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Fabrication of Asymmetric Pores for Biosensors and Transport Studies

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PAGE 1

1 FABRICATION OF ASYMMETRIC PORES FOR BIOSENSORS AND TRANSPORT STUDIES By JOHN EDWARDSON WHARTON 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 John Edwardson Wharton

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3 To my family, for their continued support and love

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4 ACKNOWLEDGMENTS I wish to acknowledge the many people who have given me advice, help, and encouragement during my years in graduate school. I would like to thank Prof. Dr. Charles R. Martin and the entire Martin group for the opportunity to work with them over the years. Prof. Martin was continuously supportive and always willing and ready to discus s some of our new ideas for resear ch. I appreciate the level of independence that Prof. Martin allowed all of his students. Undoubted ly, this created an atmosphere of confidence among the st udents where creativity flourished. I would like to thank Drs. P unit Kohli, Zuzanna Siwy, and Lane Baker for their patience and expert advice along the way. I wish to thank Fan Xu, Lindsay Sexton, Pu Jin, Lloyd Horne, Stefanie Sherrill, and Warren Mino for very impor tant contributions to some of my projects; these great students have been very patient with me and are always willing to help with very high interest and good spirits. Finally, I would like to thank my family and friends for showing confidence in me and for giving their love and sup port throughout the years.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS...............................................................................................................4 LIST OF FIGURES................................................................................................................ .........8 ABSTRACT....................................................................................................................... ............11 CHAPTER 1 INTRODUCTION AND BACKGROUND...........................................................................13 Ion Track-Etch Method.......................................................................................................... .14 Track Formation..............................................................................................................14 Acceleration and Irrad iation Facilities............................................................................15 Chemical Etching of Ion Tracks......................................................................................15 The Effect of Storage of Tracked Material......................................................................16 Effect of Etch Promoters.................................................................................................16 Etch Properties of Selected Polymers..............................................................................17 Effect of Thermal Annealing...........................................................................................17 The Effect of Temperature during Etching.....................................................................17 Effect of Detergents.........................................................................................................18 Etch Properties in Muscovite Mica.................................................................................18 Template Synthesis............................................................................................................. ....18 Template Synthesis Strategies.........................................................................................19 Electroless Deposition.....................................................................................................19 Chemical Vapor Deposition (CVD)................................................................................21 Resistive-Pulse Sensing........................................................................................................ ..21 Plasma-Based Etching........................................................................................................... .23 Asymmetric Diffusion........................................................................................................... .24 Dissertation Overview.......................................................................................................... ..25 2 A METHOD FOR REPRODUCIBLY PREPARING SYNTHETIC NANOPORES FOR RESISTIVE-PULSE BIOSENSORS.............................................................................36 Introduction................................................................................................................... ..........36 Experimental................................................................................................................... ........37 First Etch Step................................................................................................................ .37 Determination of the Diameter of the Base Opening......................................................38 Electrochemical Measurement of the Tip Diameter........................................................38 Second Etch Step.............................................................................................................38 Bovine Serum Albumin (BSA) Resistive-Pulse Sensing................................................39 Results and Discussion......................................................................................................... ..39 Conical Shaped Nanopores are Ideal Resistive-Pulse Sensor Elements.........................39 The Core Technology: The Track-Etch Method.............................................................40 The First Etch Step..........................................................................................................40

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6 Measuring the Diameter of the Ti p Opening after the First Etch....................................41 The Second Etch Step......................................................................................................42 Measuring the Diameter of the Tip Opening after the Second Etch...............................43 Reproducibly Varying the Tip Diameter.........................................................................44 The Mathematical Model................................................................................................45 Electrochemical Details...................................................................................................47 Conclusions.................................................................................................................... .........47 3 ETCH-FILL-ETCH METHOD FOR PREP ARING TAPERED PORES IN ION TRACKED MICA FILMS.....................................................................................................55 Introduction................................................................................................................... ..........55 Experimental................................................................................................................... ........57 Materials...................................................................................................................... ....57 Initial Etching of Mica Tracks to Prepare Very Small Pores..........................................58 Preparation of Tin Sensitizing Solution..........................................................................58 Preparation of Silver Plating Solution.............................................................................58 Filling the Pores with Silver Wires.................................................................................59 Etching Silver Filled Mica to form Tapered Pores..........................................................59 Making Replicas of the Tapered Pores............................................................................60 Preparation of the Carbon Tube Replicas for SEM Imaging..........................................60 Results and Discussion......................................................................................................... ..61 Conclusion..................................................................................................................... .........63 4 ELECTROLESS AU PLATI NG OF TRACK-ETCHED KAPTON POLYIMIDE NANOPOROUS MEMBRANES...........................................................................................69 Introduction................................................................................................................... ..........69 Experimental................................................................................................................... ........70 Materials...................................................................................................................... ....70 Chemical Etching............................................................................................................70 Electroless Plating of Kapton..........................................................................................71 Pore Diameter Measurement...........................................................................................71 Results and Discussion......................................................................................................... ..72 SEM and Ion Current Measurements..............................................................................72 Atomic Force Microscope Images...................................................................................72 Conclusion..................................................................................................................... .........72 5 ASYMMETRY IN DIFFUSIONAL TRAN SPORT OF MOLECULES THROUGH KAPTON CONICAL NANOPORES....................................................................................78 Introduction................................................................................................................... ..........78 Diffusional Transport Desc ribed by Ficks Laws...........................................................79 Hindered Diffusion in Cylindrical Pores.........................................................................80 Hindered Diffusion in Conical Pores..............................................................................81 Experimental................................................................................................................... ........83 Materials...................................................................................................................... ....83

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7 Kapton Polyimide Membrane..........................................................................................84 Irradiation-Track Formation............................................................................................84 Chemical Etching of Membrane Tracks..........................................................................84 Pore Diameter Measurement...........................................................................................86 Transport Measurement..................................................................................................86 Viscosity Measurements..................................................................................................87 Results and Discussion......................................................................................................... ..87 Membrane Characterization............................................................................................87 Transport Measurements of Phthalazine.........................................................................87 The Influence of Cosolute Concentration on Asymmetry...............................................89 Conclusions.................................................................................................................... .........90 6 CONCLUSION.....................................................................................................................102 LIST OF REFERENCES.............................................................................................................104 BIOGRAPHICAL SKETCH.......................................................................................................111

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8 LIST OF FIGURES Figure page 1-1 Swift heavy ions impinge on a dielectr ic material creating damaged ion tracks...............27 1-2 After irradiation, the material s are subject to chemical etchin g which preferentially removes the latent ion track.............................................................................................27 1-3 Etched pore geometry in a homogeneous is otropic medium to a first approximation, showing track etch rate, VT, and bulk at rate, VB................................................................28 1-4 Scanning electron micrographs of Au na nocones replicas of PET conical pores. ...........28 1-5 Scanning electron micrographs of etched particle tracks in single-crystal mica...............29 1-6 Scanning electron micrographs of a porous polycarbonate, alumina and mica membranes used for template s ynthesis (A-C, respectively).............................................30 1-7 Schematic diagram of Au el ectroless plating procedure....................................................31 1-8 Schematic illustration of Au nanotubes obtained from electroless gold deposition..........32 1-9 Schematic illustration of resistive-pulse sensing...............................................................33 1-10 Essential features of the staphylococcal hemolysin pore shown in a crosssection based on the crystal structure.............................................................................................34 1-11 Schematic of a conical nanopore sensor element showing the base diameter and range of tip diameters used in these studies (drawing to scale).....................................35 2-1 Schematic of a conical nanopore se nsor element and etching cell. ................................48 2-2 A typical current-voltage curve used to measure the tip diameter of the conical nanopore....................................................................................................................... ......49 2-3 Current-time transients obtained during the second etch step for three membranes that were subjected to the same first etch. ......................................................................50 2-4 Scanning electron micrograph of the base openings of two conical nanopores in a multi-track PET membrane................................................................................................51 2-5 Scanning electron micrograph of conical gold nanotubes deposited in a conical nanopore membrane...........................................................................................................52 2-6 Plot of tip diameter measured after the second etch step vs. the final nanopore ion current (If) at which the second etch was stopped. ..........................................................53

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9 2-7 Current-pulse data obtained for a protot ype protein analyte, bovine serum albumin (BSA) using PEG-modified conical nanotube sensors. ...................................................54 3-1 After irradiation, the materials are subject to chemical etching which preferentially removes the damaged ion track..........................................................................................64 3-2 Schematic of cell used to do the et ching and to make all electrochemical measurements................................................................................................................... ..65 3-3 Schematic diagram of etch-fill-etch method......................................................................66 3-4 Scanning electron micrographs of mica me mbrane that was exposed on one face to 20% HF and10% nitric acid solution at 25 C for 3 hrs. ..................................................67 3-5 Scanning electron micrographs of mica me mbrane that was exposed on one face to 40% HF and10% nitric acid solution at 25 C for 3 hrs. ..................................................67 3-6 Scanning electron micrograph of carbon tape red nanotube replica of the mica tapered pore........................................................................................................................... .........68 3-7 Low magnification SEM images of car bon tapered nanotube replicas of the mica tapered pore................................................................................................................... .....68 4-1 Definition of bulk etch rate Vb and track etch rate Vt.........................................................73 4-2 Schematic of cell used for electrochemical measurements................................................74 4-3 Pore diameters for diffe rent Au plating times....................................................................75 4-4 Pore diameter as a function of platin g time with measurements taken from SEM image (0-7.5 hrs) and ion current resistance measurements (8-12 hrs).............................76 4-5 Atomic Force micrographs of Kapton and PC membranes before and after electroless Au plating..................................................................................................................... ......77 5-1 The partitioning of a spherical molecule of radius, a in cylindrical pores of radius, r .....90 5-2 Spherical molecule of radius, a, moving within a cylindrical pore of radius, r.................91 5-3 Swift heavy ions impinge on a dielectric solid leading to damaged ion tracks witch can be chemically etched to form pores.............................................................................91 5-4 Definition of bulk etch rate Vb and track etch rate Vt.........................................................92 5-5 Conductivity cell used to prepare conical pores................................................................92 5-6 Etching curve showing moment of breakth rough with sharp increas e in ion current.......93 5-7 Kapton membran showing large op ening (base) of conical pores.....................................94

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10 5-8 Experimental set-up for transport measurements..............................................................94 5-9 Scanning electron micrograph of base side showing pore density (107 pores/cm2) of Kapton membrane..............................................................................................................95 5-10 Scanning electron micrograph of the base showing diameter of 1.68 m.........................95 5-11 A typical current-voltage curve used to measure the tip diameter of the conical nanopores...................................................................................................................... .....96 5-12 Base and tip fluxes for 1 mM phthalazine feed solution...................................................96 5-13 Base and tip fluxes for 3 mM phthalazine feed solution...................................................97 5-14 Base and tip fluxes for 10 mM phthalazine feed solution.................................................97 5-15 Base and tip fluxes for 50 mM phthalazine feed solution.................................................98 5-16 Asymmetric behavior of flux versus concentration...........................................................98 5-17 Theoretical and experimental tip flux vs concentration.....................................................99 5-18 Increase of flux through base and tip, resp ectively, with respect to the flux measured at 0.1 mM...................................................................................................................... .....99 5-19 Experimental set-up for transport meas urements of phthalazine with cosolute dextrose....................................................................................................................... .....100 5-20 Calibration curve of phthalazine, and phtha lazine with the highest concentration of dextrose cosolute used.....................................................................................................100 5-21 Viscosity of dextrose and phthalazi ne over the concentration range used......................101 5-22 Influence of dextrose cosolute concentr ations on the base and tip diffusion from 5 mM phthalazine...............................................................................................................101

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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 FABRICATION OF ASYMMETRIC PORES FOR BIOSENSORS AND TRANSPORT STUDIES By John Edwardson Wharton May 2007 Chair: Charles R. Martin Major: Chemistry The goals of this research are to develop asymmetric nanopores and nanotubes (single and multipore) in polymers and mica membranes by the track-etch method, and an extension of this technology, for sensing, nanostructure fabrication, and to investigat e the transport properties of conical pores. The first part of this work de scribes an extension of the track-etch method to make conical pores in a reproducib le fashion. We have demonstrat ed here that we can, not only reproducibly prepare trac k-etched based conical nanopore sens or elements, but that we can predict from the experimental parameters used during the second etch, what the diameter of the all-important nanopore tip will be. For these reas ons, we believe that the track-etch method will prove to be the technology of c hoice for taking artificial-nanopor e resistive-pulse sensors from the bench top to the practical protot ype-device stage of the R&D effort. The second part of this work describes a me thod to make asymmetric pores in tracked muscovite mica films using an etch-refill-etch a pproach. Tracks in the films were initially etched away with hydrofluoric acid to form na noporous membranes. These nanopores were then refilled with silver nanowires or metal tracks using an electroless plating method. One face of the membrane was then exposed to a solution of hydrofluoric acid and nitric acid, which etched the bulk material and the nanowires respectively, at two different rates. By controlling the

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12 concentration ratio of hydrofluoric acid to nitric acid, tapered pores with diamond shaped crosssection were obtained. Replicas of the asym metric pores were accomplished by carbon vapor deposition, and scanning electron microscopy was used to give evidence of the resulting nanotubes. In this study, excel lent control over tip size and cone angle was demonstrated. In the third section, electrol ess gold plating properties on the surface and pore walls of track-etched Kapton polyimide nanoporous memb ranes were studied. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and ion current measurements were used to characterize the surfaces and di mensions of the pores in th e membrane. Nanoporous Kapton polyimide membranes were elect roless gold plated over different times and the pore diameter characterized using SEM. Ion current measurements were used to measure the diameter of very small pores. AFM images show that after electroless gold plating, the gold surface layers are smooth compared to a similarly structured poly carbonate membrane. Electroless plating the membranes for 12 hours produced Au wires in the pores. Removing the membrane by oxygen plasma revealed that the plating in the walls is also relatively smooth. The final part of this work describes asymmetric diffusional transport of neutral molecular species through nanoporous polymer membranes. Membranes containing conical nanopores from polyimide (Kapton HN Dupont) foils were prepar ed by the track-etching technique, based on irradiation of the polymer with swift heavy ions and subsequent etching of the latent tracks. The transport properties (e.g., flux) of these membranes were inves tigated using UV-Vis spectroscopy. Transport experiments were perf ormed with bare polymer membranes without any modification. We report the preferential direc tion of the flow of mo lecules through conical nanopores and spatial or concentration dependence of diffusion coefficient for a particular flow direction.

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13 CHAPTER 1 INTRODUCTION AND BACKGROUND Nanopore fabrication technology has produced nanoporous materials that are potential candidates for applications in vari ous fiels, such as bionanotechnology,1, 2 gas separation,3 catalysts4 and micro-electronics2, 5 In particular, the track-etch method has become an indispensable technology for the producti on of nanoand microstructured materials.6 This technology can be applied directly to most polymers and via th e replication techniqueto a wide variety of materi als, including metals.6 Track-etch membranes were first marketed three decades ago and remain the best product for a number of biological, medical, analytical and scientific applications.6 Recently, there has been a surge of interest in developing abiotic analogues of biological nanopores as sensing elements for chemicals and biological sensors.7 We have explored the fabrication of such synthetic nanopores using the tr ack-etch method. Particular interest is in the fabrication of synthetic conical pores for resistive-pul se sensing. Resistive-pulse sensing using conical nanopores is in its infa ncy. Studies in this area coul d prove beneficial for future applications of conical po res for science and technology. Membranes and porous materials have f ound various applications in filtration and separation processes.8, 9, 10 Modern biotechnology has posed ne w challenges in the application of such membranes, and requires pores with diamet ers similar to those of molecules under study,11 (e.g., as small as several nanometers). The nanomet er scale of such pores is necessary in both achieving optimal control of the flow of biomolecu les, as well as in developing sensors for their detection. The transport properties of such na nometer scale pores are not well understood yet. The hint that nanopores behave differently from micropores, comes from Mother Nature.12

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14 Biological channels and pores have diameter of ~ 1 nm and are cr itical for functioning of living organisms. This chapter is divided into six sections wh ich provide the background information for this research. Section 1 reviews th e track-etch method, first discovered by Price and Walker, for preparation of pores in dielectric materials. The track-etch method is used in the preparation of conical pores described in Chapters 2, 3, 4 and 5. Section 2 reviews membrane-based template synthesis. This method, pioneered by the Martin group, is used in preparation of nanostructured materials described in Chapters 2 and 3. Section 3 review resistive-pulse sensing; which is the focus of the application of Chapter 2 and 3. Section 4 describes plasma based etching; a procedure necessary for the libera tion of carbon tube in mica templates in Chapter 2. In Section 5, an introduction of Asymmetric Diffusion is given. Asymmetric diffu sion is the topic of Chapter 5. Finally, Section 6 is the dissertation overview. Ion Track-Etch Method Track Formation When dielectric materials, such as poly mers, ceramics and minerals, are bombarded by swift ions, latent tracks are formed al ong the path of the ions (Figure 1-1).13, 14 Ion track materials can be divided into two categories: (a) single-tracked and (b) multiple-tracked materials. Single-tracked materials can be pr oduced by controlling the beam optics and fluence of the heavy ion beam.15 Commonly tracked materials ar e Makrofol-KG, Kapton-H, PVDF, mica films, cellulose nitrate, CR-39 and Lexan polycarbonate. Swift io n beams are produced by cyclotrons or linear accelerator and the radiation is characteri zed by extremely high linear energy transfer.6 The conventional ionization radiation sources such as radioactive isotopes or electron accelerators,16 are less sophisticated and less expensive and are mostly used in the industrial

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15 processes.6 Present-day heavy ion accel erators provide beams with i on energies in the order of 10MeV/u and even 100MeV/u which expands the treatment dept up to millimeters.6 Acceleration and Irradiation Facilities About a half dozen heavy ion accelerators em ployed for irradiation of materials on the industrial scale exist. The Tanden Van de Graaff accelerators at Brookheaven National Laboratory are used to bombard materials with ions for manufacturing and testing purposes.6 At the Grand Accelerator National dIons Lours, Fran ce, ions are produced in an electron cyclotron resonance(ECR) source.6 The cyclotron of Louvain la Neuve is a multiparticle variable energy, cyclotron capable of accelerating protons, alpha particles and hea vy ions. The RFQ + cyclotron combination at the Hahn-Meitner Institute in Be rlin, delivers intense beams of ion species such as Kr or Xe with energies from approximately 1.5-6 MeV/u17 At the Flerov Laboratory of Nuclear Reactions(Dubna) a beam line connected at the U-400 cyclotron is equipped with scanning systems which allows one to obtain a homogeneous distributi on of ion tracks on the target up to 60 cm in width and 6 cm in height.6 Additionally, a line r accelerator at GSI (Darmstadt),6 the AVF cyclotron at TRCRE JAERI (Takasaki)6 and some others are used in experiments on polymer modification.6 Chemical Etching of Ion Tracks After irradiation, the materials are subject to chemical etching which preferentially removes the latent ion track (Figure 1-2)16 This etching process results in pore formation in the material.16 Etching is the pore-size -determining and pore-shapedetermining stage of the technology. In a homogeneous isot ropic medium, mainly two influe ntial parameter describe the etch processthe bulk etch rate VB and the track etch rate VT (Figure 1-3).6 The ratio of track etch rate to the bulk etch rate is called the track-etch-ratio. When VT is >> VB, pores turn out to be cylindrical as opposed to conical. In other word s, high track-etch-ratio yields cylindrical pores,

