from the pores. These steps were done in a polyacrylic box under 30 psi nitrogen flow to limit SiCl4 polymerization by atmospheric water which occurs at ambient conditions and results in silica deposition with uncontrollable thickness. Finally, the template was soaked in CCl4/MeOH 1:1 (2 min) and EtOH (5 min) to displace CCl4, and dried in a N2 stream. Then the template was immersed in deionized water for 5 min, washed in a beaker with MeOH (2 min). After 10 deposi tion cycles the silica deposited template was cured at 100 C for 1 h. The su rface film was removed by briefly (1 min) exposing both sides of the nanopore template to a reactive-ion plasma etching system (Samco model RIE-1C). The plasma conditions were 13.56 MHz, 140 W, 20 Pa Ar pressure, Ar flow rate = 20 sccm. To liberate the nano test tube s, the nanopore alumina templa te was dissolved in 0.1 M NaOH for 3-6 h. The liberated test tubes were collected either by centr ifugation (14,000 rpm for 14 min in all experiments involvi ng centrifugation) or filtration and washed several times with water and ethanol. Transmission electron micr oscopy (TEM) samples were prepared by resuspending the liberated nano test tubes in et hanol and immersing a TEM grid into this suspension. TEM images were obtained with a Hitachi H-7000 microsc ope. Scanning electron microscopy (SEM) was also used to characterize th e alumina template and the filtered free silica nano test tubes. SEM images were obtained us ing a Hitachi S4000 FE-SEM Prior to imaging, the surface of the SEM sample was sputtered with a thin Au/Pd film using a Desk II Cold Sputter instrument (Denton Vacuum, LLC).

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92 Silica Nano Test Tube Modification with Fluorophore The labeling of silica nano test tubes with fluorophores were done while the tubes were still embedded in the alumina template. This means only the inner walls of the tubes are accessible for chemical modifications. The surface modifications were done using silanization chemistry and the structures of all silanes are show n in Figure 5-2. In ea ch case the inner tubule walls were modified with amine functional groups which are then covalently coupled to Rhodamine or Alexa Flour-488 (Figure 5-3).70,187 Briefly a solution that was 5 % APTS, 90% ethanol, and 5 % acetate buffer (50mM, pH 5.2) was hydrolyzed for 20 min and the template is immersed into this solution for 1 h. The templa te was then thoroughly washed with ethanol and cured in an oven at 100 C for 3h. Rhodamine attachment was done by immersing the amine functionalized template into a 5 mM Rhodamine B Isothiocyanate solution in dry DMF for 12 h in a desiccator. This was followed by extensive washing with DMF and EtOH. To modify the inner tube surfaces with Alexa-488, a 0.1 mg/ml solution of Alexa 488 carboxylic acidsuccinimidyl ester in 10 mM phos phate-buffered saline (PBS) buffe r (pH is adjusted to 8.1 by 0.1 M NaOH ) was prepared. The amine modified temp late was then immersed into this solution in a desiccator for 12 h and then washed with bu ffer and ethanol before the tubes were liberated from the template. A fluorescence microscopy system described previously188 was used to obtain fluorescence images of the labeled test tubes and to measure the fluorescence intensity from glass slides that are used to confirm the antibody attachment. (See antibody modifica tion.) This system combines an Axioplan 2 imaging microscope (Zeiss) with a J&M-PMT photometry system detector (SpectrAlliance), for measuring fluorescence intens ity. In addition, the system is equipped with a digital CCD camera (Zeiss) to obtain both fluor escence and optical images. The excitation source for all fluorescence measurements was a mercury lamp. A beam splitter was used to send the

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93 reflected fluorescent light from the sample to the detector and the CCD camera. The Rhodamine B was excited at 570 nm, and the emission was collected through a 590nm band-pass filter and The Alexa 488 was excited at 495 nm, and th e emission was detected through a 515-nm bandpass filter. Antibody Modification The fluorescently labeled tubes were liberated and then washed by centrifugation at 14,000 rpm three times with H20 and then three times with etha nol. The outer tube walls were functionalized with aldehyde groups by an aldehyde terminated siloxane linker.189 The aldehyde groups were then reacted by well-known Schiff-base chemistry to amine sites on the protein to be immobilized.190-192 Briefly a solution that was 5 % 3(trimethoxysilyl)propyl aldehyde, 90% ethanol, and 5 % acetate buffer (50mM, pH 5.2) was hydrolyzed for 15 min and the tubes were dispersed in this solution and reacted for 30 min with frequent vortexing. The aldehyde modified tubes were centrifuged and vorte xed three times with ethanol and then three times with 10 mM PBS, at pH 7.4. The antibodies were coupled to the aldehyde-terminate d outer tube surfaces by dispersing these tubes in the same PBS buffe r that contains 0.2 mg/ml antibody and 4 mM NaBH3CN for 12 h at 4 C with occasional vortexi ng. The tubes were either modified with Rabbit polyclonal IGF-IR (target) or IGF-IR (control) antibodies and the tube concentration was ~1010 tubes/ml. After the antibody modification, the tubes were washed three times with PBS buffer by centrifugation and dispersed in 10 mM PBS, at pH 7.4 that contains 0.2 mg/ml bovine serum albumin (BSA) and 4 mM NaBH3CN. This step is required to quench the remaining aldeyhde sites on the outer tube wall s and was done by allowing the tubes in this solution for 2 h at room temperature with vortexi ng. Finally, the tubes were washed three times

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94 with 10 mM PBS (pH=7.4) by centrifugation and dispersed in the same buffer for cell incubation studies. Covalent attachment of anti body by Schiff-base chemistry was confirmed on glass slides. Two glass slides were coated with a single layer of silica by surface sol-gel method and both slides were functionalized with aldehyde sila ne as mentioned above and dried in a vacuum desiccator for 5 h. First slide was then modified with Rabbit Ig G and the second with BSA where both proteins were 1 mg/ml in a pH 7.4, 10 mM PBS containing ~ 4 mM NaBH3CN. The slides were washed with PBS and treated with 1/5 dilu ted sea block buffer (Pierce, # 37527) for 2h. Both slides were then exposed to Alexa Flour 488 labeled goat anti-rabbit IgG (20 g/ml in PBS, pH 7.4) for ~ 10 h at 4C. After rinsing with PBS and water the slides were dried under N2 stream and their fluorescence was compared by J&M-PMT photometry system detector. Cell Incubation Studies MDA-MB-231 breast carcinoma cells (American Type Culture Collection, Manassas, VA) were maintained in Dulbeccos modification of Eagles medium (Fisher Scientific) with 10% fetal bovine serum (Invitrogen, Carlesbad, CA) and 0.5 mg/mL Gentamycin (Sigma, St. Louis, MO) at 37 C in 5% CO2/air. Cells were plated in Corning 24 well cell culture clusters and grown for 48-60 h pr ior to incubation.193 The cells were incubated with 10 mM PBS (pH=7.4) containing 0.2 mg/ml BSA solution for 30 min to prevent nonspecific binding of th e tubes to the cell surface. These cells were washed with cell media buffer and then incuba ted with the antibody-modified fluorescent silica nano test tubes (tube concentration was ~ 109 tubes/ml) for 1 h and then washed five times with cell media buffer prior to imaging. Note that two separate wells were used for the incubation of cells with the tubes; one for the target anti body-modified tubes and th e other for the control antibody-modified tubes (non-competitive).

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95 Fluorescence imaging was conducted with a conf ocal microscope setup consisting of an Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system and a tunable argon ion laser (488 nm). The imag es were taken with a 20x objective and the fluorescence was detected by a 505-525 nm band-pass filter. Microplate r eader experiment was conducted with a Tecan Safire micr oplate reader with 24 well Corn ing cell culture plates and the excess cell media buffer was removed from the pl ates prior to measurements. The excitation wavelength was 488 nm and the emission was collected at 520 nm. Results and Discussions Defect-Free Silica Nano Test Tube Preparation We have previously reported silica nano te st tube preparation using nanopore alumina templates.47 Nanopore alumina was grown only on one side of the Al foil as the other side was attached to a glass support with epoxy for stability reasons. However, when the template is dissolved, the epoxy leaches out in to the solution and contaminates the tube samples (Figure 54). Using thicker aluminum foils eliminates the need for such supports and yields alumina film on both surfaces of the Al metal (Figure 5-5, only one side is shown for simplicity.). When the conventional sol-gel method is applied to obtain sili ca test tubes from these templates, clean test tubes are obtained in larger quant ities (Figure 5-6). Note that the tube diameter reflects the template pore diameter (~ 80 nm) and the tube length reflects the template thickness (~ 1 m). Silica nano test tubes can be prepared with conventional sol-gel quite easily (< 5 min), however, the resulting tubes do not have reproducible structures (F igure 5-6C). Tubes with holes were often observed and changing the Al foil purity, hydrolysis time, TEOS concentration or dissolving conditions as well as the use of glass supported alumina templates yielded similar defective structures. These bamboo-like na nofibers were first reported by Zhang194 where they have shown that the viscosity of the gel determ ines whether the silica nanostructure will be a

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96 wire, a tube or a bamboo-like nanofiber. The de fective nanostructures in our case are observed since small variations during the sol-gel preparation (e.g. temperature or humidity) can change the viscosity of the gel. A surface sol-gel method was used to have a be tter control over the resulting silica nano test tubes. This method involves repeats of two-st ep deposition cycles, in which the adsorption of a molecular precursor (SiCl4) and the hydrolysis steps are sepa rated by a post-adsorption wash (Figure 5-1). Ideally the technique can limit each adsorption to a single monolayer, however thicker layers have been found for planar oxide films.104,106,195 Nevertheless, it allows very fine control over film thickness because a nanometer or sub-nanometer thick layer is grown on each cycle.101 Control over the atmospheric water is necessary as it rapidly polymerizes SiCl4 precursor and a silica layer deposits on the alumina template surface with uncontrollable thickness (Figure 57). This control is ach ieved by purging nitrogen stream throughout the adsorption steps. A thin layer of silica (~15 nm) is depo sited on the inner pore walls of the nanopore alumina template and on the top template surface from a SiCl4 solution (85 mol-% in CCl4) after 10 deposition cycles (Figure 5-8). The silica film on the template surface, which normally binds the nanotubes together, is removed by exposing both faces of the template to argon plasma. Figure 5-9A shows one such template after 1 min Ar-plasma treatment. When it is immersed in acid briefly, the alumina partly dissolves and re veals the protruding sili ca nanotube mouths that are not inter-connected (Figure 5-9B). Free silica nano test tubes with very smooth surface structures are obtained as the template is comp letely dissolved (Figure 5-10). Nano test tubes with different lengths can also be synthesized using alumina te mplates of various thicknesses. We have successfully varied the tube length from 100 nm to 6 m (Figure 5-10C, D). The ability

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97 to tailor the tube dimensions is an important fact or since this can affect the payload capacity of such nanotubes for delivery applications.46 Differential Modification In addition to the geometric control, the te mplate method also allo ws to independently modify the inner and outer surfaces of the tube s. When the tubes are still embedded in the template, only the inner surfaces are exposed to modifications. Once this inner surfaces is modified and the template in removed, the outer tube surfaces of the fr ee tubes are accessible, which can be further functionalized with a di fferent chemistry (Figure 5-11). A variety of functional groups can be attached to th e silica surfaces via silane chemistry196 using commercially available reagents. Previously, such differentially functionalized silica tubes are shown to selectively extract enantio meric drugs from a racemic solution.45 The motivation for making differentially function alized silica nano test tubes stems for an interest in using these tubes as drugor DNAdelivery vehicles. Th e test tube geometry is ideal for conveniently filling of the nanotube with the biomolecule of interest and by applying a cap to the open end, the biomolecule could be kept bottle d-up inside until it is ready to be delivered. We have successfully shown the capping of the t ubes with polystyrene balls using simple imine linkages.48 Potential biomedical applications will re quire that the outer surfaces of the tubes should be modified with various moieties (protein, nucleic acid s, organic functional groups) to target the nanostructures to their destinations. Template-based synthesis approach makes it possible to add these modifications afte r release from the alumina template.45,46 Proof-of-principle studies we re done where the inner tube surfaces are labeled with fluorescent tags and the outer tube surfaces ar e modified with tumor specific antibodies. Rhodamine B or Alexa Flour-488 labe led test tubes were prepared by first reacting the inner tube surfaces with APTS while the tubes were still embedded in the template. The resultant primary

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98 amine groups and then covalently coupled (Figure 5-3) to isothiocyanate or succinimidyl ester groups. Figure 5-12 shows such tube s after they have been releas ed from a 6 -thick template (same template used for the tubes in Figure 5-10 D). Since Alexa-488 is mu ch more resistant to photobleaching than other organic dyes,197 further studies only involved test tubes that are modified with this fluorophore. In order to immobilize the protein, the outer tube walls of the free fluorescent tubes are functionalized with aldehyde moieties by an aldehyde terminated siloxane linker189 (Figure 511). The aldehyde groups are then reacted by well -known Schiff-base chemistry to amine sites on the protein to be immobilized.190-192 This covalent immobilization chemistry is first confirmed with a glass slide experiment wh ere two glass slides are reacted with aldehyde silane. The first slide is then modified with ra bbit IgG and the second slide is modified with BSA. When both slides were exposed to Alexa Flour 488 labeled goat anti rabbit IgG solution; the first slide emitted distinct fluorescence at 530 nm where as the second slide showed negligible emission (Figure 5-13). This showed the successful covalent attachment of bioactive rabbit IgG on silica surface with the Schiff-base chemistry. Cell Incubation Results The cell incubation experiments were done with Alexa 488-labeled silica nano test tubes that were modified with IGF-IR or IGF-IR antibodies using Schiff-ba se chemistry for protein immobilization. IGF-IR and IGF-IR are rabbit polyclonal anti bodies raised against the and subunits of the insulin-like growth f actor-I receptor (IGF-IR), respectively.198 IGF-IR is a transmembrane protein that stimulates growth in many different cell types, blocks apoptosis, and may stimulate the growth of some types of can cer and over-expression of the IGF-IR gene has been reported in breast cancer cells.199 A recent study with MDA-MB-231 breast carcinoma cells has shown that the extracellular subunit of the IGF-IR protein showed specific activity for the

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99 IGF-IR antibody, and no activity was observed for the IGF-IR antibody.200 Consequently, to observe specific cell reaction for the silica nano te st tubes, two sets of nano test tubes were prepared. The first set wa s modified with IGF-IR (target) and the s econd set with IGF-IR (control) antibody. Figure 5-14 displays fluorescence images of two different breast carcinoma cell culture samples incubated with Alexa-488 labeled silica nano test tubes that are modi fied either with the target (Figure 5-14A) or with the control antibody (Figure 514B). Qualitative observation suggests that the tubes modified with target antibody attaches much more readily to the cell membrane surfaces than the tubes modified w ith control antibody. The tubes are generally attached to the membrane surfaces of live (elliptical) and dead (c ircular) cells and not on the well bottom. Extensive tube attachment to the well bottom was observed with tubes that are left unmodified on their outer surfaces. Further 3D sectioning studies of the confocal microscopy images are required to understa nd if any of the tube s are internalized by the carcinoma cells. We have used the same cell samples in orde r to compare the wholeplate cell fluorescence intensities using a microplate reader. The resu lt shows a fluorescence in tensity ratio of more than an order of magnitude for the cells that are incubated wi th the target antibody-modified tubes (Fl. Int. = 5495 a.u.) compared to the cells incubated with the tubes modified with control antibody (Fl. Int. = 435 a.u.). More experiments need to be conducted to verify these results. It is also important to note that these incubation studi es were carried out after the cells have been treated with BSA. When the cells were not treat ed with BSA prior to tube incubation, very similar fluorescence results were obtained from the target and control antibody-modified tubes which shows nonspecific binding of both tube types to the cell membrane surface.

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100 As a future direction, aptamer-modified nano test tubes can be used for more selective results. It has been recently reported by Tan and coworkers that aptamer-conjugated magnetic silica nanoparticles can be used for the selectiv e and sensitive detection and collection of acute leukemia cells.178 Furthermore, clever strategies need to be developed for the efficient loading and release of biomolecules into and out of these test tubes in order to use them as successful delivery devices. Conclusion We have substantiated a technique for the fa brication of uniform defect-free silica nano test tubes using alumina membrane templates. Fi rst, the advantage of us ing alumina films grown on both sides of the Al metal for having cleaner samples was shown, and then the test tube fabrication methods were compared. We have obtai ned defective test tube s with the conventional sol-gel method and this was attri buted to the small changes in th e viscosity of the gel. Uniform defect-free silica nano test tubes were prepared by layer-by-layer addition of silica through the surface sol-gel method. We have shown that argon plasma etching can be used to remove the silica film on the template surface that normally binds the nanotubes together. Using silane and Schiff-base chemistry, we have independently modi fied the inner and outer surfaces of these test tubes to investigate selective cell response via cell incubation experiments. The inner tube surfaces were first labeled with Alexa-488 fluoro phore and then the template was removed. The liberated fluorescent-tubes were modified with either a target (IGF-IR ) or a control antibody (IGF-IR ) and then incubated with breast carcino ma cells. The fluorescence imaging and the microplate reader data suggest that the tubes modi fied with target antibo dy attaches much more readily to the cell membrane surfaces than th e tubes modified with control antibody. More experiments need to be conducte d to verify these results.

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101 OH OHAlumina Pore Wall 2) Hydrolize Repeat deposition cycles Silica Gel + H2OA) B)Alumina Pore Wall OH OHAlumina Pore Wall OH OH+ HCl 1) CCl4wash] [ Si O OSiO O OH OH O Cl Si Cl O Cl Cl Si Cl O Cl Cl Si Cl Cl Cl Cl Si Cl Cl Cl] [] ] Si OH OSiO OH O O O[] ] Si O OSiO O O OO Cure Figure 5-1. Schematic of silica deposition on al umina surface by A) conventional sol-gel and B) surface sol-gel method.

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102 Figure 5-2. The structures of the silanes used for surface modifications. B) Silicon tetrachloride Cl Si Cl Cl ClA) Tetraethylorthosilicate (TEOS) H3CH2CO Si OCH2CH3OCH2CH3OCH2CH3C) 3-(amino-propyl)triethoxysilane (APTS) H3CH2CO Si OCH2CH3OCH2CH3NH2D) 3-(trimethoxysilyl)propyl aldehyde H3CO Si OCH3OCH3HC O

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103 Figure 5-3. Modification of the tube walls with fluorophore. A) Th e silica inner tube walls are functionalized with amino silane. The pr imary amine groups are then covalently coupled to B) Rhodamine B or C) Alexa Flour-488 dyes. C) + R 2 NH2 R 1 C NHR 2 =O R1= Alexa Flou r -488 =O N O R1CO OB) R 1 N = C = S + R 2 NH2 R1= Rhodamine B =S R 1 NH C NHR 2 SilicaPoreWall OH OH Hydrolyze A) Si OCH2CH3OCH2CH3CH3CH2O NH2] [ Si OH OSiO OH O NH2NH2] [ Si O OSiO O O NH2NH2Cure

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104 Figure 5-4. TEM images of test tube samples obtained from a glass supported alumina template. Epoxy resin contaminant

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105 Figure 5-5. SEM image of the cro ss-section of the alumina template.

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106 Figure 5-6. A) TEM and B,C) SEM images of the tubes obtained by conventional sol-gel method. C B 500nm A

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107 Figure 5-7. Silica deposition with surface so l-gel method without humidity control. Thick surface silica layer

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108 Figure 5-8. High resolution TEM image of the silica nano test tube with ~15 nm tube wall thickness. 100 n m

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109 Figure 5-9. SEM image of the su rface of silica deposited template A) after 1 min Ar plasma and B) after briefly dissolving the alumina template. A B

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110 Figure 5-10. SEM (A) and TEM (B,C, and D) im ages of silica nano test tubes with differe nt lengths. The templates in which these tubes are synthesized were anodized for A,B) 12 min, C) 1.5 min and D) 1 h. A B 1 m C 200 nm D 2 m

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111 Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Figure 5-11. Preparation and differential m odification of the silica nano test tubes. 3) Modify with fluorophore 1) Deposit Silica 2) Remove top Si-surface 4) Dissolve template 5) Modify outer surface with antibody Alumina Aluminum

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112 Figure 5-12. Fluorescence microscopy images of A) Rhodamine B and B) Alexa Flour-488 labeled silica nano test tubes. Scale bars ar e 10 m and acquisition time is 1.8 sec. A B

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113 Figure 5-13. Fluorescence spectra of A) Rabbit IgG and B) BSA modified glass slides after exposure to a solution containing Al exa 488tagged anti-rabbit IgG. 400450500550600650700 350 400 450 500 550 600 Fl Intensity (counts)Wavelength (nm)A400450500550600650700 400 500 600 700 800 900 1000 Fl Intensity (counts) Wavelength (nm)B

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114 Figure 5-14. Fluorescence images of two diffe rent breast carcinoma cell culture samples incubated with Alexa-488 labeled silica nano test tubes. A) test tubes are modified with target antibody B) test tubes are m odified with control antibody. Scale bars represent 200 m.

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115 CHAPTER 6 CONCLUSIONS The aim of this dissertation is to develop membrane platforms for applications in ion transport modulation and biomolecule carrier fabr ication. The use of template synthesized gold nanotube membranes and silica nano test tubes have been the common themes in this research. Chapter 1 provides background information about the template synthesis approach and its applications that are related to this work. The preparation of the tracketched polycarbonate and anodized aluminum oxide template membranes is presented in detail. Reviews of electroless gold deposition, sol-gel technology, s ilane chemistry and plasma etching that are frequently used in later chapters are then given. A brief overvie w of the delivery vehicles used in biomolecule transport is also provided. We have been interested in developing strategi es for controlling the rates of ion transport through gold nanotube membranes. Chapter 2 intr oduces a new method for electromodulated ion transport across such membranes. We have s hown that cation transport through Au nanotube membranes can be electromodulated by controlling th e extent of oxidation of a Fc-thiol attached to the Au surfaces. Electrochemical characteri zation of the Fc-thiol modified Au nanotube membranes is first examined. Surface confined cyclic volta mmograms were obtained and the stability of these voltammograms was found to depend on the redox state of Fc and the type electrolyte. We have found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic permeate species is suppressed relative to when the Fc is in its reduced state. We have defined an electromodulation selectivity coefficient for cation transport, As would be expected, higher values are obtained for membranes containing smaller inside-diameter nanotubes. For the 10 nm-diameter nanotubes a maximum value of = 9.4 was obtained. A decrease in values has

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116 been observed which is due in part to decomposition of Fc+. Membranes modified with decamethyl-ferrocene with smaller diameters are s uggested for more stable systems with higher values. The unstable nature of Fc+ has been further investigated in Chapter 3 with special interest to its decay properties in constrained geometri es. Previous studies have shown that the Fc+ decomposition is a first order decay in bulk aqueous solutions. The Fc+ decay properties of four membranes with different pore sizes were investigat ed in an aqueous electr olyte and compared to the decay for commercial gold button electrode. After the membrane samples were modified with Fc-thiol monolayer, they were exposed to argon plasma that removes Fc-thiol on Au surface films leaving only the Fc-thiol lining the Au na notube walls. The results suggest that the decay rate increases with increasing pore size and in all cases it is found to obey first order decay kinetics. Furthermore, the decay pattern resemb les a surface-like decay as the pore size of the membrane increases. These results were attributed to the mildly hydrophobic character of Fcthiol monolayer and the varying availability of counterions inside the pores as the pore dimensions change. The use of silica nano test tubes, that ar e introduced by the Mart in group, as potential universal drug delivery vehicles was exploited where these nano te st tubes could be filled with payload and then the open end corked with a chemically labile cap.48 Our long range objective with these test tubes is to impart multifunctionality through differential modification for developing a technology for cell specific biomolecu le delivery. Generally the synthesis involves deposition of silica within the pores of a nanopor e alumina template via sol-gel chemistry. Chapter 4 describes the fabrication of a uni que nanopore polymer template and its use for silica nano test tube production. A plasma etch method, using a nanopore alumina film as the

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117 mask, was used to etch a replica of the alum ina pore structure into the surface of a polymer (photoresist) film. In doing so, we created a ne w type of nanopore polymer template for use in template synthesis of nanomaterials. An appea ling feature of this new template is that the distance that the pores propagate into the su rface of the polymer film can be controlled by varying the plasma etch time. This allows fo r corresponding control over the lengths of the nano test tubes prepared by template synthesis within the pores. Via this route, we have successfully prepared silica nano test tubes that were over 100 nm shorter than the shortest tubes prepared in an alumina-film template.47 In Chapter 5, we have substantiated the fabr ication method for the pr eparation of uniform silica nano test tubes using alumina templates and then illustrated th e response of breast carcinoma cells to test tubes that have been bi ochemically modified. When conventional sol-gel method was used, defective test t ubes were obtained. This was attr ibuted to the small changes in the viscosity of the gel. Uniform defect-free si lica nano test tubes were prepared by the layer-bylayer addition of silica through the surface sol-ge l method. We have used argon plasma etching to remove the silica film on the template surf ace, which normally binds the nanotubes together. Using silane and Schiff-base chemistry, we ha ve independently modified the inner and outer surfaces of these tubes for the cell incubation stud ies. The inner tube surfaces were first labeled with a fluorophore and then the template was re moved. The liberated fluorescent-tubes were modified with either a target or a control antibody and then incubated with breast carcinoma cells. The fluorescence data suggest that the tubes modified with target antibody attaches much more readily to the cell membrane surfaces than the tubes modified w ith control antibody.

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118 LIST OF REFERENCES (1) Klabunde, K. J. Nanoscale Materials in Chemistry; Wiley-Interscience: New York, 2001. (2) Martin, C. R.; Kohli, P. Nature Rev. Drug Discov. 2003, 2, 29-37. (3) Martin, C. R. Science 1994, 266, 1961-1966. (4) Martin, C. R.; Mitchell, D. T. Anal. Chem. 1998, 70, 322A-327A. (5) Ozin, G. A. Adv. Mater. 1992, 4, 612-649. (6) Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997, 7, 1075-1087. (7) Martin, C. R.; Mitchell, D. T. Electroanal. Chem. 1999, 21, 1-74. (8) Choi, Y.; Baker, L. A.; H illebrenner, H.; Martin, C. R. Phys. Chem. Chem. Phys. 2006, 8, 4976-4988. (9) Fleischer, R. L.; Price, P. B.; Walker, R. M. Nuclear Tracks in Solids. Principles and Applications; University of Californi a Press: Berkeley, 1975. (10) Hornyak, G. L.; Patrissi, C. J.; Martin, C. R. J. Phys. Chem. B 1997, 101, 1548-1555. (11) Tonucci, R. J.; Justus, B. L.; Campillo, A. J.; Ford, C. E. Science 1992, 258, 783-785. (12) Beck, J. S.; Vartuli, J. C.; Roth, W. J.; Leonowicz, M. E.; Kresge, C. T.; Schmitt, K. D.; Chu, C. T. W.; Olson, D. H.; Sheppard, E. W.; et al. J. Am. Chem. Soc. 1992, 114, 1083410843. (13) Clark, T. D.; Ghadiri, M. R. J. Am. Chem. Soc. 1995, 117, 12364-12365. (14) Hou, S.; Wang, J.; Martin, C. R. J. Am. Chem. Soc. 2005, 127, 8586-8587. (15) Hou, S.; Wang, J.; Martin, C. R. Nano Lett. 2005, 5, 231-234. (16) Wirtz, M.; Martin, C. R. Adv. Mater. 2003, 15, 455-458. (17) Menon, V. P.; Martin, C. R. Anal. Chem. 1995, 67, 1920-1928. (18) Li, N.; Mitchell, D. T.; Lee, K.-P.; Martin, C. R. J. Electrochem. Soc. 2003, 150, A979A984. (19) Che, G.; Jirage, K. B.; Fisher, E. R.; Martin, C. R. J. Electrochem. Soc. 1997, 144, 42964302. (20) Che, G.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R. Nature 1998, 393, 346-349.

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119 (21) Patrissi, C. J.; Martin, C. R. J. Electrochem. Soc. 2001, 148, A1247-A1253. (22) Sides, C. R.; Martin, C. R. Adv. Mater. 2005, 17, 125-128. (23) Cai, Z.; Lei, J.; Liang, W.; Menon, V.; Martin, C. R. Chem. Mater. 1991, 3, 960-967. (24) Menon, V. P.; Lei, J.; Martin, C. R. Chem. Mater. 1996, 8, 2382-2390. (25) Martin, C. R. Acc. Chem. Res. 1995, 28, 61-68. (26) Cho, S. I.; Kwon, W. J.; Choi, S.-J.; Kim, P.; Park, S.-A.; Kim, J.; Son, S. J.; Xiao, R.; Kim, S.-H.; Lee, S. B. Adv. Mater. 2005, 17, 171-175. (27) Scopece, P.; Baker, L. A.; Ugo, P.; Martin, C. R. Nanotechnology 2006, 17, 3951-3956. (28) Heins, E. A.; Siwy, Z. S.; Baker, L. A.; Martin, C. R. Nano Lett. 2005, 5, 1824-1829. (29) Harrell, C. C.; Choi, Y.; Horne, L. P.; Baker, L. A.; Siwy, Z. S.; Martin, C. R. Langmuir 2006, 22, 10837-10843. (30) Mara, A.; Siwy, Z.; Trautmann, C.; Wan, J.; Kamme, F. Nano Lett. 2004, 4, 497-501. (31) Siwy, Z.; Trofin, L.; Kohli, P.; Baker, L. A.; Trautmann, C.; Martin, C. R. J. Am. Chem. Soc. 2005, 127, 5000-5001. (32) Lee, S.; Zhang, Y.; White, H. S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004, 76, 6108-6115. (33) Siwy, Z. S. Adv. Funct. Mater. 2006, 16, 735-746. (34) Jirage, K. B.; Hulteen, J. C.; Martin, C. R. Science 1997, 278, 655-658. (35) Yu, S.; Lee, S. B.; Kang, M.; Martin, C. R. Nano lett. 2001, 1, 495-498. (36) Jirage, K. B.; Hulteen, J. C.; Martin, C. R. Anal. Chem. 1999, 71, 4913-4918. (37) Steinle, E. D.; Mitchell D. T.; Wirtz, M.; Lee, S. B.; Young, V.; Martin, C. R. Anal. Chem. 2002, 74, 2416-2422. (38) Lee, S. B.; Martin, C. R. Chem. Mater. 2001, 13, 3236-3244. (39) Nishizawa, M.; Menon, V. P.; Martin, C. R. Science 1995, 268, 700-702. (40) Kang, M.; Martin, C. R. Langmuir 2001, 17, 2753-2759. (41) Lee, S. B.; Martin, C. R. J. Am. Chem. Soc. 2002, 124, 11850-11851.

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129 BIOGRAPHICAL SKETCH Fatih Buyukserin, the last of three children in the family of Husniye and Hasan Fehmi Buyukserin, was born in Konya, Turkey, on Februa ry 15th, 1980. He graduated from Bilkent University in 2001 with a Bachelor of Scie nce degree in chemistry. His interest in nanotechnology started here while he was studying the physical proper ties of silver nanoparticles under the guidance of Dr. Serdar Ozcelik. He took this to the next step by joining the research group of Dr. Charles R. Martin at the Universi ty of Florida in August 2001. He completed his research on template synthesis of nanomateri als in May 2007, obtaining a Doctor of Philosophy degree. He pursued a postdoctoral associate position at the University of Texas at Dallas working on multifunctional nanotubes for cancer diagnosis.


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Permanent Link: http://ufdc.ufl.edu/UFE0019745/00001

Material Information

Title: Template Synthesized Membranes for Ion Transport Modulation and Silica-Based Delivery Systems
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0019745:00001

Permanent Link: http://ufdc.ufl.edu/UFE0019745/00001

Material Information

Title: Template Synthesized Membranes for Ion Transport Modulation and Silica-Based Delivery Systems
Physical Description: Mixed Material
Copyright Date: 2008

Record Information

Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
System ID: UFE0019745:00001


This item has the following downloads:


Full Text









TEMPLATE SYNTHESIZED MEMBRANES FOR
ION TRANSPORT MODULATION AND SILICA-BASED DELIVERY SYSTEMS



















By

FATIH BUYUKSERIN


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

2007
































2007 Fatih Buyukserin






























To my parents, my wife Miyase, and my son Faruk Eren Buyukserin.









ACKNOWLEDGMENTS

Many individuals have been there to help me during my graduate studies. I would like to

thank Dr. Charles R. Martin for his patience, guidance and support throughout my career at the

University of Florida. He has been an excellent mentor by providing scientific discussions about

my research and at the same time a great teacher in paper writing and presentation skills.

The Martin group members have been very supportive. I am very grateful to Myungchan

Kang, Punit Kohli, Mark Wirtz, Shufang Yu and Dave Mitchell for providing insightful

discussions about my experiments and training me in surface modification, analytical detection

techniques and instrumentation. Mario Caicedo, Lane Baker, John Wharton, Lacramioara Trofin,

Damian Odom, JaiHai Wang and Fan Xu shared their experiences and provided helpful

suggestions. Miguel Mota spent many hours growing aluminum oxide for my experiments.

Heather Hillebrenner was a perfect collaborator and a great friend.

I want to thank Drs. Weihong Tan and Rick Rogers for their valued advice on my research

and career. I am also grateful to Karen Kelly and Lynda Schneider from the ICBR for their help

with SEM and TEM, to Eric Lambers from the MAIC for his help with XPS, and to Colin

Medley from the Tan research group for his help with confocal microscopy.

Finally, my family and friends provided enormous help and moral support during my

graduate career. My wife Miyase Buyukserin has always been there with her patience and

unconditional love. My parents Husniye and Hasan Fehmi Buyukserin, my brothers Mehmet and

Mustafa Buyukserin deserve most of the credit for my success as they were constant sources of

encouragement and support before and during my graduate career. I want to also thank members

of the Gainesville Turkish community, especially Fatih Gordu, Erkan Kose, Ahmet Basagalar,

Onur Kahya, Zafer Demir, Cem Demiroglu, Kaan Kececi, Nezih Turkcu and Ugur Baslanti, for

providing a very friendly, calming, home-like atmosphere.









TABLE OF CONTENTS

page

A CK N O W LED G M EN T S ................................................................. ........... ............. .....

L IS T O F T A B L E S ................................................................................. 8

LIST OF FIGURES .................................. .. ..... ..... ................. .9

A B S T R A C T ........ ........................ ............................................................ 1 1

CHAPTER

1 INTRODUCTION AND BACKGROUND ........................................ ....................... 13

In tro d u ctio n ................... ...................1...................3..........
B a c k g ro u n d ....................................................................................................................... 1 4
T em plate Synthesis................ ........... .. .................................................... .... 14
Applications in electrochemistry and sensing .....................................................15
Applications in control of ion transport and electromodulation ...........................16
Applications with silica and biomolecule nanotubes .............................................17
Track-Etched Polycarbonate Membranes ..............................................................19
Electroless plating of polymeric templates ....................................................21
Estimation of nanotube inside diameter............... ................................ 22
A nodic A lum ina Tem plates.................................................. ............................... 23
Two-step anodization m ethod ........................................................................... 24
Membrane detachment .......... ........ ......... .. ....... ............... 26
S ol-G el T echn ology ........... ....................................................................... ....... .. ....... .. 2 7
Surface Sol-G el M ethod .......... .................... ........................................ ..........................29
Silane C hem istry ....................................................... 30
Plasm a-A assisted D ry Etching ......... ................. ............................... ..................... 31
Biomolecule Delivery with Nanoparticles and Viruses ...............................................33
Chapter Summaries.......... ................. .. .... .... ..................36

2 ELECTROACTIVE NANOTUBES MEMBRANES AND REDOX-GATING .................42

In tro d u ctio n ................... ...................4...................2..........
E x p e rim e n ta l ..................................................................................................................... 4 3
M materials .........................................................................................................4 3
Electroless G old D position .................................................. ................................... 43
Membrane Sample Preparation and Thiol Modification...............................................44
Electrochem ical Experim ents .............................................. ....... ....................... 45
T ran sport E xperim ents .......................................................................... ....................4 5
R results and D iscu ssion ................. ........................................................................... .. ... 46
E lectrochem istry of the Fc-Thiol..................................................................................46
Electromodulated Transport Experiments........................ .......................... 47
C onclu sions.......... ............................... ................................................49









3 KINETICS OF FERRICINIUM DECOMPOSITION CONFINED WITHIN GOLD
NANOTUBES- EFFECT OF THE NANOSCALE ENVIRONMENT ON KINETICS.......57

In tro d u ctio n ................... ...................5...................7..........
E x p e rim e n ta l ..................................................................................................................... 5 8
M materials .........................................................................................................5 8
Electroless G old D position ..................................................................................... 58
Membrane Sample Preparation and Thiol Modification..............................................59
Surface T hiol R em ov al ......... ................. ....................................... .............................60
Electrochem ical Experim ents .......................................................... ............... 60
Results and Discussion ..................................... ................. ........ .... 61
Surface F c-T hiol R em oval ..................................................................... ...................6 1
Electrochemical Decay Studies ................................. ........ ....... ............... 62
C o n c lu sio n ............ ..................... ................. ....................... ................ 6 4

4 PLASMA-ETCHED NANOPORE POLYMER FILMS
AND THEIR USE AS TEMPLATES TO PREPARE NANO TEST TUBES ......................73

In tro d u ctio n ................... ...................7...................3..........
E x p e rim e n ta l ..................................................................................................................... 7 4
Materials ........................................................... .......... ...... .........74
Preparation of the Nanopore Alumina-Membrane Masks.............................................75
Preparation of the Nanopore Polymer-Replica Films ...................................................75
Preparation of the Silica Nano Test Tubes................................................................... 76
Results and Discussion ..................................... ................. ........ ..... 77
C onclu sions.......... ..........................................................79

5 SILICA NANO TEST TUBES AS DELIVERY DEVICES; PREPARATION AND
BIOCHEMICAL MODIFICATION ................................... .............................. 87

In tro d u ctio n ...........................................................................................................8 7
E xperim mental ........... ......................................... ................... ............... 89
Materials ......... .................. ..... .... .........................89
Preparation of the Nanopore Alumina-Membrane Templates ............. ..............90
Preparation of the Silica Nano Test Tubes............ ........... ................. .. ...............90
Silica Nano Test Tube Modification with Fluorophore ...............................................92
Antibody Modification ........... ... ........ ............................93
C ell Incubation Studies............ ... .......................................................... .......... ....... 94
R results and D iscussions........................... .... .......................... ...... ...... ..............95
Defect-Free Silica Nano Test Tube Preparation................................... ............... 95
D differential M modification ......................................................................... .................. 97
Cell Incubation Results.............. .. ............... ...................98
C on clu sion ......... ..... ............. ........................................... .................................10 0

6 CONCLUSIONS .............. ........ ..... ....................... 115

L IST O F R E F E R E N C E S ......... ................. .............................................................................118



6









B IO G R A PH IC A L SK E T C H ............................................................................... ............... ..... 129









LIST OF TABLES


Table page

2-1 Flux and electromodulated selectivity coefficients (a) for membranes containing
10-nm and 16-nm diameter nanotubes............................................... ................ 51

3-1 Fc+ decay constants for different membrane systems and for bulk aqueous solutions
of Fc compounds in phosphate solutions at neutral pH. .................................................66









LIST OF FIGURES


Figure page

1-1 The chemical structure of polycarbonate and Scanning Electron Micrograph (SEM)
of the surface of a commercial track-etched polycarbonate membrane. ...........................39

1-2 Top and cross-sectional view of PC membrane before & after the gold plating ..............40

1-3 SEM images of the surface of commercially available and home-grown alumina
m em b ran e ...............................................................................................4 1

2-1 Cyclic voltammogram for a Fc-thiol-modified Au nanotube membrane and the plot
of anodic peak current vs. scan rate from such voltammograms.....................................52

2-2 Effect of electrolyte on the stability of the Fc /Fc voltammogram ..............................53

2-3 Long-term stability of the Fc-thiol layer. ................................ ................................ 54

2-4 Plot of nanomoles of MV2+ transported across a nanotube membrane (nanotube
inside diam eter = 10 nm ) v s. tim e ............................................................................ ...... 55

2-5 Moles of electroactive Fc vs. cycle number for a membrane containing
16 nm -diam eter Au nanotubes. ................................................ ............................... 56

3-1 Finding the optimum etching time for surface Fc-thiol removal................... ..............67

3-2 XPS spectra of the Fc-thiol modified gold membrane after various argon plasma
etching periods. .............................................................................68

3-3 Cyclic voltammograms of a Fc-thiol modified membrane before (solid curve) and
after (dashed curve) 30 sec of Argon plasma etching................................ ... ................. 69