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16 where as low track-etch-ratio yiel ds conical pores. The arctangent of the inverse track-etch-ratio ( VB/ VT) yields the half cone angle of the pore. Th e bulk etch rate depends on the material, on the etchant composition and on the temperature.14 The track etch rate depends on the sensitivity of the material, irradiation conditions, post-i rradiation conditions a nd etching conditions.6, 14 The Effect of Storage of Tracked Material The most important storage factors are the atmo sphere in which the material is stored, the temperature and the illumination conditions during storage. In the presence of oxygen the latent ion tracks becomes susceptible to track etching. This is due to the oxidation of the radicals formed during irradiation. When polymers are stor ed at temperatures close their glass transition temperature, rearrangement on a molecular scale can take place which may lead to annealing of the ion tracks. Storing under i llumination may lead to photo oxidation and is able to increase the track etch ratio by orders of magn itude. It is reported that trac ks in poly(ethylene terephthalate) (PET) may be sensitized by uv radiation of 310 to 400 nm. Soaking in weak solvents, such as dimethyl formamide or water-soluble gass can sensitize ion tracks in PET.6 Effect of Etch Promoters Etch promoters are organic solven ts that accelerate the etch pr ocess when added to the etch bath. It has been observed th at track etch ratios in polycar bonate (PC) can be above ten thousand. On the other hand, track etch ratio can be dramatically decreased down to 2 to 4 in PET by the addition of solvents such as metha nol, ethanol or propanol, leading to wide cone angles. These organic solvents help to dissolv e large fragments ready to move into the liquid phase by disengaging them from their neighbors.6 Figure 1-4 shows an example of etch promotion using ethanol in PET.18

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17 Etch Properties of Selected Polymers In PET (OOC-C6H4-COO-CH2CH2), the main points of etch attack are the partially charged COOester groups, which are hydrolyz ed by alkalis. During alkaline etching the ordinary bond between carbon and oxyge n is broken which produces COOand HOat the ends of the formed fragments. For polycarbonate (OOC-O-C6H4-C(CH3)2-C6H4), the main point of etch attack is the carbonate group O-COO-. During the alkaline etching, chemical bonds are ruptured on both sides of the carbonate group, leading to the formation of carbonate ions, CO3 2-. The other product is diphenylol HO-C6H4-C(CH3)2_C6H4-OH. In Polyimide (C6H4-OC6H4), the preferential point of et ch attack is the oxygen. At high pH the imide group will be hydrolyzed. The et ching mechanism is complex because of the simultaneous factors of oxidation and alkalinity. The chemical reaction responsible for etching different polyimide can be different becau se they are made up of monomer units. 19 Effect of Thermal Annealing By increasing the temperature of the tracked material, ion tracks can be thermally annealed. For polymers, heating above the gla ss transition temperature mobilizes the polymer fragments formed along the ion path. These fr agments are sucked in to the voids of the neighboring pristine material, wi ping out the latent ion track.14 The Effect of Temperature during Etching Etch rates usually increase with temperature. Therefore, to obtain high throughput makes it necessary to work at high temperatures. It is found that by alternati ng the temperature during etching, high aspect ratio pores with large diameters can be obtained. At room temperature diffusion processes can be faster than chemical reactions of the etchant within the polymer. 14

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18 From this basis, the technique to make pores wi th large cone angles we re develop by soaking at low temperature and etching at high temperature.20 Effect of Detergents Amphiphilic detergents may increase the track-etch-ratio. These detergents have been shown to produce nanopores with cigar-like shapes and very sma ll entrance openings. It is thought that amphiphilic detergents attach to the hydrophilic surface layer, rendering it less permeable to the etchant.21 Etch Properties in Muscovite Mica Damage tracks are form in single crystal muscovite mica (KAl2(AlSiO3O10)(OH)2 from ion irradiation along the (001) directi on. Because the etching rate along the tracks is much faster than both the lateral and bulb etch rate, when the tracks are etch ed through, nanopores with small cross-section and sm all taper angle (0.02 ) are created.22 In muscovite mica, all the pores are diamond-shaped with inner angles very close to 60 and 120 (Figure 1-5). All the pores in a given sample have the same size and orientation.22 By correlating th e results of scanning electron microscopy and X-ray diffraction on et ched mica crystals, it was found that the orientation of the diamonds is exactly the same as that of the mica unit cell. The four sides of the diamonds are parallel to the four oxygen-terminated planes within the unit cell. These facts points out that the diamondshaped pores have their origin in the mica crystal structure. Also, this shows that the uniform diamond shape aris es because the oxygen-terminated planes are those with the slowest etch rate, and the pores are aligned because th e template are single crystals.22 Template Synthesis The Martin group has pioneered a general me thod for the preparation of nanostructured materials called template synthesis23-25 Template synthesis method entails depositing a desired

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19 material of interest into a porous solid. The size and shape of the nanomaterial depend on the dimentions of the nanocavities within the porous template material. Depending on the membrane and synthetic material used, nanostructu res such as solid nanofibers or nanotubes can be obtained. The method is termed general because nearly any chemical synthesis method used to prepare bulk materials can be adapted to synthesize materials. There are reports of metals,10, 26-32 polymers,33-36 carbons37-39 and semiconductors40, 41 prepared by the template synthesis method. More advance material pr eparation include composite nanostructured material, both concentric and tubular42, 43 and segmented composites nanowires.44 Template Synthesis Strategies Three commonly used templates are; porous polymers, alumina and mica membranes (Figure 1-6). Some of the more common synthetic strategies us ed to prepare nanomaterials include chemical vapor deposition,38, 45, 46 electrochemical47-49 and electroless deposition,50, 51 chemical52 and electrochemical polymerization,53 and sol-gel chemistry.40, 54 Special attentions will be devoted to the electroless deposition in side polymers and mica, and chemical vapor deposition in mica since these are the methods used for preparing templates in chapters 2, 3, and 4 respectively. Electroless Deposition The electroless deposition met hod involves the use of a chemi cal reducing agent to plate a metal from a solution onto a surface. Unlike el ectrochemical deposition, a conductive surface is not necessary. The key requirement of electrole ss deposition is arrangement of the chemistry such that the kinetics of the homogenous electr on transfer from the reducing agent to the metal ions is slow. This is essential because the metal ions would simply be reduced in the bulk solution for fast electron transfer In electroless depos ition, a catalyst needs to be coated onto the pore walls so that reduction of th e metal ion only occurs at the pore surfaces. Figure 1-7 shows

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20 the schematic representation that was used to prepare silver and gold nanowires and nanotubes within PET, Kapton and Mica track-etched membrane s. The membranes were first exposed to a sensitizer (Sn2+). This is accomplished by simply immersing the membrane for 45 minutes in a solution that is 0.026 M in SnCl2 and 0.07 M in trifluoroacitic acid in 50/50 methanol/water. The tin sensitizer binds to the pore walls and memb rane surfaces via complexation with the amine, carbonyl and hydroxyl groups. 63 After sensitization, the membra ne is rinsed thoroughly with methanol and immersed into an aqueous solu tion of ammoniac silver nitrate (0.029 M Ag (NH3)2 +) for 5 minutes. A redox reation occ ours in which the surface bound tin ( ) is oxidized to tin ( V) and the Ag+ is reduced to elemental Ag. As a result, the pore walls and the membrane surface become coated with nanoscopic silver particles. The membrane is again thoroughly rinsed with methanol. The silver coated membrane is then immersed into a gold plating bath that is 7.9 x 10-3 M in Na3Au (SO3)2, 0.127 M Na2SO3, and 0.625 M in formaldehyde at 4 C. The Au galvanically displaces the Ag particles because the reduction potential of Au is more positive than that of Ag. As a result, the pore walls and surfaces become coated with Au particles. These particles are excellent catalyti c sites for the oxidation of fo rmaldehyde and the concurrent reduction of Au (I) to Au (0).63 Without a catalyst, the kinetics of the electron transfer from the reducing agent (formaldehyde) to Au (I) is slow ; therefore, the gold plating continues on gold particles instead of in the bulk solution. The reaction can be represented as follows: 2Au (I) + HCHO + 3OH HCOO+ 2H2O + 2Au (0) (1-1) This method yields the Au nanowires or nanot ubes within pores plus Au surface layers on both face of the membrane. These structures r un through the entire thic kness of the template membrane (Figure 1-8). By controlling the platin g time, the inside diameter of the tubes can be varied because the thickness of both the Au surface films and nanotube wall increase with

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21 plating time. By controlling the plating time, the inside diameter of the nanotubes can be varied, even as low as 1 nm in diameter.28 As a result, these membranes can be used in a simple membrane permeation experiment to cleanly separa te small molecules on the basis of molecular size.28 Also, by chemisorbing appropriate thiols to the Au nanotube wall based on well known gold-thiol chemistry, the Au nanotube membrane can be made to preferentially transport cations vs. anions and hydrophobic vs. hydrophilic molecules.10, 27, 31, 55 In addition, Au nanotube membranes are electronically conductive and can be charged electrostatically in an electrolyte solution.27 This introduces ion transport selectivity, allowing the Au nanotube membranes to be electromodulated between ideal-catio n and idea-anion transport states.27 Thus these Au nanotube memebranes are ideal model systems for studying how pore size, chemistry, and charge affect the transport selectivity at the nanometer scale. Chemical Vapor Deposition (CVD) CVD is commonly used to prep are carbon nanomaterials. We38, 56 and others57, 58 have synthesized carbon nanotubes with in the porous alumina membranes using CVD. This involves placing an alumina membrane in a high-temperature furnace (ca. 700 C) and passing a gas such as ethane, propene or ethylene through the memb rane. Thermal decomposition of the gas occurs on the pore walls, resulting in th e deposition of carbon nanotubes w ithin the pores. High surface area microporous carbon with long-range order has been synthesized by using zeolite Y as a template with propylene CVD.59 Besides carbon nanostructures, other nanomaterials have been obtained by CVD. For example, the martin group has used a CVD method to coat an ensemble of gold nanotubes with concentric TiS2 outer nanotubes.43 Resistive-Pulse Sensing Resistive-pulse60 sensors for molecular and macromolecules analytes60-77 use a nanopore in a synthetic or biological membra ne as the sensor element. This method, which when applied to

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22 such analytes is sometimes called stochastic sensing,60, 69 entails mounting the membrane containing the nanopore between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ion current flowing through the electrolyte-filled nanopore. In simplest terms, when the analyte en ters and translocates th e nanopore, it transiently blocks the ion current, resulting in a downward current pulse (Fi gure 1-9). The frequency of such analyte-induced current puls es is proportional to the concen tration of the analyte, and the identity of the analyte is encoded in the magnitude and duration of the current pulse.60-77 The majority of such resistiv e-pulse biosensing data has been obtained using a biological nanopore, -hemolysin ( -HL), embedded in a supported lipid-bilayer membrane as the sensor element (Figure 1-10).60-69 This biological nanopor e sensor has two key adva ntages. First, it can be made analyte selective by using chemical or genetic-engineering methods to attach molecularrecognition agents to the nanopore. As a result numerous different analyte types including metal ions,64 DNA,65, 66 proteins,67 and small molecules68 have been selectively detected with the -HL nanopore. Second, the biological nanopore can be reproducibly prepared from the commercially available -HL protein, which is obviously of great im portance if practical, real-world, sensing devices are ultimately to be derived from this technology.78 There is however a key impediment to developing practical sensors based on the biological nanopore. This problem concerns the fragility of the supported bilayer membrane that houses the nanopore. Such membranes typically survive for periods of only hours before rupture, much too short of a time to make a practical sensing device.69 One approach for solving this problem is to replace the biological nanopore, and bilayer membrane, with an artificial nanopore embedde d in a mechanically and chemically robust synthetic membrane.69-77, 79, 80 Such artificial nanopores are often prepared by modern

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23 microlithographic methods, using for example a focused ion71 or electron72 beam to bore the nanopore into a silicon or Si3N3 membrane. We and others are exploring an alternative technology, called the track-etch method,16, 19, 81, 82 for preparing nanopores for resistive-pulse sensors.70, 75-77, 79, 80 Analytes detected with prototype track-etched nanopore sensors include small molecules,70 DNA,75, 76 proteins77 and nanoparticles.79 Furthermore, there are older reports of developing virus sensors based on track-etched nanopores.80 The sensor elements we evaluated were conically shaped nanopores70, 75-77, 79, 81 prepared by the track-etch method in polyethylene terepht halate (PET) membranes. Such conical nanopores have two openings the large-diameter (base) opening at one face of the membrane and the small-diameter (tip) opening at the opposi te face (Figure 1-11). Fabrication methods to prepare these conically shaped nanopores for resis tive pulse sensing, is addressed in chapters 1. Plasma-Based Etching Plasma etching, a dry etching process, has b ecome a very useful means of removing small quantities of material from a variety of substrates quickly and efficiently.83 Plasma processes have been used in many highly sensitive integrat ed applications to precisely remove specific materials from sample surfaces. To generate pl asma, a pair of electrode is needed; one is connected to a radio frequency (R F) voltage and the other is gr ounded. RF energy is applied to the electrodes which accelerates el ectrons to increase their kinetic energy. The electrons collide with a neutral gas to form a collection of gase ous species including ions, free radicals, electrons, photons and neutrals.83 The gaseous species can react with the surface to be etched such that reaction product is volatile and can be pumped away. There are several types of plasma etchingphysical etching (anisotropic), chemical etchi ng (isotropic) an d reactive ion (combination of physical etching and chemical etching). Usually in plasma based etching, both chemical etching and physical etching occur.83

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24 There are many applications for plasma etch ing such as photoresist removal, glass-like compound etching (e.g., SiO2) and polymer etching to produce microstructures and nanostructures. For example, large-area, well-o rdered, periodic nanopillar arrays with lateral dimensions as small as 40 nm have been developed based on a combination of colloidal lithography and plasma etching techniques.84, 85 The etching mask on silicon substrates were prepared using the close-packed structures formed by monodisperse polystyrene beads.84, 85 Polymer surfaces can also be modified by plasma treatment, for example, to improve wetting properties and to enhance the adhesi on of plasma-deposited coatings.86 Plasma etching technique is described in Chapter 2 to remove carbon surface layers from mica tracked etched membrane that was previous ly exposed to CVD. This was necessary to expose the underlying mica surface for dissolution with HF so that the carbon nanotube can be revealed. Asymmetric Diffusion In some systems, diffusive transport thr ough membranes may in some circumstances pass more readily in one direction than the othe r. This phenomenon is known as asymmetric diffusion, and is known in the contex t of transport across membranes,87-89 and in the context of osmosis.90, 91 The above asymmetric phenomena is explai ned either by the binding of particles at intra-pore sites or other electrosta tic interaction with the pore surf ace. In this research, we show that asymmetric diffusion can occur with no binding or electrostatic interaction of particle with the pore surface. It is demonstrated in Chap ter 5 that asymmetric diffusion can take place by purely geometric constraints. He re, conical nanopores, with tip di ameter comparable to that of the diffusion molecule, are the used for asym metric diffusion of neutral molecules.

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25 Dissertation Overview The goal of this research is to develo p conical nanopores and nanotube (single and multipore) in polymers and mica membranes by th e track-etch method and extension of this technology for sensing and other app lications, and to inve stigate the tr ansport properties of these conical pores. The previous part of Chapter 1 has reviewed background information for this dissertation including the ion tr ack-etch method, membrane based te mplate synthesis, electroless metal deposition, chemical vapor deposition, resi stive-pulse sensing, plasma based etching and asymmetric diffusion. In Chapter 2, an extension of the track-etch method to make conical pores in a reproducible fashion is demonstrated. We have shown here th at we can not only repr oducibly prepare tracketched based conical nanopore sens or elements, but that we can predict from the experimental parameters used during the second etch, what the diameter of the all-important nanopore tip will be. For these reasons, we believe that the track -etch method will prove to be the technology of choice for taking artificial-nanopore resistive-pulse sensors from the bench top to the practical prototype-device stage of the R&D effort. Chapter 3 describes a method to make asymme tric pores in tracked muscovite mica films using an etch-refill-etch approach. Tracks in the films were initially etched away with hydrofluoric acid to form nanoporous membranes. These nanopores were then refilled with silver nanowires or metal tr acks using an electroless pl ating method. One face of the membrane was then exposed to a solution of hydr ofluoric acid and nitric acid, which etched the bulk material and the nanowires respectively, at two different rates. By controlling the concentration ratio of hydrofluoric acid to nitric acid, tapered pores with diamond shaped crosssection were obtained. Replicas of the asym metric pores were accomplished by carbon vapor

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26 deposition, and scanning electron microscopy was used to give evidence of the resulting nanotubes. In this study, excel lent control over tip size and cone angle was demonstrated. In Chapter 4, electroless gol d plating properties on the su rface and pore walls of tracketched Kapton polyimide nanoporous membranes we re studied. Scanning electron microscopy (SEM), atomic force microscopy (AFM), and ion current measurements were used to characterize the surfaces and por e dimensions of the membrane. Nanoporous Kapton polyimide membranes were electroless gold plated over different times and the pore diameter characterized using SEM. Ion current measurements were used to measure the diameter of very small pores. AFM images show that after electroless gold plating, the gold surface layers are smooth compared to a similarly structured polycarbonat e membrane. Electroless plating the membranes for 12 hours produced Au wires in the pores Removing the membrane by oxygen plasma revealed that the plating in the walls is also relatively smooth. Finally, Chapter 5 describes asymmetric diffusi onal transport of neutral molecular species through nanoporous polymer membranes. Me mbranes containing conical nanopores from polyimide (Kapton HN Dupont) foils were prepar ed by the track-etching technique, based on irradiation of the polymer with sw ift heavy ions and subsequent etch ing of the latent tracks. The transport properties (e.g, flux) of these membranes were investigated using UV-Vis spectroscopy. Transport experiments were perf ormed with bare polymer membranes without any modification. We report the preferential direc tion of the flow of mo lecules through conical nanopores and spatial or concentration dependence of diffusion coefficient for a particular flow direction.

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27 Figure 1-1. Swift heavy ions impinge on a diel ectric material creating damaged ion tracks. Figure 1-2. After irradiation, the ma terials are subject to chemical et ching which preferentially removes the latent ion track. Etch-stop solution Etchant Etch-stop solution Etchant Etch-stop solution Etchant Etch-stop solution Etchant

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28 Figure 1-3. Etched pore geometry in a homogeneous isotropic medium to a first approximation, showing track etch rate, VT, and bulk at rate, VB. A B C A B C Figure 1-4. Scanning electron mi crographs of Au nanocones repl icas of PET conical pores. Increasing fraction of ethanol in the etch solution results in greater cone angle (AC).[Adapted from Scopece, P.; Baker, L. A.; Ugo, P.; Martin, C. R. Nanotechnology 2006 17 3951-3956.]

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29 Figure 1-5. Scanning electron micrographs of et ched particle tracks in single-crystal mica

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30 A B C A B C Figure 1-6. Scanning electr on micrographs of a porous polycarbonate, alumina and mica membranes used for template synthesis (A-C, respectively).

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31 Figure 1-7. Schematic diagram of Au electroless plating procedure

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32 Side View Electroless Gold Plating Membrane Pores Gold tubes lining pores Side View Electroless Plating Membrane Pores Au nanotubes lining the pores Electroless Gold Plating Membrane face Pore Gold surface layer Top View Top View Membrane Face Pore Electroless Plating Gold surface film Side View Electroless Gold Plating Membrane Pores Gold tubes lining pores Side View Electroless Plating Membrane Pores Au nanotubes lining the pores Electroless Gold Plating Membrane face Pore Gold surface layer Top View Top View Membrane Face Pore Electroless Plating Gold surface film Figure 1-8. Schematic illustration of Au na notubes obtained from electroless gold deposition

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33 Figure 1-9. Schematic illustrati on of resistive-pulse sensing

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34 Figure 1-10. Essential features of the staphylococcal hemolysin pore shown in a crosssection based on the crystal structure. [Adapted from Bayley, H.; Martin, C. R. Chemical Reviews (Washington, D. C.) 2000 100 2575-2594.]

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35 Figure 1-11. Schematic of a conical nanopore se nsor element showing the base diameter and range of tip diameters used in these studies (drawing to scale)

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36 CHAPTER 2 A METHOD FOR REPRODUCIBLY PREPARING SYNTHETIC NANOPORES FOR RESISTIVE-PULSE BIOSENSORS Introduction Resistive-pulse60 sensors for molecular and macromolecules analytes60-77 use a nanopore in a synthetic or biological membra ne as the sensor element. This method, which when applied to such analytes is sometimes called stochastic sensing,60-69 entails mounting the membrane containing the nanopore between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ion current flowing through the electrolyte-filled nanopore. In simplest terms, when the analyte en ters and translocates th e nanopore, it transiently blocks the ion current, resulting in a downward current pulse. The frequency of such analyteinduced current pulses is proportional to the concen tration of the analyte, and the identity of the analyte is encoded in the magnitude and duration of the current pulse.60-77 The majority of such resistiv e-pulse biosensing data has been obtained using a biological nanopore, -hemolysin ( -HL), embedded in a supported lipid-bilayer membrane as the sensor element.60-69 A key advantage of this biological-na nopore sensor element is that it can be reproducibly prepared from the commercially available -HL protein. This is of great importance if practical, real-world, sensing devi ces are ultimately to be derived from this technology.78 There is, however, a key impediment to developing practical sensors based on the biological nanopore. This problem concerns the fragility of the supported bilayer membrane that houses the nanopore. Such membranes typically survive for periods of only hours before rupture, much too short of a time to make a practical sensing device.69 One approach for solving this problem is to replace the biological nanopore, and bilayer membrane, with an artificial nanopore embedde d in a mechanically and chemically robust synthetic membrane.69-77, 79, 80 Such artificial nanopores are often prepared by microlithographic

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37 methods, using for example a focused ion71 or electron72 beam to bore the na nopore into a silicon or Si3N3 membrane. We and others are exploring an alternative technology, called the track-etch method,16, 19, 81, 82 for preparing nanopores fo r resistive-pulse sensors.70, 75-77, 79, 80 Analytes detected with prototype track-etched nanopore sensors include small molecules,70 DNA,75, 76 proteins77 and nanoparticles.79 Furthermore, there are older re ports of developing virus sensors based on track-etched nanopores.80 This field of artificial-nanopor e resistive-pulse sensing is cu rrently in its infancy. A key question that must be addressed before practical sensors can be developed is can the nanopore sensor element be prepared reproducibly, as the biological nanopore can? We address this critically important issue here. The sensor elements we evaluated were conically shaped nanopores70, 75-77, 79, 81 prepared by the track-etch method in polyethylene terepht halate (PET) membranes. Such conical nanopores have two openings the large-diameter (base) opening at one face of the membrane and the small-diameter (tip) opening at the opposite face (Figure 2-1a). We have found that the diameters of both of these opening can be contro lled with good reproducib ility using a new twostep pore-etching procedure. Furthermore, we ha ve developed a simple mathematical model that allows us to predict the diameter of the tip opening from the parameters used during pore etching. Good agreement was obtained between th e predicted and experimentally measured tip diameters. Experimental First Etch Step The tracked PET membrane (from GSI) was mo unted in cell shown in Figure 2-1b, and the etch solution (9 M NaOH) was placed in one half cell and the stop solution (1M formic acid plus 1 M KCl) in the other. A platinum wire el ectrode was placed in each solution and a Keithley

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38 6487 was used to apply a transmembrane potential difference of 1V during etching, with polarity such that the anode was in the etch solution. The electrochemical reac tions occurring at the anode and cathode are discussed in the Supplementa ry Materials. Etchin g was terminated after two hours by replacing the contents of the etch half-cell with stop-etch solution. The membrane was then rinsed with purified water (Barnstead D4641, E-pure filters). Determination of the Diamet er of the Base Opening Multi-track membranes (106 cm-2) were subjected to the same first etch step as used for the single-track membranes, and th e base openings were imaged via FESEM (JEOL JSM-6335F). The average base diameter obtained (520+ 45 nm) is associated with measurements of 50 pores in five different multi-track membrane samples. Electrochemical Measurement of the Tip Diameter The membrane was mounted in the cell, and the half cells were filled with 100 mM phosphate-buffered saline, pH = 7.0 that was also 1 M in KCl. The specific conductivity of this solution was measured using a conductometer (YSI 3200) at 25 C; a conductivity of 0.107 S cm-1 was obtained. A Ag/AgCl electrode immersed into each solution was used in conjunction with the Kiethly 6487 to obtain the current-volta ge curve for the nanopore (Figure 2-2). We have validated this electrochemical method by comparing diameters obtained via this method with diameters for the same pores measured by electron microscopy.92, 93 Second Etch Step The etch solution in this case was 1 M NaOH, and its conductivity was measured at 0.160 S cm-1 (25 C). The membrane was mounted in the cel l, and the half cells were filled with this etch solution. A platinum electro de was immersed into each ha lf-cell solution, and the Keithley 6487 was used to apply a transmembrane potenti al difference of 1V and measure the nanopore ion current as a function of time. Etching was te rminated at specified current values (Figure 2-3,

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39 Figure 2-4) by replacing the etch solution in both half cells with the stop-etch solution. The membrane remained in the stop etch for at least 30 min and was then rinsed with purified water. Bovine Serum Albumin (BSA) Resistive-Pulse Sensing Conical nanopores sensors ha ving two different tip diameters, 58 nm and 44 nm, were prepared; the base diameter fo r both sensors was 520 nm. After etching the pore walls were coated with gold nanotubes using the electr oless plating method de scribed previously.28 After electroless plating the tip diameters of the re sulting gold nanotubes were measured using the electrochemical method discussed above; tip diameters of 32 nm and 23 nm were obtained. The Au-coated nanopore walls were th en functionalized with a thio lated PEG (MW 5000 Da, Nektar Therapeutics) to prevent non-specific protein adsorption.94 This was accomplished by immersing the nanotube-containing membrane in a 0.1 mM solution of the PEG at 4 oC for ~15 hours. The membrane was then immersed in purified water for 1.5 hours to remove any unbound PEG. The tip diameters were then re-measur ed; values of 27 nm and 17 nm were obtained. The BSA (Sigma) was dissolved in 10 mM phosphate-buffered saline that was also 100 mM in KCl (pH 7.4). The concentration of BS A was 100 nM, and the BSA solution was placed on the tip side of the membrane. Buffer was placed on the base side and a transmembrane potential of 1000 mV was used to drive the protein through the nanopor e (tip to base) by electrophoresis. Results and Discussion Conical Shaped Nanopores are Ideal Resistive-Pulse Sensor Elements Resistive-pulse sensing entails mounting the nanopore membrane between the two halves of an electrolyte-filled cell (Figure 2-1 (b)) and passing an io nic current through the electrolyteflooded nanopore. In a conically shaped nanopore the voltage drop caused by this ion current is focused to the electrolyte solution in the tip opening of the nanopore.79 Indeed, the field strength

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40 in the solution within the nanopor e tip can be greater than 106 V m-1, when the total voltage drop across the nanopore membrane is only 1 V.79 A consequence of this fo cusing effect is that the nanopore ion current is extremely sensitive to anal yte species present in the nanopore tip. That is, there is an analyte sensing zone just insi de the tip, which makes conically shaped nanopores ideally suited for the resistive-pulse sensing ap plication. This has b een demonstrated with prototype conical-nanopore sensors for analyte speci es ranging in size from small molecules, to proteins, to nanoparticles.70, 75-77, 79 The Core Technology: The Track-Etch Method The track-etch method has been practiced comm ercially for decades to make polymeric nanopore membranes for filtration applications.16, 19, 81, 82 It entails passing high energy particles through the membrane, to create damage tracks, followed by chemical etching to convert these damage tracks into pores. While the commercia l process yields membranes that contain high pore densities, a method for preparing single-damage-track membranes was developed at the Gellsellschaft fur Schw erionenforschung (GSI).19 We purchased such single-track PET membranes from GSI for these studies. This is an important point with regard to the overall sensor-fabrication technology the key precu rsor material, the tracked membrane, can be obtained commercially. The First Etch Step Both etch steps used the cell shown in Figure 2-1b. Step 1 entails placing a solution that etches the damage track on one si de of the membrane and a soluti on that neutralizes this etchant on the other side.81 For PET the etchant is NaOH, and the etch-stop is formic acid. This yields a conically shaped pore (Figure 21a) with the base opening facing the etch solution and the tip opening facing the etch-stop solution. To determ ine when the etchant ha s broken through to the etch-stop solution, and a contiguous pore has been obtained, an el ectrode is placed in each

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41 solution, and a potential difference is applied across the membrane. Before breakthrough, the transmembrane ion current is zer o, and breakthrough is signaled by a sudden rise in the current.81 We previously showed that the diameter of the base opening could be controlled by varying the potential applied across the membrane during this first etch step.92 An applied transmembrane potential of 1.0 V was used in th e first etch step for all of the nanopores investigated here. To obtain a measure of the re producibility of the base diameter obtained after the first etch step, we subjected multi-track membranes (106 tracks cm-2) to the same first etch as used for the single-track membranes, and imag ed the base openings using field emission scanning electron microscopy (FESEM, Figure 2-4) We used multi-track membranes for this study because it is difficult to locate the base opening in electron micrographs of a singlenanopore membrane. We have previously shown that the pore diameter obtained for trackedetch membranes is independent of track density.95 A base diameter of 520+ 45 nm was obtained, indicating good reproducibility in base di ameter after the first etch step. However, the tip diameter varied between 1 and 7 nm and could not be reproduced from etch to etch. We reasoned that th is is an inherent feature of this anisotropic etch process. This is because the etch and etch-stop solutions ar e mixing (and neutralizing each other) in the nascent tip, which makes it difficult to control the etch rate in this critically important region of the nanopore. Measuring the Diameter of the Tip Opening after the First Etch Because the tips after the first etch are so small, they are very difficult to find and image via electron microscopy. Therefore, an el ectrochemical method described previously70, 76, 81, 92 was used to measure the diameter of the tip. Th is entailed mounting the membrane sample in the cell (Figure 2-1b), filling both half cells with an electrolyte so lution of known ionic conductivity,

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42 and obtaining a current-voltage (I-V) curve a ssociated with ion-tran sport through the nanopore (Figure 2-2). The experimental slope of this linear I-V curve is the ionic conductance, G1, (in Siemens, S) of the nanopore, which is given by81 G1 = ( KCl db dt) / 4 L (2-1) where KCl is the experimentally measured conductivity of the KCl-based electrolyte used (S cm1), L is the length of the nanopore (membrane th ickness), db is the experimentally measured diameter of the base opening, and dt is the diameter of the tip ope ning. Because all of the other parameters in Equation 1 are known, dt can be calculated. The Second Etch Step In the second step NaOH etch solution is pl aced on both sides of the membrane. Again, a transmembrane potential is ap plied, and the ion current fl owing through the nanopore is measured as a function of time during this etch. Our key innovation is that the second etch is stopped at a prescribed value of this nanopore ion current rather than at some prescribed time after starting the second etch (e.g., Figure 2-3). We adopted this approach because of the variability in tip diameter obtained after the first etch step. The c onsequence of this variability is that if we stopped the second etch at a prescribed time, we would obtain a corresponding variability in the tip diameters obtained after the second etch step. In contrast, as we will see below, there is an exact mathematically relati onship between the ion cu rrent flowing through the nanopore when the second etch is stopped (If) and the diameter of tip opening. To prove that the pores obtained after the s econd etch truly are conically shaped, we used an electroless plating method28 to deposit corres pondingly conically shaped gold nanotubes within the pores of the multipore membranes de scribed above. The PET membrane was then

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43 dissolved and the conical nanopores colle cted by filtration and imaged by FESEM.18 These images show that a nearly ideal conically shaped pore is obtained (Figure 2-5). Measuring the Diameter of the Tip Opening after the Second Etch The same electrochemical method was used, but the mathematics is slightly different. We define the diameters of the base and tip openings after the first etch step as db1 and dt1 and the diameters after the second etch as dbf and dtf. These diameters are related via dbf = (db1 + x) (2-2) dtf = (dt1 + x) (2-3) where x is the change in diameter during the se cond etch. Substituting Equations 2-2 and 3-3 for db and dt in Equation 2-1 yields G2 = ( KCl (db1 + x)(dt1 + x)) / 4 L (2-4) where G2 is the slope of the current-voltage curve us ed to determine the tip diameter after the second etch, db1 and dt1 are the experimentally determined base and tip diameters, respectively, after the first etch, KCl is the experimentally measured cond uctivity of the electrolyte used (100 mM phosphate-buffered saline, pH 7, that was also 1 M in KCl; = 0.107 S cm-1), L is the membrane thickness, and x is the change in diameter between the first and second etch steps. We define the parameter M as L MKCl4 (2-5) which allows us to write, after some simple algebraic manipulation, 2 1 1 1 1 2) ( x x d d d d M Gt b t b (2-6) Substituting (dtf dt1) for x (where dtf is the diameter of the tip opening after the second etch), and again applying some simple algebraic manipulations, yield

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44 0 ) (2 1 1 2 M G d d d dtf t b tf (2-7) This is a quadratic equation in dtf for which the solution is 2 / 4 ) ( ) (2 2 1 1 1 1M G d d d d dt b t b tf (2-8) Note that the quadratic formula has two roots; i.e., there should be a + instead of a + between the two terms in the numerator of Equation 2-8. Howe ver, the root that results when subtraction is used yields a negative valu e for the tip diameter. Because all of the parameters on th e RHS of Equation 2-8 are known, dtf can be calculated. Furthermore, because the base diameter at the start of the second etch is large (520 nm), the change in base diameter during the second etch ( x in Equation 2-2) is negligibly small for all but the very largest tip investigat ed here (60 nm, Figure 2-6). Reproducibly Varying the Tip Diameter Figure 2-6 shows a plot of nanopor e tip diameter, measured after the second etch step (Eq. 2-5), vs. the nanopore ion current at which this etch was stopped (If). We see that the tip diameter can be reproducibly vari ed over the range from 10 to 60 nm (data points in Figure 2-6). This is important because this is exactly the ra nge in tip diameters we used in our prototype protein,77 DNA,76 and nanoparticles79 sensors. The lower limit (10 nm) is determined by the tip diameter obtained after the first etch which, again, was in the range of 1 to 7 nm. However, we have shown that the walls of such na nopores can be lined with gold nanotubes,77 and that the diameter of these tubes can be c ontrolled at will down to 1 nm.96 Hence, if tip diameters smaller than 10 nm are needed, a pore with a 10 nm tip can be gold plated to reduce the tip to any desired value. Furthermore, tips larger than the 60 nm maximum shown in Figure 2-6 can be easily prepared by simply stopping the sec ond etch at larger values of If.

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45 The Mathematical Model We begin by defining a new conductance, Getch, which is the ion current at which the second etch is stopped (If) divided by the transmembrane potential applied during the second etch (Eap). With this definition, Eq uation 4 can be rewritten as If = Eap ( etch (db1 + x)(dt1 + x)) / 4 L (2-9) where etch is the experimentally meas ured conductivity of the NaOH solution used in the second etch. Equation 2-9 is agai n a quadratic Equation in dtf for which the solution is 2 / 4 ) ( ) (2 1 1 1 1K I d d d d df t b t b tf (2-10) where K = Eap etch /4L (see Derivation of Equation 2-5, be low). Equation 2-10 allows us to calculate the value of the tip diam eter after the second etch step (dtf) for any value of If at which the second etch was stopped. We noted above that the base diameter before and after the second et ch is essentially the same for all but the largest tip in Figure 2-6. This allows us to simplify Equation 2-9 to If = Eap etch db1 dtf / 4 L (2-11) which can be rearranged to dtf = If 4 L/ (Eap etch db1 ) (2-12) This obviously provides a much simpler relationship between dtf and If. Plots of dtf vs. If calculated using the simplified e quation (Equation 2-12) and the exact equation (Equation 2-10) are shown as the two solid lines in Fi gure 2-6. The tip diameters calculated by these two equations are identical for tips below ~20 nm. Furthermore, the agreement between the experimentally measured (Equation 2-8) and theoretically calculated (Equation 2-10) tip diameters is good, especially considering that there are no adjustable

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46 parameters in the calculations. For example, at If = 20 nA the experimental and calculated tip diameters differ by less than 10%, and at 40 nA they are identical. The calculated tip-diameter values are, in general, slightly smaller than the experimental values. This results from an interesting featur e of the transport properties of conical nanopores if there is charge on th e pore wall and if the tip opening is small, such nanopores act as ion current rectifiers.97 The consequence of this rectifi cation phenomenon is that the ionic conductivity of an electroly te solution within the tip of the nanopore can be lower than the value measured for a bulk sample of the same elect rolyte. Since we used the bulk-solution conductivity in our calculations, the calculated values are in genera l low. Excellent agreement is obtained between the experimental and calculate d diameters for the largest tip (Figure 2-6) because large-tip pores do not rectify the ion current.98 We believe that procedures to obviate the small disagreement between the experimental and calculated tip diameters can be developed, and we are currently pursuing this issue. To illustrate the importance of controlling the tip diameter in resistive-pulse sensing, we obtained current-pulse data for a prototype protein analyte, bovi ne serum albumin (BSA), with nanopore sensors having two different tip diameters. The sensors in this case were conical PET nanopores that had been lined with gold nanotubes28, 77 and then coated with a poly(ethylene glycol thiol) (PEG) to preven t nonspecific protein adsorption.94 The tip diameters, 17 nm and 27 nm, were measured after PEG functionalization. Figure 2-7 shows current-pulse data obtained for BSA with th ese two different sensors. The current-pulse signature can be define d by the average duration and magnitude ( I) of the current pulses. The magnitude of the current pulse is important because if I is not larger than the peak-to-peak noise in the background current, the current pulse will be undetectable. We

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47 found that I is larger for the nanopore sensor with the smaller tip opening ( I = 80+ 20 pA) than for the sensor with the larger tip opening ( I = 35+ 9 pA). This is because the roughly 4 nm x 4 nm x 14 nm94 BSA molecule more effectively blocks the ion current as it translocates the smaller, 17 nm, tip. Electrochemical Details Pt electrodes were used to a pply the transmembrane potential di fference in both of the etch steps, and the applied potential was 1.0 V in bot h cases. The half reactio n occurring at the Pt cathode was the reduction of the dissolved O2 in the solution. O2 + 4H+ + 4 e2H20 (2-13) The low (nA-level) currents, a nd the fact that the solutions were exposed to air during etching, insured that the O2 was not depleted. The half reacti on occurring at the Pt anode was the reverse of Equation 2-15. Conclusions In his review of nanowire-based chemical a nd biosensors, Lieber stresses the importance of being able to reproducibly pr epare the nanowire sensing element.78 The same is true for artificial nanopores to be used as resistive-pulse sensor elements. We have shown here that we can not only reproducibly prepare track-etched ba sed conical nanopore sensor elements, but that we can predict from the experimental parameters used during the second et ch, what the diameter of the all-important nanopore tip will be. For these reasons, we believe that the track-etch method will prove to be the technology of choice for taking artificial-nanopore resistive-pulse sensors from the bench top to the practical prototype-device stage of the R&D effort.