3-4 Cyclic voltammograms of four different gold nanotube membranes...............................70

3-5 Cyclic voltammograms of modified gold button electrode. .............. ......................71

3-6 First order kinetic plots for the loss of the Fc+ for gold nanotube membranes with
different pore diamaters and for a gold button electrode................................................72

4-1 Schematic diagrams of the preparation of closed-end nano test tubes and the
preparation of closed-end pores in a substrate material...............................................80

4-2 SEM images of the nanopore alumina-membrane mask. ............. .............. 81

4-3 Cross-sectional SEM of the Al-mask:Au/Pd-film:polymer-film assembly...................... 82

4-4 SEM images of the polymer-film surface and the cross-section of the film after 4 min
of 0 2/A r plasm a etching.......... ...................................................................................... 83









4-5 SEM images of the polymer-film after 8 min of 02/Ar plasma etching and the silica
nano test tubes synthesized in this template. ........................................ ............... 84

4-6 SEM images of the polymer-film after 10 min of 02/Ar plasma etching and the silica
nano test tubes synthesized in this template. ........................................ ............... 85

4-7 SEM images of the polymer-film surface and the cross-section of the film after
12 m in of 02/A r plasm a etching. .............................................. ............................. 86

5-1 Schematic of silica deposition on alumina surface by the conventional sol-gel and
surface sol-gel m ethods.......... .............................................. ................ .......... ....... 10 1

5-2 The structures of the silanes used for surface modifications ...........................102

5-3 M odification of the tube walls with fluorophore. ............................................................103

5-4 TEM images of test tube samples obtained from a glass supported alumina template. ..104

5-5 SEM image of the cross-section of the alumina template..................... ...........105

5-6 TEM and SEM images of the tubes obtained by the conventional sol-gel method.........106

5-7 Silica deposition with surface sol-gel method without humidity control ......................107

5-8 High resolution TEM image of the silica nano test tube with -15 nm tube wall
thickness ................................................................. ................ ..... 108

5-9 SEM images of the surface of silica deposited template after 1 min Ar plasma and
after briefly dissolving the alumina template .... ........... ...................................... 109

5-10 SEM and TEM images of silica nano test tubes with different lengths...........................110

5-11 Preparation and differential modification of the silica nano test tubes ..........................111

5-12 Fluorescence microscopy images of Rhodamine B and Alexa Flour-488 labeled
silica nano test tubes. ................................................. .. ......... .......... .. 112

5-13 Fluorescence spectra of Rabbit IgG and BSA modified glass slides after exposure to
a solution containing Alexa 488- tagged anti-rabbit IgG...................................113

5-14 Fluorescence images of two different breast carcinoma cell culture samples
incubated with Alexa-488 labeled and antibody-modified silica nano test tubes............14









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

TEMPLATE SYNTHESIZED MEMBRANES FOR
ION TRANSPORT MODULATION AND SILICA-BASED DELIVERY SYSTEMS

By

Fatih Buyukserin

May 2007

Chair: Charles R. Martin
Major Department: Chemistry

The objective of this research is to prepare membrane platforms for potential applications

in ion transport modulation and biomolecule delivery-device fabrication. Template synthesis

approach is used to obtain gold nanotube membranes and silica nano test tubes that are the two

main tools used in this dissertation. Chapter 1 provides an overview of the template synthesis

method and its applications. The preparation of the track-etched polycarbonate and anodized

aluminum oxide template membranes is provided. Reviews of deposition-modification

techniques and plasma etching that are used in later chapters are then given.

Chapter 2 describes an alternative method for electromodulating ion transport through

template synthesized Au nanotube membranes. This method entails attaching to the nanotubes a

molecule that contains a redox-active ferrocene (Fc) substituent. Using these redox-active

nanotubes, excess cationic charge can be placed on the membrane by oxidizing Fc to ferricinium

(Fc+) by external voltage. It has been found that when the nanotube-bound Fc is oxidized to Fc+,

the flux of a cationic permeate species is suppressed relative to when the Fc is in its reduced

state. Hence, with these redox-active tubes, the membrane can be gated between high and low

cation-transporting states.









In Chapter 3, the effect of constrained geometry on the decay properties of Fc is

examined. The Fc+ decay properties of four membranes with different pore sizes were

investigated in an aqueous electrolyte and compared to the decay for commercial gold button

electrode. After the membrane samples were modified with Fc-thiol monolayer, they were

exposed to argon plasma that removes Fc-thiol on Au surface films leaving only the Fc-thiol

lining the Au nanotube walls. The results suggest that the decay rate increases with increasing

pore size and in all cases it is found to obey first order decay kinetics.

Chapter 4 describes the fabrication of a unique nanopore polymer template and its use for

silica nano test tube production by sol-gel chemistry. Our objective with these test tubes was to

impart multifunctionality through differential modification for developing a technology for cell

specific biomolecule delivery. A plasma etch method, using a nanopore alumina film as the

mask, was used to etch a replica of the alumina pore structure into the surface of a polymer film.

The distance that the pores propagate into the photoresist film is determined by the duration of

the etching process. The pores in such plasma-etched nanopore photoresists films were used as

templates to prepare silica nano test tubes with lengths as small as 380 nm.

In Chapter 5, we have compared the preparation techniques for silica nano test tube

fabrication from alumina templates and then illustrated the response of breast carcinoma cells to

test tubes that have been biochemically modified. Defect-free uniform silica nano test tubes were

obtained by the surface sol-gel method. These test tubes were differentially modified with a

fluorophore on the inner surface and with an antibody (target or control) on the outer surface for

the cell incubation studies. The fluorescence data suggest that the tubes modified with the target

antibody attaches much more readily to the cell membrane surfaces than the tubes modified with

the control antibody. Chapter 6 summarizes the results and conclusions of this research.









CHAPTER 1
INTRODUCTION AND BACKGROUND

Introduction

Nanoscience, the science of small particles of materials, is one of the most important

research and development frontiers of modern science.1,2 The systems being studied in

nanoscience are measured by nanometer length scale and a nanometer is one billionth of a meter.

Materials of nanoscopic dimensions are of fundamental interest since the properties of a material,

such as optical, electronic and magnetic etc, can change in this regime of transition between the

bulk and molecular scale.3 These new material properties have led to potential technological

applications in areas as diverse as microelectronics, coatings and biotechnology.2 For instance,

one such application that is now in use involves using gold nanoparticles as visual indicators in

over-the-counter medical diagnostic kits.4

Nanomaterials can be fabricated through various methods, ranging from chemical methods

to lithographic techniques.5'6 The template method, pioneered by the Martin group, is a general

approach for preparing nanomaterials that involves the synthesis or deposition of the desired

material within the cylindrical and monodisperse pores of a nanopore membrane or other solid

surface.3'6 The applications of template synthesized nanomaterials composed of polymers,

metals, semiconductors, and carbons have been applied in chemical separation, sensing,

catalysis, electrochemistry, biomolecule extraction and delivery.3'4'6-8

Template synthesized gold nanotube membranes and silica nano test tubes are the main

scientific tools used in this research. This chapter provides background information on the

preparation and application of these tools. An overview of template synthesis is given which is

followed by past and recent important applications related to the presented research. The

preparation of the track-etched polycarbonate and anodized aluminum oxide template









membranes is examined. Reviews of electroless gold deposition, sol-gel technology, silane

chemistry and plasma etching that are used in later chapters are then given. Finally, a brief

overview of the delivery vehicles used in biomolecule transport is provided.

Background

Template Synthesis

Many methods for the fabrication of nanoparticles have been developed, ranging from

lithographic techniques to chemical methods.5'6 Our research group has pioneered a general

method called template synthesis for the preparation of nanoparticles.3'6 This method entails

synthesis or deposition of the desired material within the cylindrical and monodisperse pores of a

nanopore membrane or other solid. We have used nanopore polycarbonate filters, prepared via

the track-etch method,9 and nanopore alumina, prepared electrochemically from Al foil,10 as our

template materials. A variety of other porous materials such as glass nanochannel arrays,

zeolites, and polypeptide tubes can also be used as templates.1113 Depending on the properties of

the synthesized material and the chemistry of the pore wall, hollow nanotubes or solid nanowires

can be obtained.6

Probably the most useful feature of the template synthesis is that it is extremely general

with regard to the materials that can be prepared. For example, we have used this technique to

prepare nanotubes and nanowires composed of conductive polymers, metals, semiconductors,

carbon, Li+-intercalation materials, and biomolecules such as DNA and protein.6'14'15 Methods

used to synthesize such materials within the pores of the template membranes include electroless

and electrochemical metal deposition, chemical and electrochemical polymerization, sol-gel

deposition, chemical vapor deposition3'6 and layer- by-layer deposition.14'15 In addition, template

membranes contain cylindrical pores of uniform diameter which yields monodisperse

nanocylinders of the desired material with controllable dimensions. Finally, the resultant









nanotubes or nanowires can be assembled into a variety of architectures. The nanostructure can

remain inside the pores of the template membrane or they can be freed from the template

membrane and collected as an ensemble of free nanoparticles.6

Applications in electrochemistry and sensing

One very exciting application of the template synthesis is in the area of electrochemistry.16

The electroless deposition of chemistry allows us to routinely prepare ensembles of gold

nanodisk electrodes with diameters as small as 10 nm.17 Long plating times (24 h) results in the

deposition of Au nanowires into the pores. These nanoelectrode ensembles (NEE) can be used in

ultra trace detection of electroactive species. The signal-to-background (S/B) ratio at the NEE is

orders of magnitude larger than at a macroelectrode because the double-layer charging currents

at the NEE are orders of magnitude lower than those at a macroelectrode of equivalent geometric

area. This great increase in the S/B ratio allows detecting ultra trace amounts of electroactive

analytes.17

Nanostructured Li -intercalation materials that are synthesized by the template method

have been used to design novel Li-ion battery electrodes.18 These nanostructured electrodes have

improved rate capabilities compared to the thin film electrodes composed of the same material.
19-21 In addition, Sides and Martin demonstrated that V205 nanofibers prepared by sol-gel

synthesis in polymer templates show increased low-temperature performances compared to the

micrometer-sized V205 fibers.22

There has been a significant amount of research in the area of template synthesis of

conductive polymers.6 Such nanofibers of conducting polymers have been shown to be more

conductive than the bulk material.23'24 A detailed review of this topic can be found elsewhere in

the literature.25 Cho et al. recently fabricated well defined nanotube arrays of poly(3,4-

ethylenedioxythiophene) (PEDOT) that can be used as an extremely fast electrochromic display









(switching time less than 10 ms).26 The thin nature of the template synthesized nanotube walls

offers a short diffusion distance and results in ultrafast switching rates.

Finally, there is a great current interest in nanopores that have a conical pore shape and the

correspondingly conical nanostructures synthesized via the template method within these pores.27

A number of applications utilizing the conical pore geometry have been reported. For example,

such conically shaped nanopores can be used as the sensing element for new types of small

molecule,28 DNA,29'30 protein,31 and particle32 sensors. Conically shaped gold nanotubes

deposited within such pores can also mimic the function of voltage gated ion channels.33 The

details of the fabrication of the pore geometry and the sensing mechanism for such platforms has

been recently reviewed by Choi and Martin.8

Applications in control of ion transport and electromodulation

Ensembles of Au nanotubes are obtained in the multipore track-etched polycarbonate (PC)

templates when the electroless plating is done for shorter times. We discovered that by

controlling the Au deposition time, we could prepare Au nanotubes with inside diameters that

can be of molecular dimensions.34 We have demonstrated four transport-selectivity paradigms

with these Au nanotube membranes (Au-NTM). First, because the nanotubes can have inside

diameters of molecular dimensions (<1 nm), these membranes can be used to cleanly separate

small molecules on the basis of molecular size.34 The ability to control the tube diameter has also

been used in the separation of a mixture of protein molecules with different sizes.35 Second,

chemical transport selectivity can be introduced by chemisorbing thiols to the Au nanotube

walls.36-38 Third, by using a thiol with both acidic and basic functional groups, ion transport

across the Au-NTM can be modulated by controlling the pH of the contacting solution phases.38

Finally, because the Au nanotubes are electronically conductive, excess charge can be placed on

the nanotube walls by electrostatic charging in an electrolyte solution.39'40 This introduces ion-









transport selectivity as well, and the Au-NTM can be electromodulated between cation and anion

transporting states.

Lee and Martin described a unique way for the electromodulation of neutral molecules

across Au-NTMs.41 This approach makes use of an anionic surfactant which, when a positive

potential is applied to the Au-NTM, partitions into the nanotubes. Because of hydrophobic tail of

the surfactant, this renders the nanotubes interior hydrophobic, and the membrane preferentially

extracts and transports hydrophobic molecules.36 The anionic surfactant can then be expelled

from the nanotubes by applying a negative potential. This provides a route for reversibly

electromodulating neutral molecule transport.

We have recently been investigating an alternative method for electromodulating transport

in nanotube membranes.42 This method entails attaching to the nanotubes a molecule that

contains a redox active ferrocene (Fc) substituent. With these redox-active nanotubes, excess

cationic charge can be placed on the membrane by oxidizing Fc to ferricinium (Fc+) by external

voltage. Buyukserin et al. has shown that cation transport through Au-NTMs can be

electromodulated by controlling the extent of oxidation of Fc- thiol monolayer attached to the Au

surface.43 Miller and Martin demonstrated the control of surface charge, and thus electroosmotic

flow (EOF) in poly (vinylferrocene) coated carbon nanotube membranes.42 Reversible switching

between the neutral and polycationic forms of the redox-active polymer results in changes in the

rate and direction of EOF.

Applications with silica and biomolecule nanotubes

The use of silica nanotubes, whether still embedded within the template or freed from the

template, has been shown in a variety of applications.44-46 The preparation method is generally

sol-gel chemistry and the template material is commercial or home-made porous alumina

membranes. We have shown that silica nanotubes synthesized within the pores of a home-made









alumina template can be used to separate two enantiomers of a chiral drug.44 An antibody that

selectively binds one of the enantiomers of the drug was attached to the inner walls of the silica

nanotubes. Such membranes selectively transport the enantiomer that specifically binds to the

antibody, relative to the enantiomer that has lower affinity for the antibody.44

Ensembles of silica nanotubes are obtained when such a membrane is dissolved. The

nature of template synthesis allows independent modification of the inner and outer surfaces of

silica nanotubes.45'46 For example, silica nanotubes, that have been modified with a fluorophore

on the inside and a hydrophobic silane group on the outside, have been shown to selectively

partition into the organic phase in a mixture of aqueous/organic solvents.45 Furthermore, silica

nanotubes that have been modified with a certain antibody on both inner and outer tube surfaces

can be used to selectively extract the enantiomer that specifically binds to the antibody from a

racemic mixture of enantiomers.45

Novel nanostructures called nano test tubes have been recently introduced by the Martin

group.47-49 Silica nano test tubes are prepared by sol-gel synthesis of silica in the pores of an

alumina template that remains attached to underlying aluminum metal. Unlike the previously

mentioned nanotubes that are open on both ends, nano test tubes are closed on one end and open

on the other. The use of test tubes as potential universal drug delivery vehicles was exploited

where these nano test tubes could be filled with payload and then the open end corked with a

chemically labile cap.48 For such studies, the tube dimensions can have an important effect.

Buyukserin et al. very recently fabricated a nanopore polymer template that can be used to

prepare silica nano test tubes with lengths as small as 380 nm.49

Nanotubes composed of biomolecules such as DNA or protein have been fabricated by

Hou et al.14,15 Layer-by-layer deposition has been applied in both cases using a commercial









alumina membrane as the template. Protein nanotubes are obtained by alternately exposing the

template to a solution of the desired protein and then to a solution of glutaraldehyde, which acts

as crosslinking agent to hold the protein layers together. Biologically active tubes are collected

by removing the template and their activity depends on the number of layers deposited.15 The

DNA nanotubes, however, have an outer skin of one or more cX,w-diorganophosphonate/Zr(IV)

layers, to provide structural integrity, surrounding an inner core of multiple double-stranded

DNA layers held together by hybridization between the layers. The DNA components can be

released from the nanotube by melting of the DNA duplexes comprising the nanotubes.14

Track-Etched Polycarbonate Membranes

The use of nuclear tracks for the production of porous membranes was proposed almost

immediately after the discovery of particle track etching in thin sheets of materials.50 Progress in

this field was further accomplished through new particle sources, studies of new polymeric

materials, search for new applications and development of numerous methods of modification.5

There are two basic methods of producing latent tracks in the foils to be transformed into

porous membranes.51 The first method is based on the irradiation with fragments from the fission

of heavy nuclei such as californium or uranium.9'50 The main advantages of this technique are

the relatively low cost, good stability of a particle flux in time, and non-parallel particle flux that

enables the production of membranes with high porosity and low percent of overlapping pore

channels. The contamination of the tracked foil with the radioactive product is a major limitation

of the method which requires cooling of the material for few months. In addition, angle

distributions of pore channels and the range of fission fragments (membrane thickness) are

limited.51 The second method involves the use of ion beams from accelerators.9'5254 Thicker foils

with higher pore densities and controllable pore distributions can be obtained with higher energy









non-radioactive ions. Although the cost of irradiation is higher, the popularity of the ion-

accelerator facilities has been increased in the past decade.5

After irradiation, the material is subjected to chemical etching that preferentially removes

the latent ion tracks.51 As a result the latent ion track is transformed into a hollow channel. Pore

size and pore shape is determined during this chemical etching stage. The simplest description of

pore geometry is based on two parameters: bulk etch rate and track etch rate. The bulk etch rate

depends on the material, on the etchant and on the temperature. The sensitivity of the material,

irradiation conditions, post-irradiation conditions and etching conditions determines the track

etch rate. Cylindrical, conical, funnel-like, and cigar-like pore shapes can be made by controlling

the bulk and the track etching rates.51 Track etched membranes can be prepared from various

polymeric materials such as polycarbonate (PC),9 poly (ethylene terephthalate) (PET),51

polypropelene55 and polyimide.53'56 Track membranes are known as precise porous films with a

very narrow pore size distribution. The pore diameter can be from 10 nm to tens of micrometers.

The pore density can vary from 1 to 1010 cm-2.51

PC has been used for track membrane production for over thirty years.9 The chemical

etching of PC involves the rupture of chemical bonds on both sides of the carbonate group,

leading to the formation of carbonate ions (Figure 1-1A). PC has a high sensitivity for irradiation

which allows producing membranes with a pore diameter as small as 10 nm without UV

sensitization stage. When compared to PET, PC has a lower resistance to organic solvents and

lower wettability.51 Poly (vinylpyrrolidone) (PVP) coating can be used to render the PC

membranes hydrophilic.17 Track-etched PC filtration membranes are commercially available

from a number of companies (e.g. Whatman, Osmonics). Cylindrical pores are randomly

distributed on the membrane surface in these commercial membranes and pore diameters ranging









from 10 nm up to 20 tm and pore densities between 104 and 109 pores.cm-2 are available (Figure

1-1B).

Electroless plating of polymeric templates

The Martin group has developed a new class of synthetic membranes that consist of a

porous polymeric support that contains an ensemble of gold nanotubes.34'36-41 Monodisperse Au

nanotubes that span the complete thickness of the polymeric support can be prepared. The

support used in this work is the track-etched polycarbonate filter described above. The gold

nanotubes are prepared via electroless deposition of Au onto the pore walls; that is the pores act

as templates for the nanotubes (Figure 1-2). Electroless metal deposition, in general, involves the

use of a chemical reducing agent to plate a metal from solution onto a surface.5 The key

requirement of an electroless deposition bath is to arrange the chemistry such that the kinetics of

homogeneous electron transfer from the reducing agent to the metal ion is slow. Otherwise, the

metal ion would simply be reduced in the bulk solution. A catalyst that accelerates the rate of

metal ion reduction is then applied to the surface to be coated.17

The electroless deposition method for the preparation of gold nanotube membranes can be

summarized as follows; the template membrane is first "sensitized" by immersion into a SnC12

solution which results in deposition of Sn(II) onto all the membrane surfaces (pore walls and

membrane faces). Sn2+ adheres to the membrane because it is precoated with PVP during

production to render the membranes hydrophilic. Amine and carboxyl groups of PVP are thought

to act as "molecular anchors"'5 that bond the Sn2 to the membranes surfaces.59

The sensitized membrane is then immersed into an aqueous basic AgNO3 solution. This

causes a surface redox reaction in which the surface-bound Sn(II) is oxidized to Sn(IV) and the

Ag+ is reduced to nanoscopic metallic Ag particles on the membrane surface (Equation 1-1);

some silver oxide is also obtained.6









Sn(II),srf + 2Ag(I)aq Sn(IV)surf + 2Ag(0)surf (1-1)

The subscripts "surf and "aq" denote species adsorbed to the membranes surfaces and species

dissolved in solution, respectively. The membrane is then immersed into a commercial gold

plating solution and a second surface redox reaction occurs, to yield nanoscopic Au nanoparticles

on the surfaces.