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48 PET membrane 12 m Conical nanopore Base opening(520 nm) Tip opening(10 to 60 nm) a A PET membrane 12 m Conical nanopore Base opening(520 nm) Tip opening(10 to 60 nm) a PET membrane 12 m Conical nanopore Base opening(520 nm) Tip opening(10 to 60 nm) a A Poly(chlorotrifluoroethylene) cell Pressure plate 3.5 cm 1.0 cm 3.5 cm 1.0 cm Electrodes Membrane 3.5 cm 1.0 cm b Aluminum frame Clamping screw Solution chamber B Poly(chlorotrifluoroethylene) cell Pressure plate 3.5 cm 1.0 cm 3.5 cm 1.0 cm Electrodes Membrane 3.5 cm 1.0 cm b Aluminum frame Clamping screw Solution chamber Poly(chlorotrifluoroethylene) cell Pressure plate 3.5 cm 1.0 cm 3.5 cm 1.0 cm Electrodes Membrane 3.5 cm 1.0 cm b Aluminum frame Clamping screw Solution chamber B Figure 2-1. Schematic of a conical nanopore sensor element a nd etching cell. (A) The base diameter and range of tip diameters used in these studies (drawing not to scale). (B) Cell used to do the etching and to ma ke all electrochemical measurements.

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49 -2 -1 0 1 2 -0.2-0.100.10.2Applied transmembrane potential (V)Nanopore ion current (nA)a -2 -1 0 1 2 -0.2-0.100.10.2Applied transmembrane potential (V)Nanopore ion current (nA)a -2 -1 0 1 2 -0.2-0.100.10.2Applied transmembrane potential (V)Nanopore ion current (nA)a -2 -1 0 1 2 -0.2-0.100.10.2Applied transmembrane potential (V)Nanopore ion current (nA)a Figure 2-2. A typical current-voltage curve used to measure the tip diameter of the conical nanopore.

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50 020406080100120140 0 5 10 15 20 25 30 35 40 45 Nanopore ion current (nA)Etch time (minutes) Figure 2-3. Current-time transients obtained during th e second etch step for three membranes that were subjected to the same first et ch. The second etch was stopped in each case when a final nanopore ion current (If) of 40 nA was obtained.

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51 Figure 2-4. Scanning electron mi crograph of the base openings of two conical nanopores in a multi-track PET membrane that had been etch ed (first etch step) as per the singletrack membranes used in these studies.

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52 Figure 2-5. Scanning electron micrograph of conical gold na notubes deposited in a conical nanopore membrane.

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53 0 10 20 30 40 50 60 70 80 010203040Final nanopore ion current during second etch, If(nA)Tip diameter (nm) Figure 2-6. Plot of tip diameter measured after the second etch step vs. the final nanopore ion current (If) at which the second etch was stopped. The points are the experimentally measured tip diameters. The error bars are measurements on three different membrane samples prepared identically. The solid curves were calculated using the simplified equation (Equation 2-12, red curv e) and the exact e quation (Equation 2-10, blue curve).

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54 100 s 20 pA 100 s 20 pAa b A B 100 s 20 pA 100 s 20 pAa b 100 s 20 pA 100 s 20 pAa b A B Figure 2-7. Current-pulse data obtained for a prototype protein analyte, bovine serum albumin (BSA) using PEG-modified conical nanotube sensors. (A) Tip diameters of 17 nm and (B) Tip diameters of 27 nm. BSA concentration = 100 nM. Applied transmembrane = 1000 mV.

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55 CHAPTER 3 ETCH-FILL-ETCH METHOD FOR PREPARING TAPERED PORES IN ION TRACKED MICA FILMS Introduction The Martin research group and others have been investigating a general method for preparing nanomaterials know n as template synthesis.23, 96 This method entails the synthesis or deposition of a desired material within the po res of a nanopore membrane that serves as a template. These template membranes contain mo nodisperse pores that ar e typically cylindrical in geometry, and the pore diameter can be varied at will from tens of nanometers to tens of microns. Since typical pore geometries are cylindrical, corr espondingly cylindrical nanostructures are usually synthesized via the template method; depending on the membrane and synthetic method used, these may be solid nanowires or hollow nanotubes. 99 Recently, our research team and others have become interested in nanopores that have a conical pore shape and the correspondingly conical nanostructures synthesized via the template method within these pores. A number of applic ations can potentially benefit from conical pore geometry. For instance, it has been shown that su ch conically shaped nanopores can be used as the sensing element for new types of small molecule, 70 DNA, 75, 98, 100, 101 protein 77 and particle79 sensors. Conically shaped gold nanotubes deposited within such pores can also act as mimics of voltage gated ion channels. 102 Membranes used for separations might also benefit from a highly asymmetric pore structure. Fi nally, in addition to sensing and separations platforms, conical nanostructures prepared by more conventional methods have been proposed for use as cathodes in field-emission displays. 103 To date, the vast ma jority of conical pores have been fabricated using tracked polymers. 81, 89, 93, 95, 97, 104, 105 However, we are interested in exploring materials other than polymers to use as conical pore templates because their different properties may prove potentially superior for cert ain applications, and other applications might

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56 be realized that are not possibl e with polymers. One such material of interest is tracked muscovite mica. Because mica is an inorganic crystalline mate rial, it possesses some properties not present in polymers that might make it superior in some aspects for certain applications. For example, the surface of mica is molecularly flat, 106 and is a good candidate for platform for AFM imaging of DNA, 106 and support layer for lipid bilayers. 107 Also, mica is very chemically resistant and has high thermal and mechanical stability. 22, 108 Mica conical pores might prove much more stable for resistive pulse sensor s than polymers. Furthermore, these properties make it possible for template synthesis of materials that requir e high temperatures. Additionally, nanostructures that demand special geometry that is diffi cult to obtain by conven tional methods may be realized. For instance, tapered-shape carbon structure can provide mechanical stability yet provide very sharp tips, which may be usef ul for enhanced electron field emission. 109 We and other have shown that the well-known track-etch method 16 can be used as a starting place for prepari ng such conical nanopores. 81, 89, 93, 95, 97, 104, 105 This method entails bombarding a thin film (5-20 m) film of the material with a collimated beam of high-energy particles to create parallel damaged tracks throug h the film. To make cylindrical pores, the tracked membrane is simply immersed into a ch emical etch bath, wher e preferential etching along the damaged track converts each track into a cylindrical pore. To make conical pores, the tracked membrane is mounted in an etching cell with an etch solu tion on one side of the tracked membrane and a stop solution on the other side.81 This is shown schematically in Figure 3-1. Since the damaged track is etched at a longe r duration time and faster at the face of the membrane exposed to the etch solution than at the face of the membrane exposed to the stop solution, conically shaped nanopores are obtained.

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57 While this stop-etch approach has been successf ul in making conical na nopores in a variety of polymer materials, the method fails for the pr eparation of conical pores in mica membranes. The key impediment is due to the ratio of the trac k to bulk etching rate. The track etch rate is much faster than the bulk etch ra te in mica (about 3000 time faster), 108 so that the etch solution traverses the entire membrane before any signifi cant bulk etching takes place. This means that all parts of the membrane start etching almost at the same time in an isotropic fashion giving rise to uniform pores instead of asymmetrical pore shap e. One approach to solve this problem is to replace the tracks of the mica films with a material that is more controllably etched, for instance metal nanowires. In this study, we developed a method to independently control the solution etch rate traversing the membrane, and also the etch rate of the surrounding bulk material, to give asymmetric pores. We prove this by using car bon vapor deposition to replicate the pores, dissolving the membrane to expose the tapered tubes, and imaging the nanotubes using scanning electron microscopy. The results of th ese investigations are reported here. Experimental Materials Muscovite mica wafers (1.181X 0.0004 inches ) were purchased from Spruce Pine Co. USA. Then these bare wafers were irradiated by swift heavy U25+ ions of 2.2GeV kinetic energy with fluence of 104 to107 cm-2 (GSI Darmstadt, Germany), which produced damage tracks through the mica membranes. H ydrofluoric acid (HF, 48~51% from ACROS), for etching and dissolving mica membranes, was used as received. Anhydrous tin (II) chloride 98% (Aldrich) and hydrochloric acid (A CROS) were used as received to sensitize the mica membranes for electroless plating. Ammonium hydroxide (Fis her), silver nitrate (M allinckrodt), potassium sodium tartrate tetrahydrade (Aldrich), and magn esium sulfate (Fisher) were used as received to prepare silver plating so lution. Ethylene (30% balanced with Helium, from Praxair) was used as

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58 the CVD carbon precursor gas. Purified water wa s prepared by passing house-distilled water through a Millipore Milli-Q water purification system. Initial Etching of Mica Tracks to Prepare Very Small Pores Mica wafers, containing damaged tracks, were exposed to low concentration HF solution on both faces to create pores of about 10nm in diameter. The wafers were sandwiched between two half cells of a conductivity ce ll (Figure 3-2.) and 2% HF at 25 C were placed in each half cell for a period of 10 minutes. The etch proce ss was terminated by quickly removing the etch solution and replacing it with wa ter for 2 minutes. Fresh water was replaced several times for two minute intervals. Finally water was allo wed to sit in the cell for another two hours. Preparation of Tin Sensitizing Solution Tin (II) chloride was used to sensitize the wafe rs so that electroless plating of silver can take place on the surfaces. Tin (II) chloride crys tals (0.5 g) were placed in 100 mL water and stirred to give a cloudy appearance. 2 mL of 10 % hydrochloric acid was added using a pipette causing the mixture to become a clear ti n (II) chloride sensitizing solution. Preparation of Silver Plating Solution Filling the pores in the wafer with silver wire was done to make a more controllably etched material. A two part electroless silver plating solution was made, where solution A contains the silver ions and solution B contains a reducing agent. Solution A was made by dissolving 45.4 g of silver nitrate to 450 mL of water and th en adding ammonium hydroxide drop wise using a pipette until the solution goes from clear to da rk brown and returns to clear. Solution B was made by dissolving 159 g of potassium sodium tartrate tetrahydrate and 11.4 g magnesium sulfate to 364 mL of water. Both so lutions were stored away from light.

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59 Filling the Pores with Silver Wires The purpose of silver plating the nanopores in the mica membrane was to control the rate at which etch solution traverses the membrane relative to the lateral or bulk etch rate. First, the intended stop side of the membrane was exposed to tin by filling one half cell with the solution. The other half cell was left empty so that one face of the membrane was exposed to air. Tin solution remains in the cell for 45 minutes giving it time to properly wet and sensitize the inner walls of the pores. The membrane was then rinsed with water several times then was left to sit in water for at least one hour. Aft er removal of water from the both half cells, the membrane was now ready for silver electroless plating. A dilute solution A (0.5 mL Solution A in 45 mL water) was cooled to 4 C and then 0.5 mL of solution B was added. This mixture was then placed in the half cell that was not exposed to tin solution (sid e to be etched). The c onductivity ce ll containing the membrane was placed in a refrigerator, which was set at a temperature of 4 C, for one hour. Following electroless plating of silver, the remaining solution in the cell was removed and the cell was thoroughly rinsed with water. Figure 33 shows the schematic outline for this etch-filletch method. Etching Silver Filled Mica to form Tapered Pores The silver wire-containing mica membrane was exposed to HF/HNO3 etch solutions on one face. This etch solution of HF (variable concentration) and HNO3 (10%) etched mica and silver wire respectively at two different rates. After 3 hours th e etch solution was removed and rinsed with water several times. The me mbrane was then left to sit in 10% HNO3 for at least 3 hours to get rid of any residual s ilver. Finally, the membrane was rinsed in water several times followed by soaking for at least 3 hours.

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60 Making Replicas of the Tapered Pores Tapered carbon tubes were obtained using a chemical vapor de position (CVD) method described in detail previously. 38, 110, 111 A piece of a porous mica membrane (preparation methods were described in the previous secti on) was placed vertically into a quartz tube (diameter: 4.5 cm, length: 48 cm). This tube wa s then inserted into a high-temperature tube furnace (Thermolyne 21100) and the furnace was heated to 670oC under Ar flow. Once the temperature stabilized, the Ar gas was replaced with an ethylene gas (20 sccm), which thermally decomposed into carbon on the inner-wall and bot h faces of the mica template. After a desired deposition time, the heating was terminated, th e ethylene gas was replaced by Ar flow, and the furnace was cooled down to room temperature. Unlike our previous CVD procedure with alumina templates, 111 heat pretreatment of the template in this experiment was skipped since the mica membranes we used can withstand temperatures above 900oC without any physical deformation. The yielded carbon thickness can be controlled by vary ing the duration of deposition. Preparation of the Carbon T ube Replicas for SEM Imaging First, CVD carbon/mica membrane was put into 48~51% HF solution for 16 hr to dissolve away the mica template. Next, HF was then removed by pipette, leaving the liberated carbon nanoboxes (connected together by the carbon surface f ilm) which were rinsed with methanol and suspended in methanol. The next preparati on procedure entailed the removal of the carbon surface layer on one face of the CVD-treated mi ca membrane to expose the carbon tubes. This was accomplished by using an oxygen plasma etch procedure. A 1 cm x 1 cm hole was premade in one piece of aluminum foil. This hole de fines the area of the membrane that is exposed to the oxygen plasma. The entire assembly, with the hole-containing Al foil facing up, was then placed in the center of vacuum chamber of a plasma reactive-i on etching system (Samco, model

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61 RIE-1C). The following etch cond itions were used: power = 100 W, O2 pressure = 300 Pa, O2 flow rate = 30 sccm. After etching away the carbon surface film (determined by measuring the conductivity of the membrane su rface), the membrane was immersed into ~ 48-51 wt % HF solution to dissolve the mica template. This st ep does not result in free carbon tubes because they are held together by carbon surface film that was not exposed to oxygen plasma. Finally, the sample was rinsed with distilled water and air dried overnight. Sample imaging was conducted using JEOL 6335F field emission scanni ng electron microscope (FESEM). Prior to FESEM imaging, all samples were sputtered with Au/Pd using the Desk II Cold Sputter instrument (Denton Vacuum, LLC). The sputter current = 45 mA, Ar pressure = 75 mTorr, sputtering time = 60sec. The resu lting Au/Pd film was ~ 16 nm. Results and Discussion During chemical etching of ion-tracked membrane s, the damaged zone of the latent track is transformed into nanopores. 16 The simplest description of the etching pro cess defines two parameters: the bulk etch rate ( VB) and the track etch rate ( VT). VB depends on the material, etchant composition and temperature. VT depends on additional parame ters, such as sensitivity of the material to trackin g, post-irradiation conditi ons and etching conditions. 14 When the tracked film is exposed to an etchant on one f ace as described above, the results give conical pores. In most ion-tracked materials, without exposure to extreme conditions (like exposure to high illumination) the VB/ VT ratio defines the cone angle that is formed. 14 However, in mica VT is 3000 times faster than VB, 108 thus producing c one angles (0.02 )108 that is almost zero, giving essentially pores with almost identical cross-sect ions along the membrane thickness. To improve the cone angle in polymeric materials, the VB/ VT ratio is increased by a number of methods including increasing the etchant concentration, 108 applying a high transmembrane potential, 92

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62 and modifying the etch solution composition. 18 However, none of the above methods can work with mica since VT is orders of magnitude greater than VB. 108 To get around this problem, the etched ion tracks in mica was re placed with silver metal that can be independently etched with nitric acid to a wide variety of etch rates. Figu re 3-4a is an SEM image of a membrane that was exposed on one face to 20% HF a nd10% nitric acid solution at 25 C for 3 hrs. Here, HF and HNO3 solutions etch the bulk mica membrane and th e metal track respectively. The base side clearly shows the tapered cone shape that resulte d from this etch. The decreasing pores size is made quite evident from the progressive mica la yers going down into the cone. The opposite face of the membrane (Figure 3-4b) shows the tip to be on the order of a magnitude smaller than the base. The Martin group has been using conical pores for resistive pulse sens ing of molecules. One important feature that make conical pores idea l for sensing is, that most of the resistance is focus in a short distant of the tip. As shown by Lee et al,79 the electric field in conical pores is focused at the tip. The greater the half cone opening angle, the smaller the focus and hence a sensing zone for molecular translocation of the pore for resistive pulse sensing. Half cone opening angle depends on the VL/ VT ratio, and we changed the concentration ratio of the etchants to achieve this. Because the concentration ratio of HF (the lateral etchant) to HNO3 (the track etchant) is greater than used previously, the lateral to track etch rate increases VL/ VT resulting in greater half cone opening angle (~ 21 ). Figure 3-5 is an SEM image of a tapered mica pore that was etched with a higher percen tage of HF solution than previously in Figure 3-4. We demonstrate here that the cone angle can be c ontrolled at will, because we can independently control the solution etch rate traversing the memb rane, and also the lateral etch rate, to give asymmetric pores. This implies that the eff ective pore length (the part of the pore where

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63 resistance is focused), and hence the focus of el ectric field can be varied in these tapered mica pores. This is an important feature for resistivepulse sensing that is like ly responsible for pulse duration. To indirectly capture the entire geometry of the tapered mica pores in the membrane, a replica was done using CVD met hod. Figure 3-6 clearly shows SEM images of carbon tapered nanotubes replicas of the mica pores. The angl es are well defined and surface of the tube appears rather smooth at th e magnification shown. The half cone opening angle was calculated to be ~ 6 This is a relatively large cone angle when compared to those of polymers that are etched without any promoters (Lane) or applied high potential (chad). The half cone opening angle can be calculated as ) 2 ) (( arctan L d dt b (3-1) where L is the length of the pore, db and dt are the large and sma ll openings of the pore, respectively. For db >> dt the equation simplifies to: ) 2 ( arctan L db (3-2) Figure 3-7 is a low magnificati on image of the carbon replicas of mica tapered pores indicating that we can indeed reproduce the tapered geometry mica pore uniformly. Conclusion This study described a method to make asymmetric pores in tracked muscovite mica films using an etch-fill-etch approach. Tracks in the films were initially etched away with hydrofluoric acid to form nanoporous membranes. We demonstrated th at by controlling the concentration ratio of hydrofluoric acid to nitric acid during etch ing, tapered pores with diamond shaped cross-section can be obtai ned. Additionally, we have s hown that the cone angle of the pores can be controlled be changing the concentra tion ratio of the bulk and metal etch solutions.