Au(I)aq + Ag(0)surf Au(0)surf + Ag(I) aq (1-2)

These Au nanoparticles are excellent catalytic sites for the oxidation of formaldehyde and

the concurrent reduction of Au. As a result, Au deposition will begin at the pore walls, and Au

tubes will be obtained within the pores. In addition, the faces of the membrane become coated

with thin gold films without blocking the mouths of the nanotubes.59 The Au nanotubes can have

inside diameters of molecular dimensions (<1 nm),34 and inside diameter can be controlled at

will.36 Various applications of these membranes are presented in the template synthesis section.

Estimation of nanotube inside diameter

We use a gas-transport method to determine the effective inside diameter of the template-

synthesized Au nanotubes.36 Briefly, the tube containing membrane was placed in a gas-

permeation cell, and the upper and lower half-cells are evacuated. The upper half-cell will then

be pressurized, typically to 20 psi, with He, and the pressure-time transient associated with the

leakage of He through the tubes is measured using a pressure transducer in the lower half-cell.

The pressure-time transient was converted to gas flux (Q, mol.s-1) which is related to the radius

of the nanotubes ( r, cm) via61,62

Q = (4/3) (2 7 MRT)'2 (nr3AP /) (1-3)

where AP is the pressure difference across the membrane (dynes.cm-2), M is the molecular

weight of the gas, R is the gas constant (erg K-1 mol-1), n is the number of nanotubes in the

membrane sample, I is the membrane thickness (cm) and Tis the temperature (K). This equation









is based on the following assumptions: 1) that we know the number of tubes in the sample, 2)

that the tubes have a constant diameter down their entire length, 3) that the mechanism of gas-

transport through the membrane is Knudsen diffusion in the nanotubes.59 The presence of cigar-

shaped pores and bottlenecked tubes causes slight deviations in the first two assumptions. For

this reason, the calculated diameters are sometimes referred to as "effective inside diameters."

The current plating conditions have shown to decrease the formation of these bottlenecked tubes

and provide more uniform Au depositions.36

Gas transport through the membranes occurs via three different mechanisms; ordinary

(viscous), Knudsen or surface diffusion.63 In addition, a solution-diffusion model is adopted for

describing the transport through the non-porous solid-phase. Knudsen diffusion occurs when the

mean-free path of the gas is much larger than the average pore radius in the membrane. In our

case, equation 1-3 is predicated on Knudsen diffusion in the nanotubes. The validity of this

assumption is explored by comparing the diffusion of He/H2 and 02/N2 gas pairs through the Au

nanotubes membranes.36 The ratios of the fluxes of the two gases in each pair across membranes

of different pore sizes are compared. If the gas transport occurs via Knudsen diffusion, this ratio

is the inverse square root of the molecular weights for the two gases in each pair, and it does not

change with changing pore sizes (i.e. plating times). It has been shown that the He/H2 pair

perfectly applies the Knudsen type gas diffusion36 and He gas was used in this work to determine

the approximate inside diameter of Au nanotubes.

Anodic Alumina Templates

Anodic aluminum oxide (AAO) films formed by the electrochemical oxidation of

aluminum have been investigated and used in numerous products for more than 100 years.64-66 In

recent years, nanoporous AAO with a hexagonal arrangement of monodisperse nanopores has

become a popular template system for the synthesis of various functional nanostructures.44'45,67-69









In addition, the use of these well-ordered structures as evaporation or etching masks yields novel

nanometric materials such as nanodots, nanotubes, nanowires, nanowells and nanopores made of

metals, metaloxides and semiconductors.7-72 Nanopore arrays with interpore spacing ranging

from 50 to 400 nm, pore diameter from 10 to 200 nm, membrane thickness from 0.1 to 200 rim,

and pore density as high as 1012 pore.cm-2 can be prepared.72-74 Alumina membranes are

commercially available as 60 mrn-thick filtration membranes with pores of nominally 20, 100

and 200 nm diameters from Whatman International, Maidstone, England. Generally the pores of

commercial membranes are not uniform in size or shape (Figure 1-3A). Due to these limited and

non-uniform membrane parameters, we prepare the alumina membranes in-house (Figure 1-3B).

High purity aluminum metal (99.999%) is used in order to prepare alumina films with

highly monodisperse cylindrical pores. This metal is first mechanically polished with sand paper

(600 grit) and then electropolished at 15 V in a solution that is 95 wt% H3P04 and 5 wt % H2S04

with 20 g/L in CrO3 which prevents pitting. Using smooth electropolished aluminum surfaces is

necessary for obtaining ordered hexagonal structures.7 The aluminum is the anode, a Pb plate is

the cathode and the voltage is supplied by a variable power supply. The temperature of the

electrolyte is kept around 70 C and the polishing is done for periods of 5 minutes for at least 2

times on both surfaces for a mirror-like finish. Concentrated acid solution at high temperature is

used for immediate dissolution of alumina.7 Following the electropolishing, the Al foil is

subjected to a two step anodization process developed by Masuda and Fukuda.76

Two-step anodization method

Traditionally, the ordered pore arrangements are formed under some specific anodizing

conditions after a long anodization time, and as a result, they can only be observed on the bottom

part of the films.77 Masuda and Fukuda first showed that straight ordered nanoholes could be

formed in a thin membrane of porous alumina by striping away the thick oxides obtained from









the first long anodization and subsequently anodizing it for a short time.76 The first long

anodization allows sufficient time for self-organization and homogenization of pore size.75 Once

it is removed, an indention or pit is left in the underlying Al substrate corresponding to each

pore. The second anodization at the same voltage and in the same electrolyte results in pore

nucleation in these pits that are already highly ordered and monodisperse; thus the alumina film

grows as patterned.76 Mechanical imprinting,78 electron-beam79 and focused-ion-beam

lithographic methods80 have also been used to create nanosized indentations on the Al surface to

precisely control the pore growth process.8

Densely packed ordered hexagonal pore structure, has been reported in oxalic, sulfuric and

phosphoric acid solutions.76'82-84 We have used 5 wt % aqueous oxalic acid at 1 OC under 50 V

in both the first and the second anodization steps. The cathode is a cylindrical stainless steel tube

that supports homogenous ion flow to both surfaces of the aluminum and the solution is

vigorously stirred. The solution temperature is kept between 0 and 4 C for low reaction rates to

prevent a runaway reaction and to keep Al in contact.

The freshly electropolished Al foil is rinsed with purified water and then anodized for 12

h. This first step produces a precursor film which is then dissolved in an aqueous solution that

was 0.2 M in CrO3 and 0.4 M in H3P04 at 80 C. The same conditions were applied to this

textured Al substrate for different anodization times for the second step, and the growth rate we

obtained was 12 min anodization per 1 tm alumina film thickness. The size of the pores to be

grown is dependent on the applied potential and on the type of acid electrolyte used. In general,

smaller pores require lower voltages and highly conductive electrolytes (e.g., sulfuric acid)

where as lower conductivity electrolytes (e.g., oxalic acid) are used for larger pores.85 In









addition, immersing the resultant alumina film in dilute H3PO4 solutions can also be used to

tailor the pore dimension as it slowly reacts with alumina film and opens the pore diameter.

Membrane detachment

After the second-step anodization, the nanopore alumina can be used as a template film

while it is still attached to the underlying Al metal that gives mechanical support to the film. (See

Chapter 5.) Generally the alumina is separated from the Al base, however, and further processed

into a freestanding membrane of nanopores that is open on the top and bottom and may be used

as a base template stencil or mask for fabricating a variety of highly ordered nanostructures.72

There are three reported ways to separate the alumina film.71'73 Dissolving Al in HgCl2 solution,

alumina film separation by voltage reduction and coating an organic compound layer on the

surface of alumina to protect the original morphology from erosive CuCl2-based aluminum

removal.86 The first two methods will be discussed here.

The simplest way of separating alumina is to dissolve Al in HgCl2. Generally, thin Al foils

are most appropriate for dissolving, and the solvent does not damage alumina. Since there is a

nonporous barrier alumina layer closest to the metal surface, dissolving aluminum results in

films that are closed on one end and open on the other. The resultant film can be further

chemically etched to obtain films with pores that are open on both sides. Hazardous Hg is

produced during Al dissolution, and one foil is consumed to prepare one alumina film. The use

of progressive reduction in the anodizing voltage to create a perforation of the barrier layer and

to achieve separation of alumina film from Al is described by Furneaux et al.73 When the film

reaches the desired thickness, the voltage is reduced to about 70 % of its original value. Since the

pore size and the film thickness are dependent on the applied voltage, the pores at the barrier

layer branch to smaller sizes and the barrier layer becomes thinner. After many voltage









reduction cycles, the film/metal composite is immersed into an etchant solution. This quickly

dissolves the thin barrier layer and the alumina is detached.

In our case, total reduction process takes about 1 h, the final voltage is 15 V and the

etchant is 10 wt % H3P04. The resultant alumina film has two distinct faces; the barrier side and

the solution side. The barrier side has small branched pores that can be widened by an acid or

base etchant to have uniform pores. In the Martin group, both commercial and home-grown

alumina membranes are extensively used as templates and etching masks for the preparation of

various functional nanostructured materials. A detailed discussion is presented under the

template synthesis section.

Sol-Gel Technology

Interest in the sol-gel processing of inorganic ceramic and glass materials began as early as

the mid-1800s with Ebelmanl's87 and Graham's88 studies on silica gels. The motivation for sol-

gel processing is primarily the potentially higher purity and homogeneity and the lower

processing temperatures associated with sol-gels compared with traditional glass melting or

ceramic powder methods.89 In addition, the technique can be used to obtain homogeneous

multicomponent systems by mixing precursor solutions; this allows for easy chemical doping of

the materials prepared. Finally, the theological properties of the sol and the gel can be utilized in

processing the material, for example, by dip coating of thin films, spinning of fibers, etc.90'91

In sol-gel synthesis a soluble precursor molecule is hydrolyzed to form a dispersion of

colloidal particles (the sol). Further reaction causes bonds to form between the sol particles

resulting in an infinite network of particles (the gel).91 The gel is then typically heated to yield

the desired material.92 Organometallic compounds are used as precursor to form the colloids, and

in the case of glass, alkoxysilane precursors such as tetramethoxysilane (TMOS) and

tetraethoxysilane (TEOS) are most widely used.93'94 These alkoxysilanes readily hydrolyze in the









presence of water to form silanols (Equation 1-4). Further polycondensation reactions occur

between these silanols with other silanols (Equation 1-5, water condensation) and alkoxysilanes

(Equation 1-6, alcohol condensation).95,96

R'3 Si-O-R + H20 R'3 Si-OH +R-OH (1-4)

R'3 Si-OH + HO-SiR'3 R'3 Si-O-SiR'3 + H20 (1-5)

R'3 Si-OH + RO-SiR'3 R'3 Si-O-SiR'3 + R-OH (1-6)

Simultaneous hydrolysis and polycondensation of alkoxysilane precursors with two or

more functional groups form an interconnected 3-D silica gel network. Many factors influence

the kinetics of hydrolysis and condensation, and the systems are considerably complex as

different species are present in the solution.89 In addition, hydrolysis and condensation occur

simultaneously. Some important variables are temperature, nature and concentration of

electrolyte (acid, base), nature of the solvent and type of alkoxide precursor. Increasing

temperature and water amount increases the rate of gelation. Acid and base catalysts can be used

for rapid and complete hydrolysis so either high or low pH extremes will speed the reaction. The

nature of solvent influences the reaction rates; for example, 20 times faster rate constants were

found in acetonitrile as opposed to formamide.97 Finally, the reaction rate decreases as the

alkoxide group gets longer and bulkier.98

Hypercriticial or ambient conditions are used to convert gel into silica. When the liquid

(resultant alcohols or water) is removed as a gas phase from the interconnected solid gel network

under hypercritical conditions (critical-point drying), the network does not collapse and a low

density aerogel is produced. If the liquid is removed at or near ambient pressure by thermal

evaporation, shrinkage occurs and the monolith is called a xerogel.89 Materials with various

shapes and sizes can be obtained through molding or dip-coating of the sol since it is a liquid









form. When a template is immersed in the sol through dip-coating; a gel layer forms at the

interface of the template. This layer can be dried and converted silica that replicates the surface

topology of the template.95 Template synthesized TiO2, ZnO, WO3, MnO2, C0304,V205 and SiO2

nanotubes21'91'99'100 can be prepared with the sol-gel method.

Surface Sol-Gel Method

Precise control over the thickness and morphology of nanotubes synthesized with the

conventional sol-gel technique can be challenging.101 More reliable control over the quality of

planar thin films can be achieved by layer-by-layer deposition techniques, where colloidal

particles102'103 or molecular precursors104-106 are successively adsorbed as a layer at a time onto

the growing surface. The latter is called surface sol-gel (SSG) method and it involves repeats of

two-step deposition cycles. In this case, the adsorption of a molecular precursor and the

hydrolysis steps (for oxide film growth) are separated by a post-adsorption wash. The washing

step desorbs weakly bound molecules that form additional layers.104 The SSG technique ideally

can limit each deposition cycle to a single monolayer; however, in practice, thicker layers have

been found for planar oxide SSG films.104,106 Nevertheless, SSG allows very fine control over

film thickness because a nanometer or sub-nanometer thick layer is grown in each two-step

adsorption/hydrolysis cycle.101

Mallouk and coworkers recently reported the synthesis of silica nanotubes in anodic

aluminum oxide membranes using the SSG technique where they have achieved a sub-nanometer

control over the tube thickness.101 Furthermore, when coated on metal nanowires, this silica layer

can be a high-quality dielectric oxide coating. For this thin silica layer, they have used SiC14 as

the precursor and CC14 as the solvent/washing solution. See Chapter 5 for more details on SSG

based silica nanotubes synthesis. The same group has also demonstrated applicability of the first









layer-by layer technique to membrane substrates by preparing uniform and smooth free-standing

semiconductor/polymer nanotubes.107

Silane Chemistry

The organofunctional silanes were first introduced over 50 years ago as coupling agents for

fiberglass and have subsequently proved to be useful in various fields such as chromatography,

catalysis and polymers applications.108-110 Organosilanes form stable covalent bonds with

siliceous materials (e.g., silicates, aluminates, borates) and various metal oxides. Thus,

silanization provides a simple method for tailoring the surface chemistries of such materials. The

general formula for an organosilane (RnSiX(4-n)) indicates two classes of functionality.108 X is a

hydrolyzable group typically halogen, alkoxy, acyloxy, or amine. After hydrolysis, a reactive

silanol group is formed, which can condense with other silanol groups, for example, those on the

surface of siliceous materials. The R group is a nonhydrolyzable organic radical that may possess

a functionally that imparts desired characteristics.108 Attachment of proteins, fluorophores,

genetic material etc. can be done using this R group as reactive handles.44'45

When a monolayer of surface modification is desired, silanes with one hydrolyzable group

are used. With a single reactive group, these molecules can either bind to the surface or dimerize

and the dimers are removed by successive washing steps. Most of the widely used organosilanes

have one organic substituent.108'109 There are four steps in the reaction of these silanes and they

are analogous to the steps in sol-gel chemistry. First, hydrolysis of the three labile groups

occurs. Condensation of oligomers follows. The OH groups of the substrate then hydrogen bond

with the oligomers. Finally, a covalent linkage is formed with the substrate by the loss of water

through drying or curing. Water for hydrolysis may come from several sources. Aqueous

alcoholic silane solutions that are made acidic with acetic acid are commonly used to initiate the









formation of silanols.110 Water can also be present on the substrate surface or it may come from

atmosphere.

The degree of polymerization of the silanes is determined by the amount of water available

and the organic substituent. The concentration of the siloxane solution correlates with the

thickness of the polysiloxane layer. It has been calculated that deposition from a 0.2% silane

solution onto glass could result in eight molecular layers. These multi-layers could be either

interconnected through a loose network structure, or intermixed, or both, and are in fact formed

by most deposition techniques.108 There is a certain amount of reversibility during the formation

of covalent bonds to the surface. As water is removed by evacuation for 2 to 6 hours or by

heating to 1200 for 30 to 90 minutes, bonds may form, break and reform to relieve the internal

stress. 108

Silanes with four hydrolyzable groups provide a model for substrate reactivity and can be

utilized in surface modifications. SiC14, for example, is commercially important since it can be

hydrolyzed in the vapor phase to form amorphous fused silica.108 Organic aprotic solvents can be

used for surface treatment of chlorosilanes. Treatment from dry solvent tends to deposit a more

nearly monomolecular layer of silane than can be obtained from water.110 Chlorosilanes react

with alcohols to form alkoxysilanes which undergo most of the reactions of chlorosilanes.

Alkoxysilanes are more convenient reagents than tetrahalosilanes since they do not generate acid

on hydrolysis and are generally less reactive.108 TEOS and TMOS are common reagents used in

sol-gel based material synthesis that have four alkoxy substituents.93'94

Plasma-Assisted Dry Etching

The most important subtractive processes encountered in miniaturization science are wet

and dry etching, focused ion-beam milling, laser machining, ultrasonic drilling, electrical

discharge machining, and traditional precision machining.1ll Dry etching involves a family of









methods by which a solid surface is etched in the gas phase, physically by ion bombardment,

chemically by a chemical reaction through a reactive species at the surface, or by combined

physical and chemical mechanisms. Plasma-assisted dry etching is categorized according to

specific setup as either glow discharge (substrate and plasma are located in the same vacuum

chamber) or ion beam (substrate and plasma are in separate chambers).111

In physical etching, momentum transfer occurs between energetic ions (e.g., Ar ) and the

substrate surface. Although the selectivity is poor, directional etching patterns (anisotropic) are

obtained with this method. Some type of chemical reaction takes place in the chemical etching

method through which faster and selective etching is achieved, but the etched features are

isotropic. The most important dry etching technique is the reactive ion etching (RIE).111 It

combines physical and chemical etching mechanisms and enables profile control due to

synergistic combination of physical sputtering with the chemical activity of reactive species with

a high etch rate and high selectivity.

A plasma is an area of high energy electric or magnetic field that rapidly dissociates a

suitable feed gas to form neutrals, energetic ions, photons, electrons, and highly reactive

radicals.11 The simplest plasma reactor consists of opposed parallel-plate electrodes in a

chamber maintainable at low pressures. In argon plasma, electrical breakdown of argon gas in

this reactor will occur when electrons, accelerated in the existing electrical field, transfer an

amount of kinetic energy greater than the argon ionization potential to the argon neutrals. These

energetic collisions generate a positive ion and a second free electron for each successful strike.

Both free electrons reenergize, creating an avalanche of electrons and ions that results in a gas

breakdown emitting a characteristic glow (blue, in the case of argon). In an RF-generated

plasma, a radio-frequency voltage applied between two electrodes causes free electrons to









oscillate and collide with gas molecules, leading to a sustainable plasma. Unlike the dc plasma,

RF plasma allows etching of dielectrics as well as metals and it sustains the plasma at lower

potentials.111

There is a wide range of applications for plasma-assisted dry etching from integrated

circuit design and micro/nano machining11 to nanobatteries,18 chemical sensors70 and optical

lenses.112 In this dissertation we have used physical etching to remove Ferrocene-thiol

monolayers from the gold membrane surfaces in Chapter 3, and chemical/physical etching to

selectively remove a polymer film to fabricate silica nanostructures in Chapter 4.

Biomolecule Delivery with Nanoparticles and Viruses

The use of nanomaterials in biomolecule delivery has been shown to present various

advantages such as increased efficacy,113 protection of drugs114 or genetic materialll115'116 from

potential environmental damage and reduced drug toxicity.117 Spherical nanoparticles are almost

always used because these shapes are easier to make and can be synthesized from a diverse range

of materials, such as liposomes, lls119 polymers,120'121 dendrimers122 and various inorganic

compounds.46'115'123

Liposomes are spherical colloidal particles in which the internal aqueous cavity is

surrounded by a self-assembled lipid membrane. Due to their size, biocompatibility and

biodegradability, liposome are very promising systems for biodelivery applications.118 The

nature of the liposomes and their features are directly related to the preparation method, the

phospholipid composition and the capability of binding other chemical species. Mixtures of egg

phosphatydilcholine (PC) are primarily used because of their low cost and neutral charge

although other neutral phospholipids are also used, such as sphingomyelin and

phosphatidylethanolamine. Although liposomes could be formed spontaneously upon hydration

of lipids, they do not generally have a thermodynamically stable structure; so that external









energy, such as sonication, extrusion or homogenization, is usually required to produce

liposomes.124 They have been widely used for both drug delivery120'121'125 and gene

transfection118'120'126 after their surface is altered by adding hydrophilic substituents, such as

poly(ethylene glycol) (PEG).120 This reduces the liposome uptake by reticuloendothelial system

(RES), thereby prolonging their circulation time.127 The main drawback for the liposome based

delivery applications is the stability (either releasing the biomaterial too quickly or entrapping

too strongly).121

Polymeric micelles are self-assembling colloidal aggregates of block copolymers which

occur when the concentration reaches the crucial micelle concentration.121 The copolymer

involves a hydrophilic and a hydrophobic component where in most cases the hydrophilic

component is poly(ethylene oxide).128 There are two principal methods for the preparation of

block copolymer micelles, the direct dissolution method and the dialysis method. The direct

dissolution method simply involves adding the copolymer to water or buffer solution where as

dialysis is used for copolymers with limited water solubility.128'129 In an aqueous environment,

the hydrophobic blocks of the copolymer forms the core and the hydrophilic blocks form the

corona. These micelles are the most common vehicles for drug delivery130-132 where the

lipophilic drug is incorporated in the microenvironment of a hydrophobic micelle core. Another

polymer type used for such studies is dendrimers. Dendrimers are self-assembling synthetic

branched polymers with exquisitely tunable nanoscale dimensions133 and their application in

drug delivery134 and targeting135 has been recently investigated. Their potential for gene delivery

has also been examined where increased DNA payloads and decreased cell toxicity were

observed with these dendrimer based delivery systems.136'137 Despite various advantages,









polymeric delivery systems can present challenges for characterization and relatively low

payload capacities.121

Viral systems with highly evolved and specialized components are by far the most

effective means of DNA delivery, achieving high efficiencies (usually > 90%) for both delivery

and expression.126 Most of the recent clinical protocols involving gene therapy use recombinant

virus-based vectors for DNA delivery. However no definitive evidence has been presented for

the clinical effectiveness of any gene therapy protocol except for a few anecdotal reports of

success in individual patients.138 The impotence of current methodology is attributable to the

limitations of viral mediated delivery, including toxicity, restricted targeting of specific cell

types, limited DNA carrying capacity, production and packaging problems, recombination, and

high cost.139,140 These systems are also likely to cause unexpected cytotoxicity and

immunogenicity which hamper their routine use in basic research laboratories.116 For these

reasons, nonviral synthetic DNA delivery systems have become increasingly desirable in both

basic research laboratories and clinical settings.126

The application of some inorganic nanoparticles for biomolecule delivery has been

recently shown; gold and silica nanoparticles, for example have been employed in DNA

delivery.115'141 Unlike nanoparticles or nanorods, nanotubes have a unique hollow structure

which allows the modification of their inner surface and filling with specific biomolecules.

However, the applications of nanotubes as biomolecule carriers are still very rare.116,142 The

template method developed in Martin group allows independent modification of inner and outer

surfaces of the nanotubes through which multifunctional tubes with controllable dimensions can

be obtained.46 Multifunctionality is highly required for modern biomedical applications125 and









these differentially modified tubes are potential novel tools for such studies. See Chapter 5 for

more details on differentially modified nanotubes and nano test tubes.

Chapter Summaries

Chapter 2 describes an alternative method for electromodulating ion transport through

template synthesized Au nanotube membranes. This method entails attaching to the nanotubes a

molecule that contains a redox-active ferrocene (Fc) substituent. Electrochemical

characterization of the Fc-thiol modified Au nanotube membranes is first examined. Surface

confined cyclic voltammograms were obtained and the stability of these voltammograms was

found to depend on the redox state of Fc and the electrolyte type. Using these redox-active

nanotubes, excess cationic charge can be placed on the membrane by oxidizing Fc to ferricinium

(Fc ) by external voltage. It has been found that when the nanotube-bound Fc is oxidized to Fc+,

the flux of a cationic permeate species is suppressed relative to when the Fc is in its reduced

state. Hence, with these redox-active tubes, the membrane can be gated between high and low

cation-transporting states.

Chapter 3 examines the effect of constrained geometry on the decay properties of Fc.

Previous studies have shown that the Fc+ decomposition is a first order decay in bulk aqueous

solutions. The Fc+ decay properties of four membranes with different pore sizes were

investigated in an aqueous electrolyte and compared to the decay for commercial gold button

electrode. After the membrane samples were modified with Fc-thiol monolayer, they were

exposed to argon plasma that removes Fc-thiol on Au surface films leaving only the Fc-thiol

lining the Au nanotube walls. The results suggest that the decay rate increases with increasing

pore size and in all cases it is found to obey first order decay kinetics. Furthermore, the decay

pattern resembles a surface-like decay as the pore size of the membrane increases. These results









were attributed to the varying hydrophobic character of Fc-thiol monolayer and availability of

counterions inside the pores as the pore dimensions change.

In Chapter 4, the fabrication of a unique nanopore polymer template and its use for silica

nano test tube production is described. Our objective with these test tubes is to develop a

technology for cell specific biomolecule delivery. A plasma etch method, using a nanopore

alumina film as the mask, was used to etch a replica of the alumina pore structure into the

surface of a polymer film. The distance that the pores propagate into the photoresist film is

determined by the duration of the etching process. Hence, by controlling the etch time, we

effectively control the thickness of the nanopore layer etched into the surface of the photoresist.

The pores in such plasma-etched nanopore photoresists films were used as templates to prepare

silica nano test tubes via sol-gel chemistry. As expected the length of the test tubes is

determined by the thickness of the porous part of the photoresist film. Test tubes with lengths of

380 nm were obtained, shorter than any of the nano test tubes previously reported where the

alumina film was used as the template.

Chapter 5 compares the preparation techniques for uniform silica nano test tube fabrication

and then illustrates the response of breast carcinoma cells to test tubes that have been

biochemically modified. Defective test tubes were obtained with the conventional sol-gel method

and it was attributed to the small changes in the viscosity of the gel. Layer-by-layer addition of

silica with the surface sol-gel method allowed preparation of defect-free uniform silica nano test

tubes. We have differentially modified these test tubes for the cell studies. Before the template

was removed, the inner tube surfaces were labeled with a fluorophore. The liberated fluorescent

tubes were then modified with a target or a control antibody and then incubated with breast

carcinoma cells. The preliminary results suggest that the tubes modified with target antibody









attaches much more readily to the cell membrane surfaces than the tubes modified with control

antibody. The results and conclusions of this dissertation are summarized in Chapter 6.











0 CH3




CH3





























Figure 1-1. A) The chemical structure of polycarbonate. B) Scanning Electron Micrograph
(SEM) of the surface of a commercial track-etched polycarbonate membrane.











*Stannous Chloride solution
Polycarbonate membrane, 'Silver Nitrate Solution
Top View --
*Gold plating Solution


DnD


Gold nanotube membrane,
Top View


DDD


Polycarbonate membrane,
Cross-sectional View


Gold nanotube membrane,
Cross-sectional View


Figure 1-2. Top and cross-sectional view of PC membrane before & after the gold plating.













































Figure 1-3. SEM images of the surface of anodized aluminum oxide (alumina) membranes.
A) Commercially available alumina membrane. B) Home-grown alumina membrane.















41









CHAPTER 2
ELECTROACTIVE NANOTUBES MEMBRANES AND REDOX-GATING

Introduction

We have developed a new class of synthetic membranes that contains monodisperse Au

nanotubes with inside diameters that can be of molecular dimensions (<1 nm).34,36-41 The Au

nanotubes span the complete thickness of the membrane and can act as conduits for molecule

and ion transport between solutions placed on either side of the membrane. We have

demonstrated four transport-selectivity paradigms with these Au nanotube membranes. First,

because the nanotubes can have inside diameters of molecular dimensions (<1 nm), these

membranes can be used to cleanly separate small molecules on the basis of molecular size.34

Second, chemical transport selectivity can be introduced by chemisorbing thiols to the Au

nanotube walls.36-38 Third, by using a thiol with both acidic and basic functional groups, ion

transport across the Au nanotube membrane can be modulated by controlling the pH of the

contacting solution phases.38 Finally, because the Au nanotubes are electronically conductive,

excess charge can be placed on the nanotube walls by electrostatic charging in an electrolyte

solution.39-41 This introduces ion-transport selectivity as well, and the Au nanotube membranes

can be electromodulated between cation and anion transporting states.

We have recently been investigating an alternative method for electromodulating transport

in nanotube membranes.42 This method entails attaching to the nanotubes a molecule that

contains a redox-active ferrocene (Fc) substituent. With these redox-active nanotubes, excess

cationic charge can be placed on the membrane by using the potential applied to the membrane

to driving the following redox reaction:42'143-145

Fc Fc+ + e- (2-1)









We have found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic

permeate species is suppressed relative to when the Fc is in its reduced state. While similar

results have been achieved using membranes composed of redox-active conductive

polymers,146-148 this paradigm for gating ion transport has not been demonstrated for redox-active

nanotube membranes. We describe the results of such redox-modulated transport experiments

here.

Experimental

Materials

Polycarbonate filtration membranes (6 ntm-thick, 30 nm- and 50 nm-diameter pores, 6x108

pores cm-2) were obtained from Osmonics Inc. Commercial gold-plating solution (Oromerse SO

Part B) was obtained from Technic Inc. Na2SO3, NaHCO3, NH4OH, HNO3, KC1, methanol and

formaldehyde were obtained from Fisher and used as received. SnC12, methyl viologen

dichloride hydrate, and 1,5-naphthalene disulfonic acid disodium salt hydrate were used as

received from Aldrich, as were KC104, AgNO3 and triflouoroacetic acid from Acros Organics,

ethanol (absolute) from Aaper, and 11-ferrocenyl-l-undecanethiol from Dojindo Chemicals.

Purified water was obtained by passing house-distilled water through a Millipore, Milli-Q

system.

Electroless Gold Deposition

The electroless deposition or plating method described previously was used to deposit gold

nanotubes within the pores of the nanopore polycarbonate membranes.59 In general terms, this

entails depositing gold along the pore walls so that each pore becomes lined with a gold

nanotube. Briefly, the template membrane was first immersed into methanol for five minutes

and then immersed for 45 min into a solution that was 0.025 M in SnCl2 and 0.07 M in

trifluoroacetic acid. This yields the Sn-sensitized form of the membrane.17 The membrane was









then immersed into an aqueous ammoniacal AgNO3 solution (0.029 M Ag ) for 7.5 minutes and

then immersed in methanol for 5 minutes. The gold plating bath was prepared by mixing 0.5 ml

of the commercial gold-plating solution with 20 mL of an aqueous solution that was 0.127 M in

Na2SO3, 0.625 M in formaldehyde, and 0.025 M in NaHCO3. The bath pH was lowered to 10 by

drop wise addition of 1 M H2S04 prior to immersion of the membrane. During electroless

deposition, the temperature of the bath was maintained at 4 C. Membranes were placed in the

gold-plating bath for different periods of time to obtain nanotubes of different inside

diameters.36,149 The inside diameter of the nanotube was determined using the gas-flux

measurement described previously.36

Membrane Sample Preparation and Thiol Modification

The electroless-plating method yields the Au nanotubes lining the pore walls as well as

thin Au films covering both faces of the membrane.17 The Au films do not block the mouths of

the nanotubes at the membrane faces and can be used to make electrical contact to all of the

nanotubes in parallel.39 This was accomplished by applying a copper tape with a conductive

adhesive (3M, #1181) to the outer edge of one Au surface film.17

The membrane sample was prepared by sandwiching the nanotube membrane between two

pieces of electrically insulating plastic tape (3M Scotch brand no. 375). Each piece of tape had a

0.2 cm2-area hole punched through it, and the holes were aligned on either side of the membrane.

This insulating tape also covered the conductive tape used to make electrical contact to the

membrane. The end of the copper tape protruding from the membrane sample was used as the

electrode lead for electrochemical experiments in which the membrane sample was the working

electrode. Details of this electrode fabrication method can be found elsewhere in the literature.17

The Au surface films and Au nanotube walls were modified with the thiol 11-ferrocenyl-1-

undecanethiol, here after called Fc-thiol. This was accomplished by mounting the assembled









membrane sample between the two halves of a U-tube permeation cell34'36'39 and filling both

half-cells with a 2 mM solution of Fc-thiol dissolved in ethanol. The membrane sample was

exposed to this solution for 20 h, followed by thorough washing with ethanol.

For some membranes, the Fc-thiol on the Au surface films was removed by brief (30 sec)

exposure to a mild Ar plasma. A Samco model RIE-1C reactive-ion etch system was used. The

plasma conditions were as follows: 13.56 MHz, 50 W, 10 Pa Ar pressure, Ar flow rate =12 sccm.

Electrochemical Experiments

Electrochemical experiments were done with the membrane sample mounted in the U-

tube cell. Electrolyte solution was added to both half-cells, and the Au nanotube membrane was

made the working electrode in a conventional three-electrode experiment. The counter electrode

was a Pt wire and the reference was an Ag/AgCl electrode with 3 M NaC1. In the transport

experiments one half-cell solution, the feed half-cell, contained the permeating species and the

other half-cell received the permeating species. The reference and counter electrodes were

placed in the feed half-cell. A Solartron SI 1287 electrochemical interface module (Solartron

Analytical, Hampshire, England) connected to a PC running CorrView and CorrWare software

(Scribner Asc. Inc., NC) was used.

Transport Experiments

The same U-tube cell was used for the transport experiments. The permeating specie

investigated was the dication methylviologen (MV2+). The feed half-cell was charged with 20

mL of a 20 mM aqueous MV2+ solution, and the receiver half-cell was charged with 20 mL of

purified water. The flux of MV2+ from the feed half-cell, through the membrane and into the

receiver half-cell was obtained by continuously measuring the UV absorbance (at 260 nm) of the

receiver half-cell solution. A flow-through Agilent 8458 spectrophotometer was used.34'39'150









The data were processed as plots of moles MV2+ transported vs. time. Straight line plots were

obtained, and the flux of the permeating ion was calculated from the slope.

Results and Discussion

Electrochemistry of the Fc-Thiol

Figure 2-1A shows a cyclic voltammogram for a Fc-thiol-modified Au nanotube

membrane (nanotube inside diameter = 8 nm). The redox waves associated with the oxidation of

the Fc to Fc+ and the re-reduction back to Fc are clearly seen.145 151-153 Figure 2-1B shows that

the anodic peak current is linearly related to scan rate as would be expected for a surface-

confined voltammogram.154

It is of interest to note, however, that there are in essence two different Au surfaces in these

membranes The Au on the inside walls of the nanotubes running through the membrane and the

Au surface films on both faces of the membrane. If the number of moles of Fc obtained from the

area under the anodic wave is divided by the total Au area (tube walls plus surface films), a

coverage by Fc of 1.0x10-9 moles.cm-2 is obtained. This is about a factor of two larger than the

value calculated from the footprint of the Fc molecule on an atomically flat Au surface.145'153 The

higher value obtained experimentally here simply reflects the surface roughness of our

electrolessly deposited gold.

Figure 2-2 shows the effect of electrolyte on the stability of the Fc/Fc+ redox couple.

When KC1 was used, the voltammogram current decayed continuously with scan number (Figure

2-2A). As has been discussed previously153, this is due to nucleophilic attack of Cl on the

Fe(III) center of Fc As shown by the analogous set of 30 cyclic voltammograms in Figure 2-

2B, the redox chemistry is much more stable in 0.1 M KC104.153 This is because C104- is a

poorer nucleophile than C1-. For long-term use, however, it is best to store the Fc-thiol-modified









membrane in its reduced (Fc) state. If this is done in the KC104 solution, Fc-thiol

electrochemistry can be observed, unchanged, for periods of at least one week (Figure 2-3).

Figure 2-2 also shows that the oxidation of Fc-thiol proceeds at more negative potentials in

KC104 than in KC1. Such effects have been observed previously for ferrocene-modified

electrodes and have been attributed to the different extents to which the anions of the electrolyte

form ion-pairs with Fc+.151152 Fc+ is a lipophilic cation, present in a lipophilic monolayer film,

and therefore ion pairs preferentially with the more lipophilic C104-. This ion-pair interaction

makes the oxidation thermodynamically easier in C104 vs. C1-. The shift in the position of the

Fc-thiol voltammetric wave with time in KC1 (Figure 2-2A) has also been observed previously,

although no explanation was offered.153 We suggest that as decomposition of the lipophilic

cyclopentadienly ring occurs (with increasing scan number in KC1, Figure 2-2A) the monolayer

film becomes less lipophilic, and this allows C1- to have greater ion-pairing access to the

remaining intact Fc+ groups.

Electromodulated Transport Experiments

A solution of the cationic permeating species MV2+ was placed on one side of the Fc-thiol-

modified membrane, and the quantity of this species transported through the nanotubes and into

the receiver solution on the opposite side was measured as a function of time (Figure 2-4).

During the time interval from 0 to -1700 sec, a potential of 0.7 V was applied to the membrane.

At this potential the ferrocene is present as oxidized Fc+, yielding excess positive charge on the

nanotube walls and membrane faces. This charge causes MV2+ to be electrostatically repelled

from the membrane, yielding the low-flux state for MV2+ transport. Complete exclusion of

MV2+ is not observed because at the 20 mM salt (MVC12) concentration used in this experiment,

the electrical double layer on the walls of the 10 nm-diameter nanotube does not completely fill

the total nanotube volume. As we have discussed in detail previously,39'40 this means that there









is a region in the center of the nanotube where MV2+ is not excluded, and transport occurs in the

region.

At 1800 sec a potential of 0 V was applied to the membrane. At this potential the

ferrocene on the nanotube walls, and membrane faces, is present as neutral Fc. Because there is

now no excess positive charge on the membrane, MV2+ is not repelled, and a higher MV2+ flux

(relative to the short time data) is obtained (data points for line 2, Figure 2-4). The slopes of the

straight-line segments in Figure 2-4 provide the fluxes for MV2+ across the nanotube membrane.

We define an "electromodulation-transport cycle" as a period when 0.7 V was applied (low flux

state) followed by a period when 0 V was applied (high flux state). This allows us to define an

electromodulated-transport selectivity coefficient (a) as the flux during the high-flux state (0 V)

divided by the flux during the low-flux state (0.7 V). The larger the value of a, the greater is the

electromodulated cation-gating effect.

Table 2-1 shows flux and a values for various cycle numbers for membranes with 10 and

16 nm-diameter Au nanotubes. Considering the flux data first, we see as would be expected, that

the fluxes in the membrane with the larger-diameter nanotubes is higher. However, the

selectivity for the membrane containing these larger diameter nanotubes is lower. Again, this is

due to the fact that the electrical double layer that is responsible for repelling MV2+ fills a

smaller fraction of the total nanotube volume for the larger diameter nanotube.39'40

The electromodulated selectivity coefficient, a, decreases with increasing cycle number

(Table 2-1). Part of this decay in selectivity is due to the fact that the magnitude of the flux in

the low-flux (Fc+) state increases with each successive cycle. To understand the origins of this

effect we obtained a cyclic voltammogram after each cycle, and from the area under the anodic

wave obtained the moles of electroactive Fc remaining in the membrane (Figure 2-5). We see









that there is a steady drop in amount of electroactive Fc with cycle number. While this may at

first seem to contradict the data in Figure 2-3, the key difference is that in Figure 2-3 the

ferrocene was left in the neutral Fc state between cycles, and in Figure 2-5 the Fc was held in the

charged Fc+ for long periods (Figure 2-4) during each cycle. Because it is the Fc+ state that is

susceptible to nucleophilic attack,155'156 electroactivity decays much more quickly in Figure 2-5

than in Figure 2-3.

This steady drop in electroactive Fc in the membrane with cycle number (Figure 2-5)

explains why the selectivity decays with cycle number (Table 2-1). This is because it is the

positively charged Fc+ groups that repel MV2+, and since the quantity of Fc+ decreases with cycle

number, the selectivity decreases with cycle number. The other factor causing the selectivity to

decay with cycle number is that the magnitude of the flux in the high flux state decreases with

cycle number (Table 2-1). This suggests that membrane fouling occurs. One possible source of

membrane fouling is that the decomposition products that result from nucleophilic attack on the

Fc+ causes partial occlusion of the nanotubes.