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64 Replicas of the asymmetric pores were accomplished by carbon vapor deposition, and scanning electron microscopy was used to give evidence of the resulting nanotubes. These conical mica pores make prove more stable for resistive pulse sens ing. Because it is so easy to tailor the cone angle, this might make these mica pores more su itable sensing devises. One potential capability that might be realized in resis tive pulse sensing, is the tuning of the cone angle to control pulse duration. Etch-stop solution Etchant Etch-stop solution Etchant Damaged ion track Etch-stop solution Etchant Etch-stop solution Etchant Etch-stop solution Etchant Etch-stop solution Etchant Damaged ion track Figure 3-1. After irradiation, th e materials are subject to chemi cal etching which preferentially removes the damaged ion track

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65 Figure 3-2. Schematic of cell used to do th e etching and to make all electrochemical measurements. Poly(chlorotrifluoroethylene) cell Pressure plate 3.5 cm 1.0 cm 3.5 cm 1.0 cm Electrodes Membrane 3.5 cm 1.0 cm b Aluminum frame Clamping screw Solution chamber

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66 HF Electroless plating of Silver Mica membrane Tracks Silver Pore HF/ HNO3 Figure 3-3. Schematic diag ram of etch-fill-etch method

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67 AB AB Figure 3-4. Scanning electron micrographs of mica membrane that was exposed on one face to 20% HF and10% nitric acid solution at 25 C for 3 hrs. (A) Side exposed to the etchant (the base). (B) Side exposed to water (the tip). A B Figure 3-5. Scanning electron micrographs of mi ca membrane that was exposed on one face to 40% HF and10% nitric acid solution at 25 C for 3 hrs. (A) Side exposed to the etchant (the base). (B) Side exposed to water (the tip).

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68 Figure 3-6. Scanning electr on micrograph of carbon tapered nanotube replica of the mica tapered pore. Figure 3-7. Low magnification SEM images of carbon tapered nanotube replicas of the mica tapered pore.

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69 CHAPTER 4 ELECTROLESS AU PLATI NG OF TRACK-ETCHED KAPTON POLYIMIDE NANOPOROUS MEMBRANES Introduction Track-etched polymer membranes have found ma ny applications in industry and research as filtration and separation materials. 6 Chemical modification of the inner walls of these membranes has made them more selective and sophisticated se paration structures. 27, 28, 31 These chemical modifications have been made possible by first plating th e inner walls of the pores of these membranes with gold, 23 then attaching a desired thiol terminated functional group. 31 For reproducible results of any analytical measurem ents done using the pores of these nanoporous membranes, it is crucial that the pores remain stable and have a well define internal diameter, particularly where the pore approaches the size of the analyte molecule. One of the problems faced when using some polymers such as poly carbonate (PC) and poly (e thylene terephthalate) (PET) membranes, is that very small pores tend to temporarily bloc k during ion transport mesurement. 112 Another disadvantage discovered wh en using these membranes to measure ion current, is that possi ble dangling alkyl groups 113 render the pores from been well define and thus gives inconsistent results and lots of noise in the current measurement. 112 The polyimide Kapton on the other hand does not exhibit these disadvantages. 112 Due to its abilities of maintaining excellent physical, elect rical and mechanical properties at both low and high temperature extremes, Kapton is a very attractive polymer for use as a particle track-etch membrane for application in separation and filtration in industry and research. 16, 114, 115 It will be advantageous therefore, if lik e polycarbonate and PET, Kapton can similarly be electrolessly plated with gold to tailor pore diameter and, furt her, be modified with desired thiol terminated functional gr oups for selective separation.

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70 The objective of this study was to determine if the polyimide Kapton, a very chemically resistant and stable polymer, can be electrole ssly gold plated with similar or better quality compared to PC porous membranes. Additionally, investigate whether pla ting can be controlledthat is, tailor pore diameter of the membrane with pl ating time. It is interesting to note that to date; there is no known report of elect roless plating of Kapton membranes. Experimental Materials Kapton 50 HN foils (12.5 m thick, 107 tracks per cm2) were obtained from the linear accelerator laboratory UNI LAC at the GSI (Darmstadt, Germany). Boric Acid was obtained from Fisher and used as receiv ed. NaOCl (13% active Cl), KI, SnCl2, AgNO3, NaHCO3, were obtained from Aldrich and used as received. Trifluoroacetic acid, Na2SO3, NH4OH, formaldehyde and methanol were obtained from Mall inckrodt and used as received. Commercial gold-plating solution (Oromerse SO Part B) was obtained from T echnic Inc. Milli Q water was used to prepare all solutions and to rinse the membranes. Chemical Etching Cylindrical pores were etched in the tracks of the Kapton foils using sodium hypochlorite solution as describes in detail elsewhere. 116 The shape and size of th e pores can be tailored by the choice of etchant an d the etching conditions. 116 To obtain cylindrical pores the etching rate along the track, the-so called track etch rate Vt has to be much faster than the non-specific etching of the polymer ca lled the bulk etched rate Vb 14, 81 The relation between Vt and Vb is explained in figure 4-1. 14, 81 Studies have shown that an e fficient etching can be performed in sodium hypochlorite containing 13% active chlorine content. 116 Furthermore, it has been demonstrated that the shape of the pore can be regulated with an a ppropriate choice of pH. 116 When sodium hypochlorite is not buffered its pH ~ 12.6. At these conditions, and elevated

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71 temperature of 50C, Vb of Kapton is high (~ 0.21 ( m / h) and the pores become strongly conical. Buffering the etchant with boric acid to pH ~9 enables one to obtain cylindrical pores. It is important to note that th e etching works only at basic pH when the hydrolysis of imide bonds by OHis possible. The membrane was immersed in a Teflon container containing 400 ml of NaOCl solution at pH 9.8 and temperature of 50C. First, the container with the etchant solution is brought to 50C by plac ing it in a water bath controled by a water heater. The size of the pores increases with etching time. After etching, the membrane was rinsed with D. I. water and left to soak for two hours. The memb rane was allowed to dry in air overnight. Electroless Plating of Kapton In order to better control th e size and surface chemistry of Kapton, the membranes can be plated electrolessly with gold. 10 Tailoring of the pore size in polymer membranes by the time of performing the electroless plating with gold has been demonstrated previously. 28, 51 Since Kapton membranes possess carboxylate groups ma de available via imide hydrolysis by the etchant, it is expected that these would act as active sites for the bonding of tin ( ). Tin can then reduce silver ion which later acts as a nucleation site for the re duction of gold. The procedure followed the recipe for the electroless plating of polycarbonate. 28, 51 Pore Diameter Measurement Cylindrical pores were ch aracterized by taking scanni ng electron microscopy (SEM) images of the membrane surfaces. Also, we have chosen to use an electrochemical technique based on measuring ion current to measure pore diameter. This entailed mounting the membrane sample in the cell (Figure 4-2), filling both half cells with an electrolyt e solution of known ionic conductivity, and obtaining a curren t-voltage (I-V) curv e associated with ion-transport through the nanopore. The experimental slope of this li near I-V curve is the i onic conductance, G, (in Siemens, S) of the nanopore, which is given by

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72 G = ( N A KCl d2) / 4 L (4-1) where N is the pore density, A is the membrane area, KCl is the experimentally measured conductivity of the KCl-base d electrolyte used (S cm-1), L is the length of the nanopore (membrane thickness), d is the pore diameter. Because all of the other parameters in Equation 41 are known, d can be calculated. Results and Discussion SEM and Ion Current Measurements Figure 4-3 shows SEM images of Kapton porous membranes before and after electroless gold plating. Here we see that th e pore diameter decreases with plating time. Figure 4-4 shows a plot of pore diameter as a func tion of Au plating time from 0 to 12 hours. The two methods of measuring pore diameter gave very similar result s. This is important because only resistance measurements can be used for small pore diameter that cannot be resolved by SEM. This data indicate that one can tailor th e pore diameter with plating time down to the nanometer scale. This capability is important for different transpor t studies and in sensor research where the size of the pore with respect to the analyte is important. 10, 28, 77, 96 Atomic Force Microscope Images Figure 4-5 shows the atomic force microscope images of the Kapton membranes before and after electroless plating. The membrane s remain relatively sm ooth after gold plating compared to a similarly porous structured poly carbonate membrane after plating with gold. Conclusion Electroless gold plating propert ies on the surface and pore walls of track-etched Kapton polyimide nanoporous membranes we re studied. SEM, AFM, and ion current measurements were used to characterize the surfaces and pore dimensions of the membrane. Nanoporous Kapton polyimide membranes were electroless gold plated over different times and the pore

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73 diameter characterized using SEM. Ion curr ent measurements were used to measure the diameter of very small pores. AFM images show that after electroless gold plating, the gold surface layers are smooth compared to a sim ilarly structured polycarbonate membrane. Electroless plating the membranes for 12 hours pr oduced Au wires in the pores. Etching away the membrane with sodium hypochlorite to expose gol d tube replicas revealed that the plating in the walls is also smooth. Figure 4-1. Definiti on of bulk etch rate Vb and track etch rate Vt.[Adapted from Apel, P. Radiation Measurements 2001, 34 559-566.]

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74 Figure 4-2. Schematic of cell used for electrochemical measurements.

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75 Before AU1.5 hrs plating3 hrs plating 4.5 hrs plating7.5 hrs plating Figure 4-3. Pore diameters fo r different Au plating times.

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76 0 20 40 60 80 100 120 140 160 180 200 220 012345678910111213Time (hrs)Pore diameter (nm) Figure 4-4. Pore diameter as a function of plating time with measurements taken from SEM image (0-7.5 hrs) and ion current resistance measurements (8-12 hrs).

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77 PC membraneBefore Au Before Au Roughness ~ 13.6 Roughness ~ 5.1 Roughness ~ 6.3nm Roughness ~ 3.6nmKapton membrane After 2.5 hrs Au plating After 2.5 hrs Au plating Figure 4-5. Atomic Force micrographs of Kapton and PC membranes before and after electroless Au plating

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78 CHAPTER 5 ASYMMETRY IN DIFFUSIONAL TRANSPO RT OF MOLECULES THROUGH KAPTON CONICAL NANOPORES Introduction Membranes and porous materials have found various applications in filtration and separation processes. 8-10 Modern biotechnology has posed new challenges in th e application of such membranes, and requires pores with diamet ers similar to those of molecules under study 11 (e.g., as small as several nanometers). The nanom eter scale of such pores is necessary in both achieving optimal control of the flow of biomolecu les, as well as in developing sensors for their detection. The transport properties of such na nometer scale pores are not well understood yet. The hint that nanopores behave differently from micropores, comes from Mother Nature. 12 Biological channels and pores have diameter of ~ 1 nm and are cr itical for functioning of living organisms. Ion channels and pores exhibit tran sport properties not obser ved with larger pores, for example (i) selectivity fo r ions or molecules, (ii) rectification of ion current117 (iii) ion current for constant voltages applied across the membrane118 (iv) facilitated transport of molecules (v) transport of ions and molecules against th eir electro-chemical potential gradient. 119 It has recently been demonstrated that asym metric conical nanopores in polymer films can exhibit transport and r ectification properties similar to biochannels. For example, conical nanopores in PET and Kapton membranes are cati on selective and rectif y ion current with a preferential direction of cation flow from the tip to the base of the cone. 81, 88, 105, 120, 121 However, unlike the above cases, in this inve stigation there is no applied transmembrane potential. Also, there are no electrostatic or bi nding interactions of the molecule with the pore surface. Since the molecule under study is neutra l, transport through the membrane is by purely diffusional and geometrical constraints. Yet we found in our studies that the diffusion of molecules across these membranes, exhibit a rectif ication behaviour. It means that there was a

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79 preferential direction of diffusion flow. This effect cannot be predicted by considering classical diffusion with constant diffusion coefficients. The rectificatio n of conical nanopores is of tremendous significance for industr ial filtration processes in wh ich asymmetric membranes are often applied. One type of asymmetric membranes used in industry consists of a thin skin of nanoporous material placed on a low resistance support, which assures high fluxes and good size separation. 122 Finding an optimal direction of concentr ation gradient will improve the filtration process. Asymmetric diffusion has been observed before with multi-membrane systems and membranes with two skins on two memb rane faces of a membrane support. 123 Diffusional Transport Described by Ficks Laws Diffusional transport is desc ribed by Ficks two diffusion laws. For diffusion in one dimension, Ficks first law of diffusion describes the flux (the net number of moles of particles crossing per unit time, t, through a unit area perpendicular to the x-axis and located at x ), stating that the flux of a molecule with diffusion coefficient, D is directly proportional to the concentration gradient. x t x c D t x J , (5-1) D normally is assumed constant, however there are known examples when D depends on position x concentration c or even time t 124, 125 The diffusion coefficient determines the time it takes a solute to diffuse a given distance in a medium. The diffusion coefficient depends on the physical characteristics of the solute as well as those of the medium. An approximate bulk diffusion coefficient (like that of molecules of similar masses) was used here, because, we are more concerned about emphasizing th e preferential flux from one side of the membrane than the absolute flux values, themselves. The second of Ficks law expresses how the concentration of

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80 the species depends on time. Here again, the gene ral form of the law in one dimension, taking into account the possible forms of D is as follows: x c D x t c (5-2) Hindered Diffusion in Cylindrical Pores The effective diffusion coefficient of a solute with in a pore of comparable size is lower than that of the bulk solution value. This phenomenon is ca lled hindered diffusion and results from steric exclusion of the solute at pore opening and hydrodynamic or wall drag resistance due to the presence of the pore wall. For steady-state di ffusion through a membrane with cylindrical pores that are comparable to the size of an uncharged solute, the effective diffusion coefficient can be expressed in terms of the solute-to-pore size ratio ) ( r a by the Renking equation: 126, 127 ) 948 0 089 2 1044 2 1 ( ) 1 ( /5 3 2 D D (5-3) Here, restriction to diffusion due to steric hindrance at the entr ance to the pores is given by the partition coefficient, =2) 1 ( (5-4) As established by Ferry,128 a molecule must pass through the opening without striking the edge. Therefore the center of the solu te particle cannot be locate d at a radius that exceeds ( r a ) (Figure 5-1). The partition coefficient ther efore, is equivalent to the frac tion of the cross-sectional area of the pore that is accessible to the center of the molecule. The second factor in the Renking equation, called the inverse enhanced drag,127 ) 948 0 089 2 1044 2 1 (5 3 1 (5-5) corrects for hydrodynamic or wall drag resistance, which is th e friction between a molecule moving within a pore and its walls (Figure 5-2).

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81 The total flux, J through a membrane with cylindrical pores of length, L and pore density, can then be given as: L C D r J 1 2 (5-6) where Dis the free diffusion coefficient of the solute in the bulk solution, C is the concentration difference across the membrane, and N r2 is the membrane porosity, Hindered Diffusion in Conical Pores Equation 5-6 is for a membrane with cylindrical pores where r is constant. The pores used for this study are conical, and therefore r increases or decreases from one end of the membrane to the other, thus varying linearly with position. The porosity of conical pores is different from that of cylindrical pores, and is obtained by th e product of the pore density and the geometric mean cross-sectional pore area: 4t bd d (5-7) where bd and td are the diameters of the large and small opening of the pores respectively. There are varying hindrance effects (steric and hydrodynamic) on diffusion over the length of the membrane. The average inverse enhanced drag, K-1, is ~ 1 for conical pores because most of the wall drag resistance occurs in the region of the tip, which is a very small faction of the pore. For cylindrical pores, the partition coefficient determin es the rate of entry of the molecules into the pores, and is equal to the ratio of the solute concentration in the pore to that of the bulk solution at equilibrium. However, unlike cylindrical por es, the partition coefficient that determines molecular entry into conical pores at steady stat e diffusion is not the same as the equilibrium partition coefficient. The partition coefficient, ( r ( x )) varies with r ; which is a function of position, x and therefore changes along the length of the pores. Hence it is expected that the

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82 equilibrium concentration along a pore length is different. However, the partition coefficient and also the enhanced inverse drag within the pore, av erages to be of neglig ible contribution to the flux since only a very small fraction of the pore approaches the size of the solute molecule. The value of these coefficients therefore approximate s to 1. The asymmetry in diffusion, then, may be caused by the difference in steric hindrance at the entrance of either side of the pore. This will determine the amount of molecules that ente r and leave the pore per unit time. The above arguments suggest that molecules entering from the large opening of a conical pore will have greater flux than those that enter from the sma ller opening, provided that they can leave at the same rate. The limit to diffusion will be the pa rtition coefficient at the tip. With these assumptions, the fluxes can be written for dilute solute concentrations as L C D Jb b 1 (5-8) L C D Jt t 1 (5-9) where the subscripts b and t represents the directions of th e net flux entering the large and small opening of the pores respectively, and K-1 is equal to 1. We can also solve the diffusion problem with a constant D through a conical nanopore with opening angle openings dt and db, with solute concentraton c0 at the tip, and zero concentration of solute at the base: 1 ) (0x L d d c x cb t, L x dt cot (5-10) valid for dt<
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83 x L d d c x cb t1 ) (0, L x dt cot (5-11) When we calculate the fluxes (moles/s) in the tw o above mentioned conditions they are equal to each other: L d d c D jb t0 (5-12) These calculations indicated that classical diffusion with constant D cannot describe our experiments, unless one takes into account th e difference in boundary conditions due to the difference in the partition coefficient. It was the purpose of this study to investig ate the diffusion of neut ral molecules from both ends of conical pores of the polyimide Kapton. Here the dimension of the tip of the conical pores approaches that of the molecules. To th is end the diffusion of th e water soluble neutral molecule phthalazine was studied. Experimental Materials Kapton 50 HN foils (12.5 m thick, 107 tracks cm-2) were obtained from the linear accelerator laboratory UNILAC at the GSI (D armstadt, Germany). Sodium hypochlorite (NaOCl, 13% active Cl) and potassium iodide (KI), were obtained from Aldrich and used as received. Phthalazine, sodium chloride, sodium phosphate dibasic, sodium phosphate monobasic, sodium azide was obtaine d from Fisher and used as received. Milli Q water was used to prepare all solutions and to rinse the membranes.