Conclusions

We have shown that cation transport through Au nanotube membranes can be

electromodulated by controlling the extent of oxidation of a Fc-thiol attached to the Au surfaces.

We have defined an electromodulation selectivity coefficient for cation transport, a. As would

be expected, higher a values are obtained for membranes containing smaller inside-diameter

nanotubes. For the 10 nm-diameter nanotubes a maximum value of a= 9.4 was obtained. It is

possible to make smaller diameter nanotubes,34 and it would be of interest to see if

correspondingly higher selectivity coefficients could be obtained. Unfortunately, the

electromodulated selectivity decreases with membrane use because when the Fc is present in the

Fc+ state it is susceptible to nucleophilic attack and decomposition. It is well-known that









decamethyl-ferrocence is less susceptible to this degradation pathway,157,158 and for this reason

would be a better choice for the nanotube-bound electromodulating agent.









Table 2-1. Flux and electromodulated selectivity coefficients (a) for membranes containing
10-nm and 16-nm diameter nanotubes.
Nanotube Diamater Cycle Low Flux High Flux a
(nm) Number nmole min-cm2- nmole min cm2-
10 1 1.2 11 9.4
10 2 1.5 11 7.3
10 3 2.0 10 5.1
16 1 6.4 38 5.9
16 2 7.3 38 5.2
16 3 7.8 34 4.3
16 4 8.3 32 3.8
16 5 9.5 28 2.9

























-20-



-40--
0.0


0.2


0.4


0.6


0.8


Potential / V vs. Ag/AgCI


20 40 60 80 100


Scan Rate/ mV s


Figure 2-1. A) Cyclic voltammogram for a Fc-thiol-modified Au nanotube membrane with
nanotube inside diameter = 8 nm. Scan rate = 70 mV s-1. B) Anodic peak current
from such voltammograms vs. scan rate. The electrolyte in both half-cells was
0.1M KC1.












15- A


10-


5-


< 0


-5-


-10-


-15 i
0.0 0.2 0.4 0.6 0.8




15- B


10-


5-


< 0-


-5-


-10-


-15 i, ,
0.0 0.2 0.4 0.6 0.8
Potential/ V vs. Ag/AgCI


Figure 2-2. Effect of electrolyte on the stability of the Fc+/Fc voltammogram. The potential was
swept continuously through the voltammetric waves for 30 scans at 20 mV s-1. The
membrane contained nanotubes with inside diameter of 26 nm. A) Electrolyte was
0.1 M KC1. The arrow points in the direction of increasing scan number (scan 1 to
scan 30). B) Electrolyte was 0.1 M KC104.
















10



0



-10



-20
0.0 0.2 0.4 0.6 0.8
Potential / V vs. Ag/AgCI


Figure 2-3. Investigation of the long term stability of the Fc-thiol layer. The Au nanotube
membrane sample (nanotube inside diameter = 10 nm) was mounted in the U-tube
cell with 0.1 M KC104 in both half-cells, and voltammograms were obtained after 2
days (solid black curve), 4 days (solid gray curve), and 6 days (dashed black curve) of
storage unpotentiostated in the reduced (Fc) state. The half-cell solutions were not
degassed and the U-tube cell was not protected from light.










200
180 /
160 5
0o 140 -
S120
100 /
5 80
E0
c 60 -
z
40 2
20
0 1

0 2000 4000 6000 8000 10000 12000
Time/ sec

Figure 2-4. Plot of nanomoles of MV2+ transported across a nanotube membrane (nanotube
inside diameter = 10 nm) vs. time. Data points for lines 1, 3 and 5 were obtained with
a potential of 0.7 V vs. Ag/AgCl applied to the membrane. Data points for lines 2, 4
and 6 were obtained with a potential of 0 V vs. Ag/AgCl applied to the membrane.
The slopes of these straight lines are used calculate to the flux of MV2+. The feed
solution was 20 mM in MV2+











0.8



0.6

o U
0_0
E 0.4
0 2


0.2



0
0 1 2 3 4 5
Cycle number

Figure 2-5. Moles of electroactive Fc vs. cycle number for a membrane containing
16 nm-diameter Au nanotubes.









CHAPTER 3
KINETICS OF FERRICINIUM DECOMPOSITION CONFINED WITHIN GOLD
NANOTUBES- EFFECT OF THE NANOSCALE ENVIRONMENT ON KINETICS

Introduction

We have been investigating a general method for preparing nanomaterials called template

synthesis.3,4,6 This method entails synthesis of the desired material within the cylindrical and

monodisperse pores of a nanopore membrane or other solid. Using this method, a new class of

synthetic membrane was developed that contain monodisperse Au nanotubes with inside

diameters that can be of molecular dimensions (<1 nm).34'36-39'41 The Au nanotubes span the

complete thickness of the membrane and can act as conduits for molecule and ion transport

between solutions placed on either side of the membrane. We have been using these gold

nanotube membranes to investigate how pore size, charge and chemistry affect transport

selectivity in membranes. Of particular relevance to the work reported here, ion and chemical

transport selectivity can be successfully introduced and modulated by chemisorbing thiols to the

Au nanotube walls.36'38'41

We have recently reported an alternative method for electromodulating ion transport in Au

nanotube membranes. This method entails chemisorbing to the Au nanotubes an alkyl thiol that

contains a redox-active ferrocene (Fc) substituent. With this membrane system the charge

density on the nanotube walls can be electromodulated Faradaically by using the potential

applied to the Au nanotube membrane to control the position of equilibrium for the following

redox reaction:42'143-145

Fc Fc+ + e- (3-1)

We have found that when the nanotube-bound Fc is oxidized to Fc+, the flux of a cationic

permeate species is suppressed relative to when the Fc is in its reduced state. However, the flux

difference between these states is lost with membrane use because when the Fc is present in the









Fc+ state, it is susceptible to nucleophilic attack and decomposition.153 The extent of Fc+

decomposition is directly related to the strength of the nucleophile155 and it is a first order decay

in aqueous solutions.159

In this chapter, we report the results of nanotube pore size affect on Fc+ decomposition.

For this purpose, it was necessary to remove the Fc-thiol on Au surface films leaving only the

Fc-thiol lining the Au nanotube walls. This was accomplished by briefly (30 sec) exposing both

faces of the membrane to an argon plasma (mild conditions). The behavior of four membranes

with different pore sizes were investigated and compared to the decay in commercial gold button

electrode. The results suggest that the decay rate increases with increasing pore size and in all

cases it is found to obey first order decay kinetics. Furthermore, the decay pattern resembles a

surface-like decay as the pore size of the membrane increases.

Experimental

Materials

Polycarbonate filtration membranes (30 nm-, 50 nm-, 200 nm- and 600 nm- diameter

pores) were obtained from Osmonics Inc. Commercial gold-plating solution (Oromerse SO Part

B) was obtained from Technic Inc. Na2SO3, NaHCO3, NH4OH, HNO3, methanol and

formaldehyde were obtained from Fisher and used as received. SnC12 was used as received from

Aldrich, as were KC104, AgNO3 and triflouoroacetic acid from Acros Organics, ethanol

(absolute) from Aaper, and 11-ferrocenyl-l-undecanethiol from Dojindo Chemicals. Purified

water was obtained by passing house-distilled water through a Millipore, Milli-Q system.

Electroless Gold Deposition

The electroless deposition or plating method described previously was used to deposit gold

nanotubes within the pores of the nanopore polycarbonate membranes.59 In general terms, this

entails depositing gold along the pore walls so that each pore becomes lined with a gold









nanotube. Briefly, the template membrane was first immersed into methanol for five minutes

and then immersed for 45 min into a solution that was 0.025 M in SnC12 and 0.07 M in

trifluoroacetic acid. This yields the Sn-sensitized form of the membrane.17 The membrane was

then immersed into an aqueous ammoniacal AgNO3 solution (0.029 M Ag ) for 7.5 minutes and

then immersed in methanol for 5 minutes. The gold plating bath was prepared by mixing 0.5 ml

of the commercial gold-plating solution with 20 mL of an aqueous solution that was 0.127 M in

Na2SO3, 0.625 M in formaldehyde, and 0.025 M in NaHCO3.

The bath pH was lowered to 10 by drop wise addition of 1 M H2S04 prior to immersion of

the membrane. During electroless deposition, the temperature of the bath was maintained at

4 C. Membranes were placed in the gold-plating bath for different periods of time to obtain

nanotubes of different inside diameters.36'149 The inside diameter of the nanotube was

determined using the gas-flux measurement described previously36 where the pore diameter was

< 50 nm. For bigger pores, electron micrographs of the pores obtained via Hitachi S4000 FE-

SEM were used to calculate the pore diameter. Gold nanotube membranes with pore diameters

10 2.0, 28 2.6, 65 7.5, and 284 20 nm were used in this work.

Membrane Sample Preparation and Thiol Modification

The electroless-plating method yields the Au nanotubes lining the pore walls as well as

thin Au films covering both faces of the membrane.17 The Au films do not block the mouths of

the nanotubes at the membrane faces and can be used to make electrical contact to all of the

nanotubes in parallel.39 This was accomplished by applying a copper tape with a conductive

adhesive (3M, #1181) to the outer edge of one Au surface film.17

The membrane sample was prepared by sandwiching the nanotube membrane between two

pieces of electrically insulating plastic tape (3M Scotch brand no. 375). Each piece of tape had a

0.2 cm2-area hole punched through it, and the holes were aligned on either side of the membrane.









This insulating tape also covered the conductive tape used to make electrical contact to the

membrane. The end of the copper tape protruding from the membrane sample was used as the

electrode lead for electrochemical experiments in which the membrane sample was the working

electrode. Details of this electrode fabrication method are described elsewhere in the literature.17

The Au surface films and Au nanotube walls were modified with the thiol 11-ferrocenyl-1-

undecanethiol, here after called Fc-thiol. This was accomplished by mounting the assembled

membrane sample between the two halves of a U-tube permeation cell34'36'39 and filling both

half-cells with a 2 mM solution of Fc-thiol dissolved in ethanol. The membrane sample was

exposed to this solution for 20 h, followed by thorough washing with ethanol. A commercial

gold button electrode (Bioanalytical Systems, Inc. IN) was modified under the same conditions

after being polished with alumina nanoparticles.

Surface Thiol Removal

The Fc-thiol modified gold nanotube membrane sample was placed into the vacuum

chamber of a reactive-ion etching system (Samco model RIE-1C). The plasma conditions were -

13.56 MHz, 50 W, 10 Pa Ar pressure, Ar flow rate =12 sccm. In order to confirm the removal of

Fc monolayer from the membrane surface, we have used a Kratos Analytical Surface Analyzer

XSAM 800 with a Mg source that is normal to the sample surface. This instrument was used to

detect the surface Fe 2p3/2 peak for membranes before and after Ar plasma etching for different

etching times.

Electrochemical Experiments

After the plasma etching, the membrane was washed with ethanol and water and then

subjected to electrochemical experiments. Electrochemical experiments were done with the

membrane sample mounted in the U-tube cell. 0.1 M KC104 electrolyte solution was added to

both half-cells, equilibrated for 1-2 days and bubbled with Argon for 30 minutes before the









experiment. Argon was also purged into the system throughout the experiment. The Au

nanotube membrane was made the working electrode in a conventional three-electrode

experiment where the counter electrode was a Pt wire and the reference was an Ag/AgCl

electrode with 3 M NaC1. A Solartron SI 1287 electrochemical interface module (Solartron

Analytical, Hampshire, England) connected to a PC running CorrView and CorrWare software

(Scribner Asc. Inc., NC) was used.

In order to observe and calculate the decay in the Fc+, the membrane sample was held at

0.7 Volts for 6 hours during which cyclic voltammograms (CVs) were taken periodically. The

cathodic half cycles of these CVs were then used to calculate the amount of redox-active Fc for

each CV. The same conditions were also applied to Fc-thiol modified gold button electrode

which was not exposed to any plasma treatment.

Results and Discussion

Surface Fc-Thiol Removal

In order to study the effect of pore size on Fc+ decay, we needed a technique to remove all

Au surface Fc but do not destroy the Fc-thiol lining the Au nanotube walls. We have first used

02 plasma conditions, but it removed nonspecifically all Fc-thiol from the gold membrane even

at short times under mild conditions. Ar plasma etching, however, proved to be useful to

selectively remove the surface Fc-thiol monolayer. Figure 3-1 shows the cyclic voltammograms

of freshly modified membranes before and after the Ar plasma treatment with different etching

times. In order to find the minimum etching time that is necessary to remove Au-surface Fc-

thiols, we have used membranes that have pores filled with Au. Since these membranes can not

have any Fc-thiol inside the pores, a successful plasma removal should show no sign of Fc in the

voltammogram. This is achieved at 30 seconds (Figure 3-1B) and further proved by XPS studies

(Figure 3-2, Curve C). The Fe 2p3/2 peak at 711 ev disappears even after 5 seconds (Figure 3-2,









Curve B) although the voltammogram (Figure 3-1A) still shows some trace which indicates the

greater sensitivity of the CV method.

When these conditions were applied to a membrane with open pores, the plasma removes

surface Fc monolayer and leaves the Fc monolayer inside the Au nanotube walls. Figure 3-3

shows the voltammograms of a membrane before and after plasma treatment. This membrane has

pores with 20 nm inside pore diameter. In this case the amount of redox-active Fc is decreased

by 40 %, which is equivalent to the relative amounts of Au surface-film vs. Au nanotube-wall

surface area (assuming cylindrical pores of 10 nm radius). Voltammograms like Figure 3-3 (solid

line) were also used to calculate the surface coverage of ferrocene. The coverage for all

membrane systems were 2 times the predicted packing limitation of 4.5 x 10-10 mol/cm2,145'160

which is due to the rough surface structures of electroless plated gold membranes.161

Electrochemical Decay Studies

Figure 3-4 shows cyclic voltammograms of four membranes with different inside pore

diameters that are subjected to 0.7 Volts for 6 hours. The same conditions were also applied to

a Fc-thiol modified commercial gold button electrode to compare the Fc decomposition for a flat

surface with no pores (Figure 3-5). The spiky peaks observed in Figure 3-5 suggest that there are

strong attractive interactions in this environment.162 Examination of CVs in Figures 3-4 and 3-5

indicates that the bigger the pore size the faster the decay and the more it resembles a flat-

surface-like behavior. For pore sizes < 65 nm, there is clearly a negative shift with increasing

time which is most pronounced for R = 10 nm (Figure 3-4A). We suspect that the mild

hydrophobicity of Fc-thiol is responsible for this observation. We and others163 have obtained

contact angles (0) < 800 for Fc terminated alkane thiol monolayers on gold surfaces where as

SAMs formed by long-chain alkane thiols have 0 values of 1150.164









This shift in the CVs to more negative potentials as it decays indicates that the

environment around the Fc groups becomes more hydrophilic with increasing scan number. This

has been observed before, and indicates that with prolonged scanning the Fc/Fc+ groups in the

monolayer film become more accessible to water and counterions.165 The hydrophobicity is most

pronounced with the smallest pore because the volume of the Fc-thiol that is filling the pore has

the biggest ratio in the R =10 nm case. As the pore size gets bigger this ratio gets smaller. There

is no clear shift where R = 284 nm (Figure 3-4D). In this case the hydrophobic contribution is

minimal and the Fc groups are already accessible to water and counterions as there is no clear

shift just similar to the flat surface gold electrode.

In order to compare the decay constants, semi logarithmic plots of normalized cathodic

charge against time166 were examined (Figure 3-6). Linear plots were obtained for each system,

obeying the first order decay kinetics that is previously observed in aqueous solutions for

ferricinium.159,166 Studies in aqueous solution have shown that ferricinium cations (Fc+)

decompose through an exchange of cyclopentadienyl anions (Cp-) with another nucleophile.

(e.g., OH-, C1-, N03-)153,155,156 The rate of exchange increases with the donor strength of the

nucleophile. The decomposition of Fc+ can be summarized as follows:155

FeCp2+ + n L K FeLn3+ + 2 Cp- (3-2)

assuming that in a primary step ligand exchange around the Fe (III) ion occurs. In this reaction L

can be a solvent molecule, a neutral nucleophilic agent or a monovalent anion. The Cp- can then

reduce undissociated FeCp2+ to FeCp2 in a follow-up reaction and Cp radicals form.

Fc+ decomposition is observed in electrolytes containing perchlorate anion.153,163 It is

found that increasing the pH increases the extent of decomposition substantially which is due to

the increased concentration of hydroxide ion.153 In the current system, both C104- and OH- can









initiate the Fc decomposition although the latter has a much smaller concentration ([C104-] = 0.1

M and [OH-] = 2.0 x 10-6 M). Table 3-1 shows the increase in decay constants with increasing

pore size. This constant approaches to that of a flat gold surface for R = 285 nm. As mentioned

above, the increasing accessibility of water and counterions to Fc groups with increasing pore

size should be a factor in such an observation.

More importantly, the tendencies of C104- vs. OHf towards an alkane-like environment

are different. Extraction of ion-pairing complexes of perchlorate into organic phases is a well

defined technique to detect trace amounts of perchlorate in aqueous samples.167-169 In this case,

perchlorate being a weak lipophilic nucleophile is the dominant anion inside the alkane-like

environment of the small pores which results in slower decay rates. As the pore gets larger and

more hydrophilic, OH- (strong nucleophile) partitioning into that pore increases and thus the rate

constant gets bigger. Other potential nucleophile in this system is water, but its donor strength is

insufficient for Fc+ decomposition. 155,170 It is also interesting to note that the decay constants of

ferrocene and 1,1-dimethyl ferrocene molecules in bulk aqueous phosphate buffer has similar

values166 as the Fc monolayers studied in this work (Table 3-1).

Conclusion

Recently, we have shown the affect of Fc+ decomposition on electromodulating ion

transport through gold nanotube membranes.43 In this paper, we have elucidated the nanotube

pore size affect on Fc+ decomposition. Fc-thiol monolayers on Au surface film were successfully

removed by briefly exposing both surfaces of the membrane to argon plasma. The

decomposition of Fc+ inside the Au nanotube walls were then studied for four membranes with

different pore sizes and compared with a flat surface electrode. The results suggest that the decay

rate increases with increasing pore size and in all cases it is found to obey first order decay









kinetics. Furthermore, the decay pattern resembles a surface-like decay as the pore size of the

membrane increases.

We suspect that limited accessibility of the counterions inside the small pores and their

different tendencies towards a lipophilic environment are responsible for the slower decay rate.

This is due to the constrained geometry of these small pores and the more pronounced

hydrophobic character of Fc- thiol monolayers. As the pore size gets bigger, both of these affects

are lost and the membrane behaves just like a flat-surface electrode. The negative shift in the

voltammograms was also more pronounced for smaller and more hydrophobic pores. This shift

in the CVs to more negative potentials as it decays indicates that the environment around the Fc

groups becomes more hydrophilic with increasing scan number.









Table 3-1. Fc decay constants for different membrane systems and for bulk aqueous solutions
of Fc compounds in phosphate solutions at neutral pH.166
Case Studied Decay Constant
(sec1)
R= 10 nm 0.7 x 105
R = 28 nm 0.9 x 105
R= 65 nm 1.4 x 105
R = 284 nm 1.9 x 105
Gold Button Electrode 2.1 x 10-
Ferrocene 1.4 x 105
1,1'-dimethyl ferrocene 0.6 x 10-5













4-

2-


r -2
--- -2 ---- --- /-

-4

-6-
0.0 0.2 0.4 0.6 0.8



B
6-

4-

2-

i 0 -------------- ^-- -
-------------- -------
S-2

-4-

-6-

0.0 0.2 0.4 0.6 0.8
Potential (V vs. Ag/ AgCI)

Figure 3-1. Finding the optimum etching time for surface Fc-thiol removal. Cyclic
voltammograms of Fc -thiol modified gold nanotube membranes before (solid
curves) and after (dashed curves) Argon plasma etching. The electrolyte is
0.1 M KC104 and the membranes have pores filled with gold. Increasing the Argon
etching time from A) 5 sec to B) 30 sec removes all Surface-Fc.










230000


C"


220000
210000
200000 -
190000 -


0
8 180000
170000


160000
150000


140000


Binding Energy (ev)


Figure 3-2. XPS spectra of the Fc-thiol modified gold membrane after A) 0 sec, B) 5 sec and
C) 30 sec of Argon plasma etching. The Fe 2p3/2 peak is detected at 711 eV and it
disappears even after 5 second etching. A Kratos XSAM surface analyzer with a Mg
source normal to the membrane surface has been used. The gold membrane is plated
overnight to fill the pores with gold completely.


.h

Vlt~Sv~


....














5-


i 0- -----


-5-


-10
0.0 0.2 0.4 0.6 0.8
Potential (V vs. Ag/ AgCI)

Figure 3-3. Cyclic voltammograms of a Fc-thiol modified membrane before (solid curve) and
after (dashed curve) 30 sec of Argon plasma etching. The membrane has pores with
20 nm inside pore diameter. The dashed curve corresponds to Fc-thiol monolayer
lining only inside the nanotube walls.

















5-

0-


-5-

-10-


I0.I 0.I 0I I I -1 I I
0.0 0.2 0.4 0.6 0.8 0.0 0.2 0.4 0.6 0.8


C 20-


10-


0.0 0.2 0.4 0.6 0.8

Potential (V vs. Ag/AgCI)


-30 1.. .
0.0 0.2 0.4 0.6 0.8

Potential (V vs. Ag/AgCI)


Figure 3-4. Cyclic voltammograms of four different gold nanotube membranes with pore
diameters A) R = 10 nm, B) R = 28 nm, C) R = 65 nm, and D) R = 284 nm. Scans
were recorded sequentially after holding Eapp at 0.7 V for 0 min (black), 35 min (red),
105 min (blue), 175 min (green), 245 min (violet) and 315 min (orange). Scan rate is
20 mV/sec.


30-

20-
-

10-

0o-

-10-
-

-20-

-30-










400-


300

200

100

0-

-100

-200

-300

0.0 0.2 0.4 0.6 0.8
Potential (V vs. Ag/AgCI)

Figure 3-5. Cyclic voltammograms of modified gold button electrode. Scans were recorded
sequentially after holding Eapp at 0.7 V for 0 min (black), 35 min (red), 105 min
(blue), 175 min (green), 245 min (violet) and 315 min (orange).
Scan rate is 20 mV/sec.










0.45

0.40 E
.D
0.35 -

0
5 0.30
C
m 0.25 -

3 0.20 B
a B
-c 0.15 -

0.10

0.05 -

0.00
0 50 100 150 200 250 300 350
Time (min)

Figure 3-6. First order kinetic plots for the loss of the Fc+ for A) R = 10 nm, B) R = 28 nm,
C) R = 65 nm, D) R = 284 nm and E) gold button electrode.









CHAPTER 4
PLASMA-ETCHED NANOPORE POLYMER FILMS
AND THEIR USE AS TEMPLATES TO PREPARE NANO TEST TUBES

Introduction

We recently introduced a new class of tubular nanostructures called nano test tubes.47'48

Unlike conventional nanotubes, which are open at both ends, nano test tubes are open on one end

and closed on the other. They are made by the template-synthesis method, in which the pores in

a nanopore material are used as templates to prepare nanotubes.3'4'6 The key to obtaining nano

test tubes is using a template in which the pores are closed on one end (Figure 4-1A). When the

tube-forming material is deposited within such pores, both the pore walls and the closed pore end

get coated with this material, and closed-end test tubes are obtained. The outside diameter of

these nano test tubes is determined by the pore diameter of the template, and the length of the

tubes is determined by the template thickness.47

Nanopore alumina films, prepared by electrochemical oxidation of Al metal,76'171have

pores that are closed on one end, provided the alumina is not removed from the underlying Al

surface.18 In our prior work, we used such alumina films as templates to prepare silica nano test

tubes.47 There is, however, a limitation with regard to the dimensions of the nano test tubes that

can be obtained with these nanopore alumina templates. Specifically, it is difficult to obtain

short (<500 nm long) test tubes. This is because such short nano test tubes require ultra-thin

alumina templates, which means that very brief anodization times must be used. However at

very short times, anodization of aluminum shows irregular growth patterns and the resulting

alumina film does not have a regular pore structure.

Our motivation for making smaller nano test tubes comes from our interest in investigating

uptake of such tubes by living cells, with the ultimate goal of using these tubes as drug- or DNA-

delivery vehicles. We believe that for such applications it would be advantageous to have tubes









that are small in length relative to the dimensions of the cell. Because of this limitation with the

alumina templates, we have been investigating methods for preparing thinner nanopore templates

so that shorter nano test tubes might be obtained. One such method builds on Masuda's concept

of using a nanopore alumina membrane as a plasma etch mask.172'173 This technology entails

removing a nanopore alumina film from the underlying Al surface so that the pores are open at

both faces of the resulting alumina membrane. The free-standing alumina membrane is then

placed on a substrate, and a plasma is used to etch a replica of the alumina pore structure into the

surface of the substrate (Figure 4-1B). We have used this method to prepare nanopore carbon

anodes for battery applications18 and nanowell glass surfaces for applications in analytical

chemistry.70,174

We have recently modified this mask/etch technology so that it can be used to produce

pores in an underlying polymer photoresistt) film, as opposed to the harder materials (glass,70'174

diamond,172'173 graphite18) etched previously. Furthermore, we have shown that with this

modified mask/etch method the distance that the pores propagate into the photoresist film can be

controlled by varying the etch time. Hence, by controlling the etch time, we effectively control

the thickness of the nanopore layer etched into the surface of the photoresist. We have used such

plasma-etched nanopore photoresist films as templates to prepare silica nano test tubes. As

expected the length of the test tubes is determined by the thickness of the porous photoresist

layer, and test tubes with lengths of 380 nm were obtained, shorter than any test tubes obtained

using an alumina template.47 We report preliminary results of these investigations here.

Experimental

Materials

Aluminum foil (99.99%) was obtained from Alfa Aesar, and microscope premium finest

glass slides from Fisher. PMGI SF 15, a polydimethylglutarimide-based positive photoresist,









was purchased from MicroChem Corp. Ethanol (absolute, Aaper), tetraethyl orthosilicate

(Aldrich), HC1 (Fisher), and 1165 Microposit Remover (a 1-methyl-2-pyrolidinone-based system

for dissolving the PMGI photoresist, Shipley) were used as received. Purified water was

obtained by passing house-distilled water through a Millipore, Milli-Q system.

Preparation of the Nanopore Alumina-Membrane Masks

The nanopore alumina membranes were prepared in house using the well-known two-step

electrochemical anodization method.18 Briefly, after annealing and polishing the aluminum foil,

a nanopore alumina film was formed across the Al surface by anodization. This film was then

dissolved in acidic Cr03, and a second anodized alumina film was formed. This film was

removed from the underlying Al surface using the voltage-reduction method.175 The resulting

free-standing nanopore alumina membrane has two faces the one that was exposed to the

solution, and the one that was adjacent to the Al substrate, during anodization. These faces are

not identical,18 and we delineate them, here, as the solution-side and the Al-side faces. The pore

diameter, as determined from scanning electron microscopic (SEM) images of the solution-side

face (Figure 4-2A), was 79+7 nm. The alumina membrane thickness was -1.5 [m (Figure 4-2B).

SEMs were obtained using a Hitachi S4000 FE-SEM. Prior to imaging, the surface of the SEM

sample was sputtered with a thin Au/Pd film using a Desk II Cold Sputter instrument (Denton

Vacuum, LLC).

Preparation of the Nanopore Polymer-Replica Films

Glass microscope slides (2 cm x 2 cm) were washed with copious amounts of ethanol and

blown dry with nitrogen. A Model 6700 spincoater (Speedline Technologies, IN) was used to

coat one surface of the slide with the PMGI SF 15 photoresist; -2 ml of the photoresist were

dispensed, the terminal spin speed was 10,000 rpm, and the spin time was 45 sec. The resulting

polymer film (-4 [i thick) was cured in air at 190 OC for 15 minutes.









As per our prior work,18'70 the general strategy was to place the nanopore alumina-

membrane mask onto the surface of the polymer film, and use a plasma-etch method to "burn" a

replica of the alumina pore structure into the polymer surface (Figure 4-1B). However, we

discovered that when the alumina mask was placed directly on top of the polymer film, a replica

of the alumina pore structure could not be obtained; instead, large diameter (-500 nm) pits were

burned into the surface of the polymer film. In order to obtain a faithful replica, it proved

necessary to sputter-coat the polymer film with a thin metal film, and then place the nanopore

alumina-membrane mask on this metal film (Figure 4-3). Three different metals Au, Ag, and

Au/Pd were investigated, with the best results obtained with Au/Pd. The Au/Pd films were

sputtered using the Desk II Cold Sputter instrument, with 45 mA sputtering current, 75 mTorr Ar

pressure, and 60 sec sputtering time. The film thickness was -30 nm.

The alumina-membrane mask was placed on top of the Au/Pd-coated polymer film with

the solution-side face of the membrane facing down. The masked substrate was placed into the

vacuum chamber of a reactive-ion etching system (Samco model RIE-1C) and subjected to two

plasma-etch treatments. The first was a 2-minute Ar-plasma etch (physical etch, 1). The plasma

conditions were 13.56 MHz, 140 W, 10 Pa Ar pressure, Ar flow rate =12 sccm. The second

etch was a chemical etch'1,176using an 02/Ar- plasma. The plasma conditions were 13.56

MHz, 140 W, 10 Pa 02 pressure, 02 flow rate = 10 sccm, 10 Pa Ar pressure, Ar flow rate = 12

sccm.

Preparation of the Silica Nano Test Tubes

A key objective of this work was to show that the pores in these nanopore polymer-replica

films could be used as templates to prepare nano test tubes. To demonstrate this, a sol-gel

method described previously47 was used to deposit silica nano test tubes within the pores of the

polymer-replica films. Briefly, a 50/5/1 (by volume) mixture of ethanol, tetraethyl orthosilicate









and 1M HC1 was prepared and allowed to hydrolyze for 30 min. The nanopore polymer-replica

film was immersed into this sol (PMGI SF 15 is insoluble in ethanol) with sonication for 30 sec

and then kept under vacuum in a desiccator for 5 more minutes. The sol-impregnated film was

dried in air, and then oven cured for -5 h at 100 OC, to yield silica nano test tubes47 within the

pores of the polymer-replica film.

To liberate the nano test tubes, the nanopore polymer-replica film was dissolved by

overnight immersion in the 1165 Microposit Remover solution. The liberated test tubes were

collected by filtration and rinsed with copious amounts of the remover and ethanol.

Transmission electron microscopy (TEM) samples were prepared by re-suspending the liberated

nano test tubes in ethanol and immersing a TEM grid into this suspension. TEM images were

obtained with a Hitachi H-7000 microscope.

Results and Discussion

As noted above, it proved necessary to coat the surface of the polymer film with a thin

Au/Pd layer prior to applying the alumina etch mask and plasma etching. The alumina-

mask:Au/Pd:polymer-film assembly (Figure 4-3) was then first etched with an Ar plasma

(physical etch).11 This brief Ar-plasma etch removes the portions of the Au/Pd film beneath the

pores in the nanopore alumina mask. Put another way, the Ar plasma creates a replica of the

alumina pore structure in the Au/Pd film, and thus exposes the portions of the polymer film in

the regions beneath the alumina pores. The assembly (Figure 4-3) was then subjected to an

02/Ar plasma (chemical etch)11 to remove the exposed portions of the polymer film beneath the

alumina pores; i.e., the O2/Ar plasma is responsible for replicating the pore structure of the mask

in the polymer film.

Figure 4-4 shows surface and cross-sectional images of the polymer film after four minutes

of etching with the O2/Ar-plasma. Some reproduction of the pore structure of the alumina-mask









can be seen in the surface image (Figure 4-4A), but the pores propagate only a very small

distance into the polymer film (Figure 4-4B). Analogous images after 8 minutes of etching with

the O2/Ar-plasma show that the pore structure has been faithfully reproduced in the surface of

the polymer film (Figure 4-5A), and that the pores obtained propagate, with uniform diameter,

-380 nm into the upper surface of the film (Figure 4-5B). The pore diameter is 817 nm

identical to the diameter of the pores in the alumina mask. When the pores in this polymer film

were used as templates to prepare silica nano test tubes, tubes with diameters of 838 nm and

lengths of 38024 nm were obtained (Figure 4-5C). As would be expected,47 not only are the

diameters equivalent to the pore diameter, but the length is equivalent to the thickness of the

porous part of the polymer film.

By controlling the 02/Ar-plasma etch time; the distance that the pores propagate into the

upper surface of the polymer film can be varied. For example, a film that was etched for 10 min

had 858 nm diameter pores (Figure 4-6A) that propagated -1 im into the polymer film (Figure

4-6B). Correspondingly, the nano test tubes synthesized within the pores of this film were -100

nm in diameter and 1000+105 nm in length (Figure 4-6C and D). In this case obtaining an

accurate value for the tube diameter is problematic, because as can be seen in Figure 4-6B, the

pore is wider at the mouth than at the bottom. As a result the outside diameter of the tubes is

likewise larger at the mouth (Figures 4-6C and D). Note that the metal film is still present on top

of the polymer film (Figure 4-6B).

When longer etch times (e.g., 12 min) were used, much larger scale damage is produced in

the polymer film, and faithful reproduction of the pores in the alumina mask is no longer

achieved (Figure 4-7). This is because for such long etch times the metal film on the surface of

polymer film is damaged and partly removed and, as a result, the pores merge at the polymer









film surface (Figure 4-7A). This damage could be detected with the naked eye, as the faint black

color of the Au/Pd coating could no longer be observed. Hence, again, we see the essential role

played by the metal film in producing a faithful replica of the alumina mask in the underlying

polymer.

Conclusions

We have extended the alumina-mask, plasma-etch concept to a new substrate material

- a photoresist polymer film. In so doing we created a new type of nanopore polymer template

for use in template synthesis of nanomaterials. An appealing feature of this new template is that

the distance that the pores propagate into the surface of the polymer film can be controlled by

varying the plasma etch time. This allows for corresponding control over the lengths of the nano

test tubes prepared by template synthesis within the pores. Via this route, we have successfully

prepared silica nano test tubes that were over 100 nm shorter than the shortest tubes prepared in

an alumina-film template.47 It is also of interest to note that this general procedure can be thought

of as a relatively high throughput nanotube synthesis technology. This is because there are -1010

pores per cm2 of template area; so for example, with 10 cm2 of template, we can make 1011 nano

test tubes.

Another appealing feature of these new polymer-film templates is that they can be used for

both aqueous-based (including both acidic and basic solution) and organic-based (including most

aliphatic alcohols, ketones and ethers) template synthesis. Nevertheless, these films can be

dissolved, when needed, in the photoresist remover solution to liberate the nano test tubes

synthesized within the pores. We are currently further exploring the plasma-etch process in

attempts to make even thinner nanopore polymer replica films.














Porous material with
closed-end pores


Alumina etch mask


Deposit tube-
forming material


Nano test tubes


Closed-end pores
I I I


Plasma etch and
remove alumina


Nonporous material
to be etched


Figure 4-1. Schematic diagrams of A) the concept of using a template with closed-end pores to
prepare correspondingly closed-end nano test tubes, and B) the alumina-mask
plasma-etch method to prepare closed-end pores in an underlying substrate material.








I
Ii IIn


Figure 4-2. SEM images of the nanopore alumina-membrane mask; A) Top view; B) cross-
sectional view.






































Figure 4-3. Cross sectional SEM of the Al-mask:Au/Pd-film:polymer-film assembly.
























Figure 4-4. SEM images of A) the polymer-film surface and B) the cross-section of the film
after 4 min of 02/Ar plasma etching.






































Figure 4-5. SEM images of A) the polymer-film surface and B) the cross-section of the film
after 8 min of 02/Ar plasma etching. C) SEM images of silica nano test tubes
synthesized in the pores of this polymer film.





































Figure 4-6. SEM images of A) the polymer-film surface and B) the cross-section of the film
after 10 min of 02/Ar plasma etching. C) SEM and D) TEM images of silica nano test
tubes synthesized in the pores of this polymer film.
























Figure 4-7. SEM images of A) the polymer-film surface and B) the cross-section of the film
after 12 min of 02/Ar plasma etching.









CHAPTER 5
SILICA NANO TEST TUBES AS DELIVERY DEVICES; PREPARATION AND
BIOCHEMICAL MODIFICATION

Introduction

The application of nanomaterials such as nanoparticles, nanotubes, nanorods, and

nanowires in biological systems has attracted great interest in the fields of materials science and

biochemistry.2'177 Because of their dimensions, which make them suitable for application in

biological systems, the potential of nanomaterials for biodetection,178-181 bioseperation,45 and

biomolecule delivery118'120121121'12126'142 has been explored.116 In particular, the use of

nanomaterials in biomolecule delivery has been shown to present various advantages such as

increased efficacy,113 protection of drugs114 or genetic material115'116 from potential

environmental damage and reduced drug toxicity.117 Spherical nanoparticles are almost always

used because these shapes are easier to make and can be synthesized from a diverse range of

materials, such as liposomes,118'119 polymers,120'121 dendrimers122 and various inorganic

compounds.46'115'123 Unlike nanospheres, nanotubes have unique hollow structures however their

use as biomolecule carriers are still very rare.116'142'182

We have pioneered a technology, called template synthesis, for preparing monodisperse

nanotubes of nearly any size and composed of nearly any material.3'183'184 These nanotubes have

a number of attributes that make them potential candidates for biomolecule delivery applications.

First, nanotubes have larger inner diameters than nanoparticles which allow nanotubes to carry a

correspondingly larger payload. In addition, the template method allows independent

modification of the distinct inner and outer surfaces of the tubes. Multifunctional delivery

vehicles can be obtained by this differential modification scheme. Such delivery tools attracted

great interest in biomedical applications, for example, multifunctional nanomaterials are









considered to be ideal units for the cancer-specific therapeutic and imaging agents.125 Finally, the

tubes can be synthesized from various materials and their dimensions are easily controlled.45

We have shown the application of differentially modified silica nanotubes as smart

nanophase extractors for enantiomeric drug molecules.45 Chen and colleagues demonstrated the

preparation of fluorescent silica tubes for gene delivery.116 After the attachment of quantum dots,

the tubes were loaded with green fluorescent protein (GFP) plasmid and incubated with monkey

kidney COS-7 cells. The loaded tubes are shown to be non-toxic to the cells, they initiate

approximately 10-20 % of the cells to express GFP and they also act as physical shields to

protect the genetic material form enzymatic degradation. The tubes, however, lack differential

modification and capping as they are necessary for targeted delivery46'47 and the tube size is

controlled by physical polishing which is inappropriate for obtaining tubes with lengths < 1 im.

Novel nanostructures called nano test tubes have been recently introduced by the Martin

group.47'48 Silica nano test tubes are prepared by sol-gel synthesis of silica in the pores of

alumina template that remains attached to underlying aluminum metal. Unlike the previously

mentioned nanotubes that are open on both ends, nano test tubes are closed on one end and open

on the other. The use of test tubes as potential universal drug delivery vehicles was exploited

where these nano test tubes could be filled with a payload and then the open end corked with a

chemically labile cap.48 We have developed a capping strategy that involves the Schiff s base

reaction to form imine linkages between the test tubes and the aldehyde-modified polystyrene

corks.48 Lee and coworkers have described a selective partial functionalization method using

controlled gold nanoparticle diffusion in nanotubes and prepared Au-capped silica nano test

tubes by seed-mediated gold-growth.185 The same group has also introduced magnetic nano test

tubes that has a layer of Fe304 prepared by dip-coating.186









In our earlier work, we have used the conventional sol-gel method to obtain silica nano test

tubes in the pores of alumina template.46'47 Although the procedure is easy, it can be challenging

to control the thickness and morphology.101 This chapter compares the preparation techniques for

silica nano test tube fabrication using the conventional and surface sol-gel methods and

illustrates the subsequent differential tube modification strategy for their use in cell incubation

studies. Defective test tubes were obtained with the conventional sol-gel method and it was

attributed to the small changes in the viscosity of the gel. Layer-by-layer addition of silica with

the surface sol-gel method allowed the preparation of defect-free uniform silica nano test tubes.

We have differentially modified these test tubes using silane and Schiff-base chemistry to impart

biochemical functionality for the cell studies. Before the template was removed, the inner tube

surface was labeled with a fluorophore. The liberated fluorescent-tubes were then modified with

a target or a control antibody and then incubated with breast carcinoma cells. The preliminary

results suggest that the tubes modified with the target antibody attaches much more readily to the

cell membrane surfaces than the tubes modified with the control antibody.