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84 Kapton Polyimide Membrane We used polyimide foils to prepare nanoporous membranes. Polyimide possesses a unique combination of properties that are ideal for a vari ety of applications in many different fields. The film maintains excellent physical, electrical, and mechanical prope rties over a wide temperature range. Polyimide also has very good chemical resistance and does not dissolve in organic solvents. We used 12.5 m thick commercially available Kapton 50 HN, produced by DuPont. Irradiation-Track Formation For the preparation of membranes we used th e-so called track etchi ng technique. It is based on irradiating a dielectr ic film with swift heavy i ons and subsequent chemical development (etching) of the damaged ion tracks. A unique feature of heavy ion irradiation is single-particle recording. That is to say, one swift heavy ion wh ich penetrates the foil produces one damaged track. Therefore, counting the number of ions used for irradiation enables one to prepare membranes with tailored number of pores from the range 1 up to 1010 ions/cm2. Kapton foils, which we used for these experiments, were irradiated with uranium ions of energy of 11.4 MeV/u, at the heavy ion accelerat or UNILAC at the Institute fo r Heavy Ions Research, (GSI) Darmstadt, Germany. We used foils irradiated with the fluencies 107 and 108 ions/cm2. The range of the ions is in all cases larger than th e thickness of the polyimide membranes. The ions penetrate the membranes at normal incidence, cr eating damaged tracks, which is then followed by chemical etching to form pores (Figure 5-3). Chemical Etching of Membrane Tracks After irradiation of the Kapton foil with heavy ions, the latent tracks have to be chemically etched. The shape and size of the pores can be tailored by the choice of etchant and the etching conditions. To obtain cylindrical pores the etching rate along the track, the-so called track etch

PAGE 85

85 rate Vt has to be much faster than the non-specific etching of the polymer called the bulk etched rate Vb The relation between Vt and Vb is explained in Figure 5-4. To obtain conical pores, one needs to choose etching c onditions which assure high vb. Preparation of conical pores is normally performed in a cond uctivity cell with etchant placed only on one side of the membrane. The other side of the membrane is in contact with a stopping medium, which neutralizes the etchant as soon as the pore is etched throug h. For example, if NaOH is used as an etchant, we use an aci dic stopping medium. The chemical stopping is further supported by an electric stopping. The etching is performed under voltage with electrodes arranged in such a fashi on that the anode is on the side of etchant, which retracts ions active in the etching process (e.g. OH-) out from the pore ( Figure5-5). Etching with an applied voltage and measuring electric curren t affords a method to monitor the process. At the beginning of etching the cu rrent is zero, because the two chambers of the conductivity cell are no t connected with one another. When the pore is etched through, the current value is finite and increases in time, i ndicating an increase of the pore diameter (Figure 5-6). Kapton is very resistant chemically, therefore, development of latent tracks has to be performed by a very aggressive etchant and at elevated temperatures. Previous studies have shown that an efficient etching can be perfor med in sodium hypochlorite containing 13% active chlorine content. Furthermore, it has been de monstrated that the shape of the pore can be regulated with an appropriate c hoice of pH. When sodium hypochl orite is not buffered its pH ~ 12.6. At these conditions, and elevated temperature of 50C, Vb of Kapton is high (~ 0.42 ( m / h) and the pores become strongly conical. Bufferi ng the etchant with boric acid to pH ~9 enables one to obtain cylindrical pores. It is important to note that the etching works only at basic pH when the hydrolysis of imide bonds by OHis possible.

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86 To obtain conical pores in Kapton, the ir radiated samples were placed between two chambers of a conductivity cell and etched from one side in sodium hypochlo rite. The other half of the cell is filled with 1 M potassium iodide (KI) solution as a stopping medium for the OClions of the etchant. As soon as the etchant co mpletely penetrates the membrane, iodide ions reduce OClto Clions: 112, 130 OCl+ 2H+ + 2II2 + Cl+ H2O 5-13 Via this reaction, the etching process stops immediately afte r the breakthrough, allowing the preparation of extremely narrow pores. Pore Diameter Measurement SEM was used to characterize the opening diam eters of conical pores, especially the big opening, which we call the base (Figure 5-7). The small opening of conical pores, called the tip is below resolution of SEM, therefore, we have to use another technique for its size estimation. We have chosen to use an elec trochemical technique ba sed on measuring ion current. The ionic conductance of a conical pore is related to its diameter by the following equation: 5-14 where n is the number of pores, is the conductivity of electrolyte, L is the length of the pore (or the equivalent membrane thickness), and db and dt are the diameters at the base and tip of the cone respectively. Transport Measurement Measurements of neutral molecular trans port through conical nanopor es were performed using UV-Vis spectrometry (Agi lent 8453). A U-cell set-up cont ained the permeate molecule solution on one side and a buffer, in our case PBS (in which the permeate was prepared) on the other side of the cell (Figure 5-8). The me mbrane was sandwiched between two transparent L d d n Gt b4

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87 tapes that have holes in the center that defined the transport area. This tape-membrane composite was clamped between the U-cell. Th e permeate solution was removed at certain time intervals and the permeate concentration was meas ured be UV-Vis spectrometry. Feed solution concentrations were varied in 4 ml PBS (pH 7.2) and the permeate side was only 4 ml PBS. The solution on both sides of the U-tube was stirre d using stir bars and stir-plate set-up. Viscosity Measurements The viscosities of phthalazine and dext rose were measured using Cannon-Fenske viscometer No. 75 (model P200; Cannon Instruments) Results and Discussion Membrane Characterization Figure 5-9 is an SEM image of the base side of a typical porous Kapton membrane used for the transport study. The average pore density taken from approximately five hundred pores in five different locations of th e image under low magnification, was 107/cm2. Figure 5-10 is an SEM image of the base s howing a diameter of 1.68 m. The size of the tip, which is below the resolution of SEM, was calculated from ionic co nductance measurements (Figure 5-11) using equation 5-14. The average tip size used in this study was ~ 2 nm. The average diameter of the molecule for transport was ~ 0.7 nm. Transport Measurements of Phthalazine From equation 5-4, the partiti on coefficients at the base a nd tip approximate to 1 and 0.42 respectively. These values put th e partition coefficient at the base about 2 times that at the tip. From the proposed equations, Figures 5-8 and 5-9 fo r base and tip fluxes, it is expected that at low concentrations the flux from base to tip should be about twice that in the opposite direction. Indeed, Figures 5-12 and 5-13 show that base fluxes are about twice that of tip fluxes for 1 and 3 mM solute concentrations. However, base fluxes approach those of tip fluxes as the

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88 concentration of the solute increases, and bot h base and tip fluxes are similar at higher concentrations (Figures 5-14 a nd 5-15). Figure 5-16 shows this asymmetric behavior over a wider rage of concentrations. The higher flux from base to tip direction can be explained by a lower ac cess resistance of the solute to the pore. Figure 5-17 shows a comp arison between the theoretical flux obtain from equation 5-9, and the experimental flux from tip to base. We see that these are in good agreement over the wide range of concentration used The experimental flux from base to tip, on the other hand, deviates from theoretical values obtained from equation 5-8, particularly at higher concentrations. To determ ine whether the base flux or tip flux behaves in a classical way, a plot showing increase of flux through base and tip, respectively, w ith respect to the flux measured at 0.1 mM was generated (Figure 5-18 ). The straight line: Flux(c)/Flux(0.1mM) = c/0.1 obtained for the tip flux indicates that tran sport through tip behaves according to the Fick's law with a constant D. Transport through base is hindered for higher co ncentrations indicating that diffusion coefficient is concentration dependent. The flux (mol/s) through an aperture of diameter dt with boundary conditions c0 is given by: .0 limDc d jt ited This value is smaller than the flux as given by eq. 5-12 The flux through the tip is limited by the value of.limitedj, while the flux through the base is not. The value of flux through a conical nanopore as gi ven by eq. 5-12 can be easily obt ained when the solute passes from base to tip. The open question however remains, why the ratio of fluxes is concentration dependent. In order to answer this question, we examined behavior of the flux from base and tip side, respectively for different con centrations of the solute.

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89 The Influence of Cosolute Concentration on Asymmetry We investigated whether molecular crowding / jamming had any influence in the behavior of the base flux at higher concentrations. To do th is, we used a constant concentration (5 mM) of phthalazine plus varying concentra tions (0 to 100 mM) of the coso lute dextrose on the feed side of the cell. We then placed identical total c oncentrations on the permeat e side of the transport cell using dextrose (Figure 5-19). This procedure was done to ensu re that there was no osmotic influence on transport. Figure 5-20 is a calib ration curve for phthalazine, and phthalazine with dextrose cosolute, generated from UV-visible absorption measurements. The two plots were placed together to show that the addition of dextrose as a cosolute to phthalazine does not affect the absorbance to any significant degree. Also to address the issue of viscosity, kinematic viscosity measurements were done on phthalazi ne and dextrose over the concentration range used in this study. Figure 5-21 in dicates that there is negligible change in viscosity over the range of concentrations (0 to 100 mM) used for this study. Figure 5-22 shows the influence of varying concentrations of the co solute dextrose on the flux of pht halazine, which is kept at a constant concentration of 5 mM. The tip flux is not affected over the entire range of concentrations. However, the base flux is sharply decreased to that of the tip flux when dextrose is added to the feed solution. Even though phtha lazine concentration wa s kept constant, base flux approached the tip flux in a very simila r way when phthalazine concentrations were increased. The increase in total concentration on the base side of the membrane causes base flux to approach the limit of tip flux. Proper mathem atical modeling of this effect is needed, taking into account dependence of diffusion coefficient on concentration of the solute and position in the channel. We would like to me ntion that theoretical studies were reported in which two sizes gas molecules were placed on the base side of co nical pores, and one size molecule was too large to pass through the tip. In this situation a total jamming was found, causing the diffusion of the

PAGE 90

90 small molecules to be zero over time. As exp ected, the rate of such jamming increased with concentration. On the other hand, when the mo lecules were placed on the tip side, no jamming occurred from that entrance. 131 We think that in our case, an increase in total concentration of the solutes might lead to similar partial molecular jamming of diffusion from base to tip. Decrease of the base flux provides ev idence that it is the exit rate of the solute from the pore that limits the diffusion transport in the direction from base to tip. Conclusions Diffusion rates through a membrane can be asymmetric due to molecular binding, electrostatic interaction, and difference in osmotic potential. In this study we have demonstrated that asymmetric diffusion can also occur by purel y geometric constrains of conical pores on the diffusing particles, where the tip of the cone is comparable to the dimension of the molecules. We show that asymmetric behavi or of diffusion is concentration dependent and there appear to be some sort of partial jamming effect related to the increase in concentration of molecules from the large opening of the pores. To further shed more light on this interesting phenomenon, proper mathematical modeling of this effect is needed, taking into account dependence of diffusion coefficient on concentration of the solute and position in the channel. Figure 5-1. The partitioning of a spherical molecule of radius, a, in cylindrical pores of radius, r.[Adapted from Davidson, M. G.; Deen, W. M. Macromolecules 1988, 21, 34743481.]

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91 = a/r Figure 5-2. Spherical molecule of radius, a, moving within a cylindrical pore of radius, r.[Adapted from Davidson, M. G.; Deen, W. M. Macromolecules 1988, 21, 34743481.] A B A B Figure 5-3. Swift heavy ions impinge on a dielec tric solid leading to damaged ion tracks witch can be chemically etched to form pores(A and B, respectively)

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92 Figure 5-4. Definiti on of bulk etch rate Vb and track etch rate Vt.[Adapted from Apel, P. Radiation Measurements 2001, 34, 559-566.] Figure 5-5. Conductivity cell used to prepare conical pores.

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93 Figure 5-6. Etching curve s howing moment of breakthrough with sharp increase in ion current.[Adapted from Siwy, Z.; Apel, P.; Dobrev, D.; Neumann, R.; Spohr, R.; Trautmann, C.; Voss, K. Nuclear Instruments & Met hods in Physics Research, Section B: Beam Interacti ons with Materials and Atoms 2003, 208, 143-148.]

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94 Figure 5-7. Kapton membran showing la rge opening (base) of conical pores Figure 5-8. Experimental setup for transport measurements

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95 Figure 5-9. Scanning electron micrograph of base side showing pore density (107 pores/cm2) of Kapton membrane. Figure 5-10. Scanning electron micrograph of the base showing diameter of 1.68 m

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96 I = 0.0077E R2 = 0.9918-0.00020 -0.00015 -0.00010 -0.00005 0.00000 0.00005 0.00010 0.00015 0.00020 -0.02-0.015-0.01-0.00500.0050.010.0150.02E (V)I (A) Figure 5-11. A typical current-vol tage curve used to measure th e tip diameter of the conical nanopores. 0 10 20 30 40 50 60 70 80 90 100 01002003004005006007008009001000Time (min)Nanomoles transported/cm2 Base Tip Figure 5-12. Base and tip fluxes fo r 1 mM phthalazine feed solution.

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97 Figure 5-13. Base and tip fluxes fo r 3 mM phthalazine feed solution. 0 20 40 60 80 100 120 050100150200250300350400450Time (min)Nanomoles transported/cm2 Base to tip Tip to base Figure 5-14. Base and tip fluxes fo r 10 mM phthalazine feed solution. 0 5 10 15 20 25 30 35 40 45 50 0100200300400500Time (min)Nanomoles transported/cm2 Base Tip

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98 0 50 100 150 200 250 050100150200250 Time (min)Nanomoles transported/cm2 Tip Base Figure 5-15. Base and tip fluxes fo r 50 mM phthalazine feed solution. 0 0.5 1 1.5 2 2.5 051015202530Concentration (mM)Base Flux/Tip Flux Figure 5-16. Asymmetric behavior of flux versus concentration.

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99 0 0.1 0.2 0.3 0.4 0.5 0.6 051015202530Concentration (mM)Flux (nmoles/cm2 min)Theoretical Experimental Figure 5-17. Theoretical and experi mental tip flux vs concentration 0 50 100 150 200 250 300 350 0100200300 c [mM]/0.1 [mM]Flux (c)/Flux (0.1 mM) from tip from base Figure 5-18. Increase of flux through base a nd tip, respectively, with respect to the flux measured at 0.1 mM.

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100 Stir bar Conical nanopore membrane Phthalazine+ Dextrose+ PBS (pH 7.2) PBS (pH 7.2) + Dextrose U-cell Stir bar Conical nanopore membrane Phthalazine+ Dextrose+ PBS (pH 7.2) PBS (pH 7.2) + Dextrose U-cell Figure 5-19. Experimental set-up for transport measurements of phthalazine with cosolute dextrose y = 0.003021x R2 = 0.999811 y = 0.003058x R2 = 0.998450 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 05101520253035CONCENTRATION (micrmolar)ABSORBANCE (a.u.) Phthalazine Phthalazine with 490 mM Dextrose y = 0.003021x R2 = 0.998406 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 00.20.40.60.811.2 CONCENTRATION (micromolar)ABSORBANCE (a.u. y = 0.003021x R2 = 0.999811 y = 0.003058x R2 = 0.998450 0 0.01 0.02 0.03 0.04 0.05 0.06 0.07 0.08 0.09 0.1 05101520253035CONCENTRATION (micrmolar)ABSORBANCE (a.u.) Phthalazine Phthalazine with 490 mM Dextrose y = 0.003021x R2 = 0.998406 0 0.0005 0.001 0.0015 0.002 0.0025 0.003 0.0035 00.20.40.60.811.2 CONCENTRATION (micromolar)ABSORBANCE (a.u. Figure 5-20. Calibration curve of phthalazine, and phthalazine with the highest concentration of dextrose cosolute used

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101 0 0.2 0.4 0.6 0.8 1 1.2 1.4 050100150200250300350Concentration (mM)Viscosity ( mm2/ sec) Dextrose Phthalazine Figure 5-21. Viscosity of dextrose and phtha lazine over the concentr ation range used 0 0.02 0.04 0.06 0.08 0.1 0.12 0.14 0.16 020406080100Dextrose Concentration ( mM)Flux (nmoles/(cm2 min) Base Flux Tip Flux Figure 5-22. Influence of dextrose cosolute conc entrations on the base and tip diffusion from 5 mM phthalazine

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102 CHAPTER 6 CONCLUSION The goals of this research were to deve lop conical nanopores and nanotube (single and multipore) in polymers and mica membranes by th e track-etch method and extension of this technology for sensing and other app lications, and to inve stigate the tr ansport properties of these conical pores. Chapter 1 reviewed background info rmation for this dissert ation including the ion track-etch method, membrane based template synt hesis, electroless metal deposition, chemical vapor deposition, resistive-pulse sensing, plasma based etch ing and asymmetric diffusion. In Chapter 2, an extension of the track-etch method to make conical pores in a reproducible fashion was demonstrated. We have shown he re that we can not only reproducibly prepare track-etched based conical nanopor e sensor elements, but that we can predict from the experimental parameters used during the second etch, what the diameter of the all-important nanopore tip will be. For these reasons, we belie ve that the track-etch method will prove to be the technology of choice for taki ng artificial-nanopore resistive-pul se sensors from the bench top to the practical prototype-device stage of the R&D effort. A method to make asymmetric pores in tracked muscovite mica films using an etch-refilletch approach was the topic of Chapter 3. By controlling the concentra tion ratio of hydrofluoric acid to nitric acid, tapered pores with diamond shap ed cross-section were obtained. Here, we see that a two dimensional control of the etch process was developed. Replicas of the asymmetric pores were accomplished by carbon vapor depos ition, and scanning electron microscopy was used to give evidence of the resulting nanotubes. In this study, excellent control over cone angle was demonstrated. This control over cone angle has great potential to make the sensing zone of the conical pores limited to a very short distance from the tip. This is ideal for single molecule detection.

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103 In Chapter 4, electroless gol d plating properties on the su rface and pore walls of tracketched Kapton polyimide nanoporous membranes we re studied. SEM, AFM, and ion current measurements were used to characterize the su rfaces and dimensions of the pores in the membrane. AFM images showed that after electr oless gold plating, the gold surface layers are smooth compared to a similarly structured polycarbonate membra ne. It is important that membranes remain relatively smooth after gold plat ing in order to have a well define pore area for transport and sensing. These properties sugg est that Kapton might be a better candidate than PC for use in transport studi es and molecular sensing. Finally, asymmetric diffusiona l transport of neutral mol ecular species through Kapton conical pores was described in Chapter 5. Membranes containing conical nanopores from polyimide (Kapton HN Dupont) foils were prepared by the track-etching tec hnique. We report the preferential direction of th e flow of molecules through conical nanopores from the base side at low concentration. However, there is con centration dependence from the base side of the pores which causes the flux to appr oach that of the tip as conc entration increases. Study of transport with a cosolute suggested that there may also be some sort of partial jamming effect with concentration from the base entry. It is hoped that further mathematical modeling of this system would shed some light on th is interesting transport phenomenon

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104 LIST OF REFERENCES (1) Sotiropoulou, S.; Vamvakaki, V.; Chaniotakis, N. A. Biosensors & Bioelectronics 2007, 22, 1566. (2) Heo, K.; Yoon, J.; Jin, K. S.; Jin, S.; Ree, M. IEE Proceedings: Nanobiotechnology 2006, 153, 121-128. (3) Hinds, B. J.; Chopra, N.; Rantell, T.; Andrews, R.; Gavalas, V.; Bachas, L. G. Science (Washington, DC, United States) 2004, 303, 62-65. (4) Lev, O.; Tsionsky, M.; Rabinovich, L.; Glezer V.; Sampath, S.; Pankratov, I.; Gun, J. Analytical Chemistry 1995, 67, 22A-30A. (5) Ree, M.; Yoon, J.; Heo, K. Journal of Materials Chemistry 2006, 16, 685-697. (6) Apel, P. Nuclear Instruments & Methods in Ph ysics Research, Section B: Beam Interactions with Materials and Atoms 2003, 208, 11-20. (7) Choi, Y.; Baker, L. A.; H illebrenner, H.; Martin, C. R. Physical Chemistry Chemical Physics 2006, 8, 4976-4988. (8) Van der Bruggen, B.; Everaert, K.; Wilms, D.; Vandecasteele, C. Journal of Membrane Science 2001, 193, 239-248. (9) Gilron, J.; Gara, N.; Kedem, O. Journal of Membrane Science 2001, 185, 223-236. (10) Jirage, K. B.; Hulteen, J. C.; Martin, C. R. Analytical Chemistry 1999, 71, 4913-4918. (11) Desai, T. A.; West, T.; Cohen, M.; Boiarski, T.; Rampersaud, A. Advanced Drug Delivery Reviews 2004, 56, 1661-1673. (12) Cooper, G. M. In The CellA Molecular Approach, 2 ed.; Sinauer Associates, i., Ed., 2000, pp 81-84; 476-491. (13) Fischer, B. E.; Spohr, R. Interdisciplinary Science Reviews 1984, 9, 329-335. (14) Apel, P. Radiation Measurements 2001, 34, 559-566. (15) Virk, H. S.; Kaur, S. A.; Randhawa, G. S. Indian Journal of Environmental Protection 2001, 21, 529-533. (16) Fleischer, R. L.; Price, P. B.; Walker, R. M. Nuclear Tracks in Solids: Principles and Applications, 1975.