Experimental

Materials

Aluminum foil (99.99%) was obtained from Alfa Aesar. Microscope premium finest glass

slides, methanol, chromium trioxide, oxalic acid, NaOH, H3PO4, H2S04 and HC1 were obtained

from Fischer and used as received. Tetraethylorthosilicate(TEOS), silicon tetrachloride, carbon

tetrachloride, 3-(amino-propyl)triethoxysilane(APTS), Rhodamine B Isothiocyanate, sodium

cyanoborohydride, IgG from Rabbit serum, and Albumin Bovine Serum were used as received

from Sigma-Aldrich as were ethanol (absolute) from Aaper, N,N- Dimethylformamide from

Acros, Alexa 488 carboxylic acid-succinimidyl ester and Alexa Flour 488 labeled goat anti-

rabbit IgG from Invitrogen, and 3-(trimethoxysilyl)propyl aldehyde from UCT Chemicals. IGF-









IRa and IGF-IRP rabbit polyclonal antibodies were obtained from Santa Cruz Biotechnology,

Inc. Purified water was obtained by passing house-distilled water through a Millipore, Milli-Q

system.

Preparation of the Nanopore Alumina-Membrane Templates

The nanopore alumina membranes were prepared in house using the well-known two-step

electrochemical anodization method. 18171 Briefly, after annealing and polishing the aluminum

foil, a nanopore alumina film was formed across the Al surface by anodization. This film was

then dissolved in acidic Cr03, and a second anodized alumina film was formed using oxalic acid

electrolyte. This yields the desired ordered nanopore alumina film on both surfaces of the

aluminum film. Unlike the work described in the previous chapter, the alumina film is not

detached from the aluminum so the template remains attached to the underlying Al metal. It is

also important to note that in the first work we reported the preparation of silica nano test tubes;

we have attached a glass substrate to one surface of Al with epoxy for stability reasons, which

yielded alumina growth only on one side of Al metal.47

Preparation of the Silica Nano Test Tubes

Two different sol-gel methods were used to deposit silica nano test tubes within the pores

of the nanopore alumina template (Figure 5-1). In the conventional sol-gel method:47'48 a 50/5/1

(by volume) mixture of ethanol, tetraethyl orthosilicate and 1M HC1 was prepared and allowed to

hydrolyze for 30 min. The alumina template was immersed into this sol with sonication for 30

sec and then kept under vacuum in a desiccator for 5 more minutes. The sol-impregnated

template was dried in air, and then oven cured for -5 h at 100 OC, to yield silica nano test

tubes47'48 within the pores of the nanopore alumina template. The surface film was removed by

wiping the membrane surface with a laboratory tissue soaked in EtOH.









In the surface sol-gel method;101 two-step deposition cycles, in which the adsorption of a

molecular precursor (SiC14) and the hydrolysis steps are separated by a post-adsorption wash. An

alumina template was immersed in SiC14 solution in CC14 (85 mol-%) for 2 min and quickly

soaked in a CC14 beaker. The template was then washed with CC14 and immersed in a second

CC14 beaker for 15 min to remove unbound SiC14 from the pores. These steps were done in a

polyacrylic box under 30 psi nitrogen flow to limit SiC14 polymerization by atmospheric water

which occurs at ambient conditions and results in silica deposition with uncontrollable thickness.

Finally, the template was soaked in CCl4/MeOH 1:1 (2 min) and EtOH (5 min) to displace CC14,

and dried in a N2 stream. Then the template was immersed in deionized water for 5 min, washed

in a beaker with MeOH (2 min). After 10 deposition cycles the silica deposited template was

cured at 100 OC for 1 h. The surface film was removed by briefly (1 min) exposing both sides of

the nanopore template to a reactive-ion plasma etching system (Samco model RIE-1C). The

plasma conditions were 13.56 MHz, 140 W, 20 Pa Ar pressure, Ar flow rate = 20 sccm.

To liberate the nano test tubes, the nanopore alumina template was dissolved in 0.1 M

NaOH for 3-6 h. The liberated test tubes were collected either by centrifugation (14,000 rpm for

14 min in all experiments involving centrifugation) or filtration and washed several times with

water and ethanol. Transmission electron microscopy (TEM) samples were prepared by re-

suspending the liberated nano test tubes in ethanol and immersing a TEM grid into this

suspension. TEM images were obtained with a Hitachi H-7000 microscope. Scanning electron

microscopy (SEM) was also used to characterize the alumina template and the filtered free silica

nano test tubes. SEM images were obtained using a Hitachi S4000 FE-SEM. Prior to imaging,

the surface of the SEM sample was sputtered with a thin Au/Pd film using a Desk II Cold Sputter

instrument (Denton Vacuum, LLC).









Silica Nano Test Tube Modification with Fluorophore

The labeling of silica nano test tubes with fluorophores were done while the tubes were

still embedded in the alumina template. This means only the inner walls of the tubes are

accessible for chemical modifications. The surface modifications were done using silanization

chemistry and the structures of all silanes are shown in Figure 5-2. In each case the inner tubule

walls were modified with amine functional groups which are then covalently coupled to

Rhodamine or Alexa Flour-488 (Figure 5-3).70'187 Briefly a solution that was 5 % APTS, 90%

ethanol, and 5 % acetate buffer (50mM, pH 5.2) was hydrolyzed for 20 min and the template is

immersed into this solution for 1 h. The template was then thoroughly washed with ethanol and

cured in an oven at 100 OC for 3h. Rhodamine attachment was done by immersing the amine

functionalized template into a 5 mM Rhodamine B Isothiocyanate solution in dry DMF for 12 h

in a desiccator. This was followed by extensive washing with DMF and EtOH. To modify the

inner tube surfaces with Alexa-488, a 0.1 mg/ml solution of Alexa 488 carboxylic acid-

succinimidyl ester in 10 mM phosphate-buffered saline (PBS) buffer (pH is adjusted to 8.1 by

0.1 M NaOH) was prepared. The amine modified template was then immersed into this solution

in a desiccator for 12 h and then washed with buffer and ethanol before the tubes were liberated

from the template.

A fluorescence microscopy system described previously188 was used to obtain fluorescence

images of the labeled test tubes and to measure the fluorescence intensity from glass slides that

are used to confirm the antibody attachment. (See antibody modification.) This system combines

an Axioplan 2 imaging microscope (Zeiss) with a J&M-PMT photometry system detector

(SpectrAlliance), for measuring fluorescence intensity. In addition, the system is equipped with a

digital CCD camera (Zeiss) to obtain both fluorescence and optical images. The excitation source

for all fluorescence measurements was a mercury lamp. A beam splitter was used to send the









reflected fluorescent light from the sample to the detector and the CCD camera. The Rhodamine

B was excited at 570 nm, and the emission was collected through a 590-nm band-pass filter and

The Alexa 488 was excited at 495 nm, and the emission was detected through a 515-nm band-

pass filter.

Antibody Modification

The fluorescently labeled tubes were liberated and then washed by centrifugation at 14,000

rpm three times with H20 and then three times with ethanol. The outer tube walls were

functionalized with aldehyde groups by an aldehyde terminated siloxane linker.189 The aldehyde

groups were then reacted by well-known Schiff-base chemistry to amine sites on the protein to

be immobilized.190-192 Briefly a solution that was 5 % 3-(trimethoxysilyl)propyl aldehyde, 90%

ethanol, and 5 % acetate buffer (50mM, pH 5.2) was hydrolyzed for 15 min and the tubes were

dispersed in this solution and reacted for 30 min with frequent vortexing. The aldehyde modified

tubes were centrifuged and vortexed three times with ethanol and then three times with 10 mM

PBS, at pH 7.4. The antibodies were coupled to the aldehyde-terminated outer tube surfaces by

dispersing these tubes in the same PBS buffer that contains 0.2 mg/ml antibody and 4 mM

NaBH3CN for 12 h at 4 C with occasional vortexing. The tubes were either modified with

Rabbit polyclonal IGF-IRa (target) or IGF-IRj (control) antibodies and the tube concentration

was -1010 tubes/ml. After the antibody modification, the tubes were washed three times with

PBS buffer by centrifugation and dispersed in 10 mM PBS, at pH 7.4 that contains 0.2 mg/ml

bovine serum albumin (BSA) and 4 mM NaBH3CN. This step is required to quench the

remaining aldeyhde sites on the outer tube walls and was done by allowing the tubes in this

solution for 2 h at room temperature with vortexing. Finally, the tubes were washed three times









with 10 mM PBS (pH=7.4) by centrifugation and dispersed in the same buffer for cell incubation

studies.

Covalent attachment of antibody by Schiff-base chemistry was confirmed on glass slides.

Two glass slides were coated with a single layer of silica by surface sol-gel method and both

slides were functionalized with aldehyde silane as mentioned above and dried in a vacuum

desiccator for 5 h. First slide was then modified with Rabbit IgG and the second with BSA where

both proteins were 1 mg/ml in a pH 7.4, 10 mM PBS containing 4 mM NaBH3CN. The slides

were washed with PBS and treated with 1/5 diluted sea block buffer (Pierce, # 37527) for 2h.

Both slides were then exposed to Alexa Flour 488 labeled goat anti-rabbit IgG (20 tg/ml in PBS,

pH 7.4) for 10 h at 40C. After rinsing with PBS and water the slides were dried under N2

stream and their fluorescence was compared by J&M-PMT photometry system detector.

Cell Incubation Studies

MDA-MB-231 breast carcinoma cells (American Type Culture Collection, Manassas, VA)

were maintained in Dulbecco's modification of Eagle's medium (Fisher Scientific) with 10%

fetal bovine serum (Invitrogen, Carlesbad, CA) and 0.5 mg/mL Gentamycin (Sigma, St. Louis,

MO) at 37 C in 5% C02/air. Cells were plated in Coming 24 well cell culture clusters and

grown for 48-60 h prior to incubation.193

The cells were incubated with 10 mM PBS (pH=7.4) containing 0.2 mg/ml BSA solution

for 30 min to prevent nonspecific binding of the tubes to the cell surface. These cells were

washed with cell media buffer and then incubated with the antibody-modified fluorescent silica

nano test tubes (tube concentration was ~ 109 tubes/ml) for 1 h and then washed five times with

cell media buffer prior to imaging. Note that two separate wells were used for the incubation of

cells with the tubes; one for the target antibody-modified tubes and the other for the control

antibody-modified tubes (non-competitive).









Fluorescence imaging was conducted with a confocal microscope setup consisting of an

Olympus IX-81 inverted microscope with an Olympus Fluoview 500 confocal scanning system

and a tunable argon ion laser (488 nm). The images were taken with a 20x objective and the

fluorescence was detected by a 505-525 nm band-pass filter. Microplate reader experiment was

conducted with a Tecan Safire microplate reader with 24 well Coming cell culture plates and the

excess cell media buffer was removed from the plates prior to measurements. The excitation

wavelength was 488 nm and the emission was collected at 520 nm.

Results and Discussions

Defect-Free Silica Nano Test Tube Preparation

We have previously reported silica nano test tube preparation using nanopore alumina

templates.47 Nanopore alumina was grown only on one side of the Al foil as the other side was

attached to a glass support with epoxy for stability reasons. However, when the template is

dissolved, the epoxy leaches out into the solution and contaminates the tube samples (Figure 5-

4). Using thicker aluminum foils eliminates the need for such supports and yields alumina film

on both surfaces of the Al metal (Figure 5-5, only one side is shown for simplicity.). When the

conventional sol-gel method is applied to obtain silica test tubes from these templates, clean test

tubes are obtained in larger quantities (Figure 5-6). Note that the tube diameter reflects the

template pore diameter (- 80 nm) and the tube length reflects the template thickness (- 1 pm).

Silica nano test tubes can be prepared with conventional sol-gel quite easily (< 5 min),

however, the resulting tubes do not have reproducible structures (Figure 5-6C). Tubes with holes

were often observed and changing the Al foil purity, hydrolysis time, TEOS concentration or

dissolving conditions as well as the use of glass supported alumina templates yielded similar

defective structures. These "bamboo-like nanofibers" were first reported by Zhang194 where they

have shown that the viscosity of the gel determines whether the silica nanostructure will be a









wire, a tube or a bamboo-like nanofiber. The defective nanostructures in our case are observed

since small variations during the sol-gel preparation (e.g. temperature or humidity) can change

the viscosity of the gel.

A surface sol-gel method was used to have a better control over the resulting silica nano

test tubes. This method involves repeats of two-step deposition cycles, in which the adsorption of

a molecular precursor (SiC14) and the hydrolysis steps are separated by a post-adsorption wash

(Figure 5-1). Ideally the technique can limit each adsorption to a single monolayer, however

thicker layers have been found for planar oxide films.104,106'195 Nevertheless, it allows very fine

control over film thickness because a nanometer or sub-nanometer thick layer is grown on each

cycle.101 Control over the atmospheric water is necessary as it rapidly polymerizes SiC14

precursor and a silica layer deposits on the alumina template surface with uncontrollable

thickness (Figure 5- 7). This control is achieved by purging nitrogen stream throughout the

adsorption steps.

A thin layer of silica (-15 nm) is deposited on the inner pore walls of the nanopore

alumina template and on the top template surface from a SiC14 solution (85 mol-% in CC14) after

10 deposition cycles (Figure 5-8). The silica film on the template surface, which normally binds

the nanotubes together, is removed by exposing both faces of the template to argon plasma.

Figure 5-9A shows one such template after 1 min Ar-plasma treatment. When it is immersed in

acid briefly, the alumina partly dissolves and reveals the protruding silica nanotube mouths that

are not inter-connected (Figure 5-9B). Free silica nano test tubes with very smooth surface

structures are obtained as the template is completely dissolved (Figure 5-10). Nano test tubes

with different lengths can also be synthesized using alumina templates of various thicknesses.

We have successfully varied the tube length from 100 nm to 6 mr (Figure 5-10C, D). The ability









to tailor the tube dimensions is an important factor since this can affect the payload capacity of

such nanotubes for delivery applications.46

Differential Modification

In addition to the geometric control, the template method also allows to independently

modify the inner and outer surfaces of the tubes. When the tubes are still embedded in the

template, only the inner surfaces are exposed to modifications. Once this inner surfaces is

modified and the template in removed, the outer tube surfaces of the free tubes are accessible,

which can be further functionalized with a different chemistry (Figure 5-11). A variety of

functional groups can be attached to the silica surfaces via silane chemistry196 using

commercially available reagents. Previously, such differentially functionalized silica tubes are

shown to selectively extract enantiomeric drugs from a racemic solution.45

The motivation for making differentially functionalized silica nano test tubes stems for an

interest in using these tubes as drug- or DNA- delivery vehicles. The test tube geometry is ideal

for conveniently filling of the nanotube with the biomolecule of interest and by applying a cap to

the open end, the biomolecule could be kept "bottled-up" inside until it is ready to be delivered.

We have successfully shown the capping of the tubes with polystyrene balls using simple imine

linkages.48 Potential biomedical applications will require that the outer surfaces of the tubes

should be modified with various moieties (protein, nucleic acids, organic functional groups) to

target the nanostructures to their destinations. Template-based synthesis approach makes it

possible to add these modifications after release from the alumina template.45'46

Proof-of-principle studies were done where the inner tube surfaces are labeled with

fluorescent tags and the outer tube surfaces are modified with tumor specific antibodies.

Rhodamine B or Alexa Flour-488 labeled test tubes were prepared by first reacting the inner tube

surfaces with APTS while the tubes were still embedded in the template. The resultant primary









amine groups and then covalently coupled (Figure 5-3) to isothiocyanate or succinimidyl ester

groups. Figure 5-12 shows such tubes after they have been released from a 6 i-thick template

(same template used for the tubes in Figure 5-10D). Since Alexa-488 is much more resistant to

photobleaching than other organic dyes,197 further studies only involved test tubes that are

modified with this fluorophore.

In order to immobilize the protein, the outer tube walls of the free fluorescent tubes are

functionalized with aldehyde moieties by an aldehyde terminated siloxane linker189 (Figure 5-

11). The aldehyde groups are then reacted by well-known Schiff-base chemistry to amine sites

on the protein to be immobilized.190-192 This covalent immobilization chemistry is first confirmed

with a glass slide experiment where two glass slides are reacted with aldehyde silane. The first

slide is then modified with rabbit IgG and the second slide is modified with BSA. When both

slides were exposed to Alexa Flour 488 labeled goat anti rabbit IgG solution; the first slide

emitted distinct fluorescence at 530 nm where as the second slide showed negligible emission

(Figure 5-13). This showed the successful covalent attachment of bioactive rabbit IgG on silica

surface with the Schiff-base chemistry.

Cell Incubation Results

The cell incubation experiments were done with Alexa 488-labeled silica nano test tubes

that were modified with IGF-IRa or IGF-IRP antibodies using Schiff-base chemistry for protein

immobilization. IGF-IRa and IGF-IRP are rabbit polyclonal antibodies raised against the a and P

subunits of the insulin-like growth factor-I receptor (IGF-IR), respectively.198 IGF-IR is a

transmembrane protein that stimulates growth in many different cell types, blocks apoptosis, and

may stimulate the growth of some types of cancer and over-expression of the IGF-IR gene has

been reported in breast cancer cells.199 A recent study with MDA-MB-231 breast carcinoma cells

has shown that the extracellular a subunit of the IGF-IR protein showed specific activity for the









IGF-IRa antibody, and no activity was observed for the IGF-IRP antibody.200 Consequently, to

observe specific cell reaction for the silica nano test tubes, two sets of nano test tubes were

prepared. The first set was modified with IGF-IRa (target) and the second set with IGF-IRP

(control) antibody.

Figure 5-14 displays fluorescence images of two different breast carcinoma cell culture

samples incubated with Alexa-488 labeled silica nano test tubes that are modified either with the

target (Figure 5-14A) or with the control antibody (Figure 5-14B). Qualitative observation

suggests that the tubes modified with target antibody attaches much more readily to the cell

membrane surfaces than the tubes modified with control antibody. The tubes are generally

attached to the membrane surfaces of live (elliptical) and dead (circular) cells and not on the well

bottom. Extensive tube attachment to the well bottom was observed with tubes that are left

unmodified on their outer surfaces. Further 3D sectioning studies of the confocal microscopy

images are required to understand if any of the tubes are internalized by the carcinoma cells.

We have used the same cell samples in order to compare the whole-plate cell fluorescence

intensities using a microplate reader. The result shows a fluorescence intensity ratio of more

than an order of magnitude for the cells that are incubated with the target antibody-modified

tubes (Fl. Int. = 5495 a.u.) compared to the cells incubated with the tubes modified with control

antibody (Fl. Int. = 435 a.u.). More experiments need to be conducted to verify these results. It is

also important to note that these incubation studies were carried out after the cells have been

treated with BSA. When the cells were not treated with BSA prior to tube incubation, very

similar fluorescence results were obtained from the target and control antibody-modified tubes

which shows nonspecific binding of both tube types to the cell membrane surface.









As a future direction, aptamer-modified nano test tubes can be used for more selective

results. It has been recently reported by Tan and coworkers that aptamer-conjugated magnetic

silica nanoparticles can be used for the selective and sensitive detection and collection of acute

leukemia cells.178 Furthermore, clever strategies need to be developed for the efficient loading

and release of biomolecules into and out of these test tubes in order to use them as successful

delivery devices.

Conclusion

We have substantiated a technique for the fabrication of uniform defect-free silica nano

test tubes using alumina membrane templates. First, the advantage of using alumina films grown

on both sides of the Al metal for having cleaner samples was shown, and then the test tube

fabrication methods were compared. We have obtained defective test tubes with the conventional

sol-gel method and this was attributed to the small changes in the viscosity of the gel. Uniform

defect-free silica nano test tubes were prepared by layer-by-layer addition of silica through the

surface sol-gel method. We have shown that argon plasma etching can be used to remove the

silica film on the template surface that normally binds the nanotubes together. Using silane and

Schiff-base chemistry, we have independently modified the inner and outer surfaces of these test

tubes to investigate selective cell response via cell incubation experiments. The inner tube

surfaces were first labeled with Alexa-488 fluorophore and then the template was removed. The

liberated fluorescent-tubes were modified with either a target (IGF-IRa) or a control antibody

(IGF-IRP) and then incubated with breast carcinoma cells. The fluorescence imaging and the

microplate reader data suggest that the tubes modified with target antibody attaches much more

readily to the cell membrane surfaces than the tubes modified with control antibody. More

experiments need to be conducted to verify these results.