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106 (37) Che, G.; Lakshmi, B. B.; Martin, C. R.; Fisher, E. R. Langmuir 1999, 15, 750-758. (38) Che, G.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R. Nature (London) 1998, 393, 346349. (39) Kyotani, T.; Tsai, L.-f.; Tomita, A. Chemical Communications (Cambridge) 1997, 701702. (40) Lakshmi, B. B.; Dorhout, P. K.; Martin, C. R. Chemistry of Materials 1997, 9, 857-862. (41) Lakshmi, B. B.; Patrissi, C. J.; Martin, C. R. Chemistry of Materials 1997, 9, 2544-2550. (42) Cepak, V. M.; Hulteen, J. C.; Che, G.; Jirage, K. B.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R. Journal of Materials Research 1998, 13, 3070-3080. (43) Cepak, V. M.; Hulteen, J. C.; Che, G.; Jirage, K. B.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R.; Yoneyama, H. Chemistry of Materials 1997, 9, 1065-1067. (44) Martin, B. R.; Dermody, D. J.; Reiss, B. D.; Fang, M.; Lyon, L. A.; Natan, M. J.; Mallouk, T. E. Advanced Materials (Weinheim, Germany) 1999, 11, 1021-1025. (45) Bowes, C. L.; Malek, A.; Ozin, G. A. Chemical Vapor Deposition 1996, 2, 97-103. (46) Shelimov, K. B.; Moskovits, M. Chemistry of Materials 2000, 12, 250-254. (47) Foss, C. A., Jr.; Hornyak, G. L.; Stockert, J. A.; Martin, C. R. Journal of Physical Chemistry 1994, 98, 2963-2971. (48) Blondel, A.; Meier, J. P.; Doudin, B.; Ansermet, J. P. Applied Physics Letters 1994, 65, 3019-3021. (49) Schwarzacher, W. Electrochemical Society Interface 1999, 8, 20-24. (50) Ang, L.-M.; Hor, T. S. A.; Xu, G.-Q.; Tung, C.-h.; Zhao, S.; Wang, J. L. S. Chemistry of Materials 1999, 11, 2115-2118. (51) Menon, V. P.; Martin, C. R. Analytical Chemistry 1995, 67, 1920-1928. (52) Parthasarathy, R. V.; Phani, K. L. N.; Martin, C. R. Advanced Materials (Weinheim, Germany) 1995, 7, 896-897. (53) Cepak, V. M.; Martin, C. R. Chemistry of Materials 1999, 11, 1363-1367. (54) Patrissi, C. J.; Martin, C. R. Journal of the Electrochemical Society 1999, 146, 31763180. (55) Lee, S. B.; Martin, C. R. Anal Chem FIELD Full Journal Title:Analytical chemistry 2001, 73, 768-775. (56) Miller, S. A.; Martin, C. R. Journal of Electroanalytical Chemistry 2002, 522, 66-69.

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PAGE 111

111 BIOGRAPHICAL SKETCH John Edwardson Wharton was born in St. Kitts, West I ndies. He spent two years at the St. Kitts-Nevis Teachers College, earning a diploma in education in 1985. After graduation, he taught for two and a half years, before taki ng up employment at the St. Kitts Biomedical Research Foundation, where he worked for a few y ears. He later went on to pursue a B.S. in chemistry with physics at the University of th e Virgin Island from 1992 to 1996. In August, 1999, John began his graduate work in analyti cal chemistry under the guidance of Prof. Dr. Charles R. Martin. He earned his M.S. in 2003 and continued in the Martin group in pursuit of his Ph.D. He completed his research in the sp ring of 2007, obtaining a Doctor of Philosophy in analytical chemistry in the area of nanostructu red asymmetric pore design and fabrication for biosensor applications.


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Title: Fabrication of Asymmetric Pores for Biosensors and Transport Studies
Physical Description: Mixed Material
Copyright Date: 2008

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FABRICATION OF ASYMMETRIC PORES FOR BIOSENSORS AND TRANSPORT
STUDIES





















By

JOHN EDWARD SON WHARTON


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




































O 2007 John Edwardson Wharton


































To my family, for their continued support and love









ACKNOWLEDGMENTS

I wish to acknowledge the many people who have given me advice, help, and

encouragement during my years in graduate school.

I would like to thank Prof. Dr. Charles R. Martin and the entire Martin group for the

opportunity to work with them over the years. Prof. Martin was continuously supportive and

always willing and ready to discuss some of our new ideas for research. I appreciate the level of

independence that Prof. Martin allowed all of his students. Undoubtedly, this created an

atmosphere of confidence among the students where creativity flourished.

I would like to thank Drs. Punit Kohli, Zuzanna Siwy, and Lane Baker for their patience

and expert advice along the way. I wish to thank Fan Xu, Lindsay Sexton, Pu Jin, Lloyd Horne,

Stefanie Sherrill, and Warren Mino for very important contributions to some of my proj ects;

these great students have been very patient with me and are always willing to help with very high

interest and good spirits.

Finally, I would like to thank my family and friends for showing confidence in me and for

giving their love and support throughout the years.












TABLE OF CONTENTS


page

ACKNOWLEDGMENTS .............. ...............4.....


LIST OF FIGURES .............. ...............8.....


AB S TRAC T ............._. .......... ..............._ 1 1..


CHAPTER


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


Ion Track-Etch Method............... ...............14.
Track Form ation ................. ... ........... ...............14.......
Acceleration and Irradiation Facilities .............. ...............15....
Chemical Etching of lon Tracks ................. ...............15........... ...
The Effect of Storage of Tracked Material............... ...............16
Effect of Etch Promoters ................. ...............16.......... ....
Etch Properties of Selected Polymers ................. ...............17........... ...
Effect of Thermal Annealing .................. ...............17................
The Effect of Temperature during Etching .............. .....................17
Effect of Detergents ................... ...............18................
Etch Properties in Muscovite Mica ................. ......... ...............18.....
Template Synthesis................ ..............1
Template Synthesis Strategies ................. ...............19........... ....
Electroless Deposition ................... .......... ...............19.......
Chemical Vapor Deposition (CVD) .............. ...............21....
Resistive-Pulse Sensing ................. ...............21.......... ......
Plasma-Based Etching .............. ...............23....
Asymmetric Diffusion .............. ...............24....
Dissertation Overview .............. ...............25....


2 A METHOD FOR REPRODUCIBLY PREPARING SYNTHETIC NANOPORES
FOR RESISTIVE-PULSE BIOSENSORS ................. ...............36........... ....


Introducti on ................. ...............36.................

Experimental ................. ...............37.................
First Etch Step .............. ..... ... .. .......3
Determination of the Diameter of the Base Opening ................. .......... ...............3 8
Electrochemical Measurement of the Tip Diameter ................. .......... ................3 8
Second Etch Step ................... ..... ....... ..... ........ ...........3
Bovine Serum Albumin (BSA) Resistive-Pulse Sensing .............. .....................3
Results and Discussion .............. ........... .... .. .. .. ............3
Conical Shaped Nanopores are Ideal Resistive-Pulse Sensor Elements .........................39
The Core Technology: The Track-Etch Method .............. ...............40....
The First Etch Step .............. ...............40....












Measuring the Diameter of the Tip Opening after the First Etch ................. ................41
The Second Etch Step.................. .... .... .. .... ..... .......4
Measuring the Diameter of the Tip Opening after the Second Etch .............. .... ...........43
Reproducibly Varying the Tip Diameter ................. ...............44........... ...
The Mathematical Model .............. ...............45....
Electro chemical Detail s............... ...............47
Conclusions............... ..............4


3 ETCH-FILL-ETCH METHOD FOR PREPARING TAPERED PORES IN ION
TRACKED MICA FILMS .............. ...............55....


Introducti on ................. ...............55.................

Experimental ................. ...............57.................
M materials ................. .. .... ..... ... .. .......... .. ... ..........5
Initial Etching of Mica Tracks to Prepare Very Small Pores ................ ............... ....58
Preparation of Tin Sensitizing Solution .............. ...............58....
Preparation of Silver Plating Solution ................. ...............58........... ...
Filling the Pores with Silver Wires .................. ...............59..
Etching Silver Filled Mica to form Tapered Pores ................. ................ ......... .59
Making Replicas of the Tapered Pores ................... .......... ............... 60 ...
Preparation of the Carbon Tube Replicas for SEM Imaging ................. ............... ....60
Results and Discussion .............. ...............61....
Conclusion ................ ...............63.................


4 ELECTROLESS AU PLATING OF TRACK-ETCHED KAPTON POLYIMIDE
NANOPOROU S MEMBRANE S ................. ...............69.......... ......


Introducti on ................. ...............69.................

Experim ental ................. ...............70.......... ......
M material s ................. ...............70.......... .....
Chemical Etching .............. ...............70....
Electroless Plating of Kapton ................. ...............71.......... ....
Pore Diameter Measurement ................. ...............71................
Results and Discussion .............. .... ...............72
SEM and lon Current Measurements .............. ...............72....
Atomic Force Microscope Images............... ...............72.
Conclusion ................ ...............72.................


5 ASYMMETRY IN DIFFUSIONAL TRANSPORT OF MOLECULES THROUGH
KAPTON CONICAL NANOPORES .............. ...............78....


Introducti on .............. .. .... .... .. ....... ..._ ... ...........7
Diffusional Transport Described by Fick's Laws ................. .............................79
Hindered Diffusion in Cylindrical Pores ................. ...............80........... ...
Hindered Diffusion in Conical Pores .............. ...............81....

Experim ental ................. ...............83.......... ......
M material s ................. ...............83.......... .....













Kapton Polyimide Membrane............... ...............84
Irradiation-Track Formation ............_...... .__ ...............84...

Chemical Etching of Membrane Tracks .....__.....___ ..........._ ...........8
Pore Diameter Measurement ............. ...... ...............86...

Transport Measurement ............_...... ...............86....
Viscosity Measurements ............_...... ...............87....
Results and Discussion .............. ...............87....
Membrane Characterization .............. ...............87....

Transport Measurements of Phthalazine .............. ......___.....__ ............8
The Influence of Cosolute Concentration on Asymmetry ....._____ ..... .. ....__..........89
Conclusions............... ..............9


6 CONCLUSION................ ..............10


LIST OF REFERENCES ............_ ..... ..__ ...............104..


BIOGRAPHICAL SKETCH ............_...... ._ ...............111...










LIST OF FIGURES


Figure page

1-1 Swift heavy ions impinge on a dielectric material creating damaged ion tracks. ..............27

1-2 After irradiation, the materials are subj ect to chemical etching which preferentially
removes the latent ion track. .............. ...............27....

1-3 Etched pore geometry in a homogeneous isotropic medium to a first approximation,
showing track etch rate, VT, and bulk at rate, Va ...._.................... ................ ..2

1-4 Scanning electron micrographs of Au nanocones replicas of PET conical pores. ...........28

1-5 Scanning electron micrographs of etched particle tracks in single-crystal mica.............. .29

1-6 Scanning electron micrographs of a porous polycarbonate, alumina and mica
membranes used for template synthesi s (A-C, respectively) ................. ........_._. .......30O

1-7 Schematic diagram of Au electroless plating procedure............... ...............3

1-8 Schematic illustration of Au nanotubes obtained from electroless gold deposition ..........32

1-9 Schematic illustration of resistive-pulse sensing ................. ...............33...............

1-10 Essential features of the staphylococcal a -hemolysin pore shown in a cross- section
based on the crystal structure. .............. ...............34....

1-11 Schematic of a conical nanopore sensor element showing the base diameter and
range of tip diameters used in these studies (drawing to scale) .........._.. ..........._......3 5

2-1 Schematic of a conical nanopore sensor element and etching cell. ........... ..................48

2-2 A typical current-voltage curve used to measure the tip diameter of the conical
nanopore. .........._._.. ...._ ... ...............49.....

2-3 Current-time transients obtained during the second etch step for three membranes
that were subj ected to the same first etch. .......... ...............50......

2-4 Scanning electron micrograph of the base openings of two conical nanopores in a
multi-track PET membrane ................. ...............51........... ....

2-5 Scanning electron micrograph of conical gold nanotubes deposited in a conical
nanopore membrane ................. ...............52.................

2-6 Plot of tip diameter measured after the second etch step vs. the final nanopore ion
current (If) at which the second etch was stopped. .......... ...............53......











2-7 Current-pulse data obtained for a prototype protein analyte, bovine serum albumin
(BSA) using PEG-modified conical nanotube sensors. ........... ....... ............... 5

3-1 After irradiation, the materials are subj ect to chemical etching which preferentially
removes the damaged ion track. ........... ......__ ...............64...

3-2 Schematic of cell used to do the etching and to make all electrochemical
measurements ................. ...............65.................

3-3 Schematic diagram of etch-fill-etch method ................. ...............66...............

3-4 Scanning electron micrographs of mica membrane that was exposed on one face to
20% HF andl0% nitric acid solution at 250C for 3 hrs. ........... ......................6

3-5 Scanning electron micrographs of mica membrane that was exposed on one face to
40% HF andl0% nitric acid solution at 250C for 3 hrs. .......... .......................6

3-6 Scanning electron micrograph of carbon tapered nanotube replica of the mica tapered
pore. ............. ...............68.....

3-7 Low magnification SEM images of carbon tapered nanotube replicas of the mica
tapered pore............... ...............68..

4-1 Definition of bulk etch rate Vb and track etch rate Vt ................. ................ ........ .73

4-2 Schematic of cell used for electrochemical measurements............... ..............7

4-3 Pore diameters for different Au plating times............... ...............75.

4-4 Pore diameter as a function of plating time with measurements taken from SEM
image (0-7.5 hrs) and ion current resistance measurements (8-12 hrs). ............................76

4-5 Atomic Force micrographs of Kapton and PC membranes before and after electroless
Au plating............... ...............77

5-1 The partitioning of a spherical molecule of radius, a, in cylindrical pores of radius, r.....90

5-2 Spherical molecule of radius, a, moving within a cylindrical pore of radius, r. ................91

5-3 Swift heavy ions impinge on a dielectric solid leading to damaged ion tracks witch
can be chemically etched to form pores............... ...............91.

5-4 Definition of bulk etch rate Vb and track etch rate Vt ........._..... ...._... ........_.......92

5-5 Conductivity cell used to prepare conical pores. ............. ...............92.....

5-6 Etching curve showing moment of breakthrough with sharp increase in ion current. ......93

5-7 Kapton membran showing large opening (base) of conical pores ................. ................. 94











5-8 Experimental set-up for transport measurements .............. ...............94....

5-9 Scanning electron micrograph of base side showing pore density (10' pores/cm2) Of
Kapton membrane. ........._.._.. ...._... ...............95....

5-10 Scanning electron micrograph of the base showing diameter of 1.68 pm ................... ......95

5-11 A typical current-voltage curve used to measure the tip diameter of the conical
nanopores. ............. ...............96.....

5-12 Base and tip fluxes for 1 mM phthalazine feed solution. ............. .....................9

5-13 Base and tip fluxes for 3 mM phthalazine feed solution. ............. .....................9

5-14 Base and tip fluxes for 10 mM phthalazine feed solution. ............. .....................9

5-15 Base and tip fluxes for 50 mM phthalazine feed solution. ............. .....................9

5-16 Asymmetric behavior of flux versus concentration ......._._............_ ........._..__....98

5-17 Theoretical and experimental tip flux vs concentration. ........._..... ....._... ............99

5-18 Increase of flux through base and tip, respectively, with respect to the flux measured
at 0.1 m M .............. ...............99....

5-19 Experimental set-up for transport measurements of phthalazine with cosolute
dextrose. .............. ...............100....

5-20 Calibration curve of phthalazine, and phthalazine with the highest concentration of
dextrose cosolute used. ............. ...............100....

5-21 Viscosity of dextrose and phthalazine over the concentration range used ...................... 101

5-22 Influence of dextrose cosolute concentrations on the base and tip diffusion from 5
mM phthalazine .............. ...............101....









Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

FABRICATION OF ASYMMETRIC PORES FOR BIOSENSORS AND TRANSPORT
STUDIES

By

John Edwardson Wharton

May 2007

Chair: Charles R. Martin
Major: Chemistry

The goals of this research are to develop asymmetric nanopores and nanotubes (single and

multipore) in polymers and mica membranes by the track-etch method, and an extension of this

technology, for sensing, nanostructure fabrication, and to investigate the transport properties of

conical pores. The first part of this work describes an extension of the track-etch method to

make conical pores in a reproducible fashion. We have demonstrated here that we can, not only

reproducibly prepare track-etched based conical nanopore sensor elements, but that we can

predict from the experimental parameters used during the second etch, what the diameter of the

all-important nanopore tip will be. For these reasons, we believe that the track-etch method will

prove to be the technology of choice for taking artificial-nanopore resistive-pulse sensors from

the bench top to the practical prototype-device stage of the R&D effort.

The second part of this work describes a method to make asymmetric pores in tracked

muscovite mica films using an etch-refill-etch approach. Tracks in the films were initially

etched away with hydrofluoric acid to form nanoporous membranes. These nanopores were then

refilled with silver nanowires or "metal tracks" using an electroless plating method. One face of

the membrane was then exposed to a solution of hydrofluoric acid and nitric acid, which etched

the bulk material and the nanowires respectively, at two different rates. By controlling the









concentration ratio of hydrofluoric acid to nitric acid, tapered pores with diamond shaped cross-

section were obtained. Replicas of the asymmetric pores were accomplished by carbon vapor

deposition, and scanning electron microscopy was used to give evidence of the resulting

nanotubes. In this study, excellent control over tip size and cone angle was demonstrated.

In the third section, electroless gold plating properties on the surface and pore walls of

track-etched Kapton polyimide nanoporous membranes were studied. Scanning electron

microscopy (SEM), atomic force microscopy (AFM), and ion current measurements were used to

characterize the surfaces and dimensions of the pores in the membrane. Nanoporous Kapton

polyimide membranes were electroless gold plated over different times and the pore diameter

characterized using SEM. Ion current measurements were used to measure the diameter of very

small pores. AFM images show that after electroless gold plating, the gold surface layers are

smooth compared to a similarly structured polycarbonate membrane. Electroless plating the

membranes for 12 hours produced Au wires in the pores. Removing the membrane by oxygen

plasma revealed that the plating in the walls is also relatively smooth.

The Einal part of this work describes asymmetric diffusional transport of neutral molecular

species through nanoporous polymer membranes. Membranes containing conical nanopores

from polyimide (Kapton HN Dupont) foils were prepared by the track-etching technique, based

on irradiation of the polymer with swift heavy ions and subsequent etching of the latent tracks.

The transport properties (e.g., flux) of these membranes were investigated using UV-Vis

spectroscopy. Transport experiments were performed with bare polymer membranes without

any modification. We report the preferential direction of the flow of molecules through conical

nanopores and spatial or concentration dependence of diffusion coefficient for a particular flow

direction.









CHAPTER 1
INTRODUCTION AND BACKGROUND

Nanopore fabrication technology has produced nanoporous materials that are potential

candidates for applications in various fiels, such as bionanotechnology, 1, 2 gas separation,3

catalysts4 and micro-electronics2, 5 In particular, the track-etch method has become an

indispensable technology for the production of nano- and microstructured materials.6 This

technology can be applied directly to most polymers and via the replication technique- to a

wide variety of materials, including metals.6 Track-etch membranes were first marketed three

decades ago and remain the best product for a number of biological, medical, analytical and

scientific applications.6

Recently, there has been a surge of interest in developing abiotic analogues of biological

nanopores as sensing elements for chemicals and biological sensors.' We have explored the

fabrication of such synthetic nanopores using the track-etch method. Particular interest is in the

fabrication of synthetic conical pores for resistive-pulse sensing. Resistive-pulse sensing using

conical nanopores is in its infancy. Studies in this area could prove beneficial for future

applications of conical pores for science and technology.

Membranes and porous materials have found various applications in filtration and

separation processes.8 9, 10 Modern biotechnology has posed new challenges in the application of

such membranes, and requires pores with diameters similar to those of molecules under study,ll

(e.g., as small as several nanometers). The nanometer scale of such pores is necessary in both

achieving optimal control of the flow of biomolecules, as well as in developing sensors for their

detection. The transport properties of such nanometer scale pores are not well understood yet.

The hint that nanopores behave differently from micropores, comes from Mother Nature.12









Biological channels and pores have diameter of ~ 1 nm and are critical for functioning of living

organism s.

This chapter is divided into six sections which provide the background information for this

research. Section 1 reviews the track-etch method, first discovered by Price and Walker, for

preparation of pores in dielectric materials. The track-etch method is used in the preparation of

conical pores described in Chapters 2, 3, 4 and 5. Section 2 reviews membrane-based template

synthesis. This method, pioneered by the Martin group, is used in preparation of nanostructured

materials described in Chapters 2 and 3. Section 3 review resistive-pulse sensing; which is the

focus of the application of Chapter 2 and 3. Section 4 describes plasma based etching; a

procedure necessary for the liberation of carbon tube in mica templates in Chapter 2. In Section

5, an introduction of Asymmetric Diffusion is given. Asymmetric diffusion is the topic of

Chapter 5. Finally, Section 6 is the dissertation overview.

Ion Track-Etch Method

Track Formation

When dielectric materials, such as polymers, ceramics and minerals, are bombarded by

swift ions, latent tracks are formed along the path of the ions (Figure 1-1).13, 14 I0n track

materials can be divided into two categories: (a) single-tracked and (b) multiple-tracked

materials. Single-tracked materials can be produced by controlling the beam optics and fluence

of the heavy ion beam." Commonly tracked materials are Makrofol-KG, Kapton-H, PVDF,

mica films, cellulose nitrate, CR-39 and Lexan polycarbonate. Swift ion beams are produced by

cyclotrons or linear accelerator and the radiation is characterized by extremely high linear energy

transfer.6 The conventional ionization radiation sources, such as radioactive isotopes or electron

accelerators,16 are less sophisticated and less expensive and are mostly used in the industrial









processes.6 Present-day heavy ion accelerators provide beams with ion energies in the order of

10MeV/u and even 100MeV/u which expands the treatment dept up to millimeters.6

Acceleration and Irradiation Facilities

About a half dozen heavy ion accelerators employed for irradiation of materials on the

industrial scale exist. The Tanden Van de Graaff accelerators at Brookheaven National

Laboratory are used to bombard materials with ions for manufacturing and testing purposes.6 At

the Grand Accelerator National d'lons Lours, France, ions are produced in an electron cyclotron

resonance(ECR) source.6 The cyclotron of Louvain la Neuve is a multiparticle variable energy,

cyclotron capable of accelerating protons, alpha particles and heavy ions. The RFQ + cyclotron

combination at the Hahn-Meitner Institute in Berlin, delivers intense beams of ion species such

as Kr or Xe with energies from approximately 1.5-6 MeV/ul7 At the Flerov Laboratory of

Nuclear Reactions(Dubna) a beam line connected at the U-400 cyclotron is equipped with

scanning systems which allows one to obtain a homogeneous distribution of ion tracks on the

target up to 60 cm in width and 6 cm in height.6 Additionally, a liner accelerator at GSI

(Darmstadt),6 the AVF cyclotron at TRCRE JAERI (Takasaki)6 and some others are used in

experiments on polymer modification.6

Chemical Etching of lon Tracks

After irradiation, the materials are subj ect to chemical etching which preferentially

removes the latent ion track (Figure 1-2)16 This etching process results in pore formation in the

material.16 Etching is the pore-size-determining and pore-shape-determining stage of the

technology. In a homogeneous isotropic medium, mainly two influential parameter describe the

etch process-the bulk etch rate VI3 and the track etch rate yr (Figure 1-3).6 The ratio of track etch

rate to the bulk etch rate is called the track-etch-ratio. When Vr is >> VI3, pores turn out to be

cylindrical as opposed to conical. In other words, high track-etch-ratio yields cylindrical pores,









where as low track-etch-ratio yields conical pores. The arctangent of the inverse track-etch-ratio

(VI3/VT) yields the half cone angle of the pore. The bulk etch rate depends on the material, on the

etchant composition and on the temperature.14 The track etch rate depends on the sensitivity of

the material, irradiation conditions, post-irradiation conditions and etching conditions.6~1

The Effect of Storage of Tracked Material

The most important storage factors are the atmosphere in which the material is stored, the

temperature and the illumination conditions during storage. In the presence of oxygen the latent

ion tracks becomes susceptible to track etching. This is due to the oxidation of the radicals

formed during irradiation. When polymers are stored at temperatures close their glass transition

temperature, rearrangement on a molecular scale can take place which may lead to annealing of

the ion tracks. Storing under illumination may lead to photo oxidation and is able to increase the

track etch ratio by orders of magnitude. It is reported that tracks in poly(ethylene terephthalate)

(PET) may be sensitized by uv radiation of 310 to 400 nm. Soaking in weak solvents, such as

dimethyl formamide or water-soluble gass can sensitize ion tracks in PET.6

Effect of Etch Promoters

Etch promoters are organic solvents that accelerate the etch process when added to the etch

bath. It has been observed that track etch ratios in polycarbonate (PC) can be above ten

thousand. On the other hand, track etch ratio can be dramatically decreased down to 2 to 4 in

PET by the addition of solvents such as methanol, ethanol or propanol, leading to wide cone

angles. These organic solvents help to dissolve large fragments ready to move into the liquid

phase by disengaging them from their neighbors.6 Figure 1-4 shows an example of etch

promotion using ethanol in PET.l









Etch Properties of Selected Polymers

In PET (OOC-C6H4-COO-CH2CH2), the main points of etch attack are the partially

charged -COO- ester groups, which are hydrolyzed by alkalis. During alkaline etching the

ordinary bond between carbon and oxygen is broken which produces -COO- and HO- at the ends

of the formed fragments.

For polycarbonate (OOC-O-C6H4-C(CH3)2- 6H4), the main point of etch attack is the

carbonate group -O-COO-. During the alkaline etching, chemical bonds are ruptured on both

sides of the carbonate group, leading to the formation of carbonate ions, CO32-. The other

product is diphenylol HO-C6H4-C(CH3)2_ 6H4-OH.

In Polyimide (C6H4-O- C6H4), the preferential point of etch attack is the oxygen. At high

pH the imide group will be hydrolyzed. The etching mechanism is complex because of the

simultaneous factors of oxidation and alkalinity. The chemical reaction responsible for etching

different polyimide can be different because they are made up of monomer units. 19

Effect of Thermal Annealing

By increasing the temperature of the tracked material, ion tracks can be thermally

annealed. For polymers, heating above the glass transition temperature mobilizes the polymer

fragments formed along the ion path. These fragments are sucked into the voids of the

neighboring pristine material, wiping out the latent ion track.14

The Effect of Temperature during Etching

Etch rates usually increase with temperature. Therefore, to obtain high throughput makes

it necessary to work at high temperatures. It is found that by alternating the temperature during

etching, high aspect ratio pores with large diameters can be obtained. At room temperature

diffusion processes can be faster than chemical reactions of the etchant within the polymer. 14









From this basis, the technique to make pores with large cone angles were develop by soaking at

low temperature and etching at high temperature.20

Effect of Detergents

Amphiphilic detergents may increase the track-etch-ratio. These detergents have been

shown to produce nanopores with cigar-like shapes and very small entrance openings. It is

thought that amphiphilic detergents attach to the hydrophilic surface layer, rendering it less

permeable to the etchant.21

Etch Properties in Muscovite Mica

Damage tracks are form in single crystal muscovite mica (KAl2(AI SiO3010)(OH)2 fTOm ion

irradiation along the (001) direction. Because the etching rate along the tracks is much faster

than both the lateral and bulb etch rate, when the tracks are etched through, nanopores with small

cross-section and small taper angle (0.020) are created.22 In muscovite mica, all the pores are

diamond-shaped with inner angles very close to 600 and 1200 (Figure 1-5). All the pores in a

given sample have the same size and orientation.22 By correlating the results of scanning

electron microscopy and X-ray diffraction on etched mica crystals, it was found that the

orientation of the diamonds is exactly the same as that of the mica unit cell. The four sides of the

diamonds are parallel to the four oxygen-terminated planes within the unit cell. These facts

points out that the diamond-shaped pores have their origin in the mica crystal structure. Also,

this shows that the uniform diamond shape arises because the oxygen-terminated planes are

those with the slowest etch rate, and the pores are aligned because the template are single

crystals.22

Template Synthesis

The Martin group has pioneered a general method for the preparation of nanostructured

materials called template synthesis23-2 Template synthesis method entails depositing a desired









material of interest into a porous solid. The size and shape of the nanomaterial depend on the

dimentions of the nanocavities within the porous template material. Depending on the

membrane and synthetic material used, nanostructures such as solid nanofibers or nanotubes can

be obtained. The method is termed "general" because nearly any chemical synthesis method

used to prepare bulk materials can be adapted to synthesize materials. There are reports of

metals,10, 26-32 pOlymers,33-36 carbons37-39 and semiconductors40, 41 prepared by the template

synthesis method. More advance material preparation include composite nanostructured

material, both concentric and tubular42, 43 and segmented composites nanowires.44

Template Synthesis Strategies

Three commonly used templates are; porous polymers, alumina and mica membranes

(Figure 1-6). Some of the more common synthetic strategies used to prepare nanomaterials

include chemical vapor deposition,38, 45, 46 electrochemical47-49 and electroless deposition,50, 51

chemical and electrochemical polymerization,53 and sol-gel chemistry.40, 54 Special attentions

will be devoted to the electroless deposition inside polymers and mica, and chemical vapor

deposition in mica since these are the methods used for preparing templates in chapters 2, 3, and

4 respectively.

Electroless Deposition

The electroless deposition method involves the use of a chemical reducing agent to plate a

metal from a solution onto a surface. Unlike electrochemical deposition, a conductive surface is

not necessary. The key requirement of electroless deposition is arrangement of the chemistry

such that the kinetics of the homogenous electron transfer from the reducing agent to the metal

ions is slow. This is essential because the metal ions would simply be reduced in the bulk

solution for fast electron transfer. In electroless deposition, a catalyst needs to be coated onto the

pore walls so that reduction of the metal ion only occurs at the pore surfaces. Figure 1-7 shows









the schematic representation that was used to prepare silver and gold nanowires and nanotubes

within PET, Kapton and Mica track-etched membranes. The membranes were first exposed to a

sensitizer (Sn2+). This is accomplished by simply immersing the membrane for 45 minutes in a

solution that is 0.026 M in SnCl2 and 0.07 M in trifluoroacitic acid in 50/50 methanol/water. The

tin sensitizer binds to the pore walls and membrane surfaces via complexation with the amine,

carbonyl and hydroxyl groups. 63 After sensitization, the membrane is rinsed thoroughly with

methanol and immersed into an aqueous solution of ammoniac silver nitrate (0.029 M Ag

(NH3)2 ) for 5 minutes. A redox reaction occours in which the surface bound tin (II) is oxidized

to tin (IV) and the Ag' is reduced to elemental Ag. As a result, the pore walls and the membrane

surface become coated with nanoscopic silver particles. The membrane is again thoroughly

rinsed with methanol. The silver coated membrane is then immersed into a gold plating bath that

is 7.9 x 10-3 M in Na3Au (SO3)2, 0. 127 M Na2SO3, and 0.625 M in formaldehyde at 40C. The Au

galvanically displaces the Ag particles because the reduction potential of Au is more positive

than that of Ag. As a result, the pore walls and surfaces become coated with Au particles. These

particles are excellent catalytic sites for the oxidation of formaldehyde and the concurrent

reduction of Au (I) to Au (0).63 Without a catalyst, the kinetics of the electron transfer from the

reducing agent (formaldehyde) to Au (I) is slow; therefore, the gold plating continues on gold

particles instead of in the bulk solution. The reaction can be represented as follows:

2Au (I) + HCHO + 30H 4 HCOO- + 2H20 + 2Au (0) (1-1)

This method yields the Au nanowires or nanotubes within pores plus Au surface layers on

both face of the membrane. These structures run through the entire thickness of the template

membrane (Figure 1-8). By controlling the plating time, the inside diameter of the tubes can be

varied because the thickness of both the Au surface films and nanotube wall increase with









plating time. By controlling the plating time, the inside diameter of the nanotubes can be varied,

even as low as 1 nm in diameter.28 As a result, these membranes can be used in a simple

membrane permeation experiment to cleanly separate small molecules on the basis of molecular

size.28 Also, by chemisorbing appropriate thiols to the Au nanotube wall based on well known

gold-thiol chemistry, the Au nanotube membrane can be made to preferentially transport cations

vs. anions and hydrophobic vs. hydrophilic moleculeS.10, 27, 31, 55 In addition, Au nanotube

membranes are electronically conductive and can be charged electrostatically in an electrolyte

solution.27 This introduces ion transport selectivity, allowing the Au nanotube membranes to be

electromodulated between ideal-cation and idea-anion transport states.27 Thus these Au nanotube

memebranes are ideal model systems for studying how pore size, chemistry, and charge affect

the transport selectivity at the nanometer scale.

Chemical Vapor Deposition (CVD)

CVD is commonly used to prepare carbon nanomaterials. We38, 56 and others57 have

synthesized carbon nanotubes within the porous alumina membranes using CVD. This involves

placing an alumina membrane in a high-temperature furnace (ca. 7000C) and passing a gas such

as ethane, propene or ethylene through the membrane. Thermal decomposition of the gas occurs

on the pore walls, resulting in the deposition of carbon nanotubes within the pores. High surface

area microporous carbon with long-range order has been synthesized by using zeolite Y as a

template with propylene CVD.59 Besides carbon nanostructures, other nanomaterials have been

obtained by CVD. For example, the martin group has used a CVD method to coat an ensemble

of gold nanotubes with concentric TiS2 Outer nanotubes.43

Resistive-Pulse Sensing

Resistive-pulse60 SCHSOTS for molecular and macromolecules analytes60-77 USe a nanopore in

a synthetic or biological membrane as the sensor element. This method, which when applied to









such analytes is sometimes called stochastic sensing,60, 69 entails mounting the membrane

containing the nanopore between two electrolyte solutions, applying a transmembrane potential

difference, and measuring the resulting ion current flowing through the electrolyte-filled

nanopore. In simplest terms, when the analyte enters and translocates the nanopore, it transiently

blocks the ion current, resulting in a downward current pulse (Figure 1-9). The frequency of

such analyte-induced current pulses is proportional to the concentration of the analyte, and the

identity of the analyte is encoded in the magnitude and duration of the current pulse.60-77

The maj ority of such resistive-pulse biosensing data has been obtained using a biological

nanopore, a-hemolysin (a-HL), embedded in a supported lipid-bilayer membrane as the sensor

element (Figure 1-10).60-69 This biological nanopore sensor has two key advantages. First, it can

be made analyte selective by using chemical or genetic-engineering methods to attach molecular-

recognition agents to the nanopore. As a result numerous different analyte types including metal

ions,64 DNA,65, 66 prOteins,67 and small molecules68 have been selectively detected with the a-HL

nanopore. Second, the biological nanopore can be reproducibly prepared from the commercially

available a-HL protein, which is obviously of great importance if practical, real-world, sensing

devices are ultimately to be derived from this technology.' There is however a key impediment

to developing practical sensors based on the biological nanopore. This problem concerns the

fragility of the supported bilayer membrane that houses the nanopore. Such membranes typically

survive for periods of only hours before rupture, much too short of a time to make a practical

sensing device."

One approach for solving this problem is to replace the biological nanopore, and bilayer

membrane, with an artificial nanopore embedded in a mechanically and chemically robust

synthetic membrane.69-77, 79, 80 Such artificial nanopores are often prepared by modern









microlithographic methods, using for example a focused ion" or electron72 beam to bore the

nanopore into a silicon or Si3N3 membrane. We and others are exploring an alternative

technology, called the track-etch method,16, 19, 81, 82 for preparing nanopores for resistive-pulse

sensorS.70, 75-77, 79, 80 Analytes detected with prototype track-etched nanopore sensors include

small molecules,70 DNA,75, 76 prOteins7 and nanoparticles.79 Furthermore, there are older reports

of developing virus sensors based on track-etched nanopores.so

The sensor elements we evaluated were conically shaped nanoporeS70, 75-77, 79, 81 prepared

by the track-etch method in polyethylene terephthalate (PET) membranes. Such conical

nanopores have two openings the large-diameter (base) opening at one face of the membrane

and the small-diameter (tip) opening at the opposite face (Figure 1-11). Fabrication methods to

prepare these conically shaped nanopores for resistive pulse sensing, is addressed in chapters 1.

Plasma-Based Etching

Plasma etching, a dry etching process, has become a very useful means of removing small

quantities of material from a variety of substrates quickly and efficiently.83 Plasma processes

have been used in many highly sensitive integrated applications to precisely remove specific

materials from sample surfaces. To generate plasma, a pair of electrode is needed; one is

connected to a radio frequency (RF) voltage and the other is grounded. RF energy is applied to

the electrodes which accelerates electrons to increase their kinetic energy. The electrons collide

with a neutral gas to form a collection of gaseous species including ions, free radicals, electrons,

photons and neutrals.83 The gaseous species can react with the surface to be etched such that

reaction product is volatile and can be pumped away. There are several types of plasma etching-

physical etching (anisotropic), chemical etching isotropicc) and reactive ion (combination of

physical etching and chemical etching). Usually in plasma based etching, both chemical etching

and physical etching occur.83









There are many applications for plasma etching such as photoresist removal, glass-like

compound etching (e.g., SiO2) and polymer etching to produce microstructures and

nanostructures. For example, large-area, well-ordered, periodic nanopillar arrays with lateral

dimensions as small as 40 nm have been developed based on a combination of colloidal

lithography and plasma etching techniques.84, 85 The etching mask on silicon substrates were

prepared using the close-packed structures formed by monodisperse polystyrene beads.84, 85

Polymer surfaces can also be modified by plasma treatment, for example, to improve wetting

properties and to enhance the adhesion of plasma-deposited coatings.86

Plasma etching technique is described in Chapter 2 to remove carbon surface layers from

mica tracked etched membrane that was previously exposed to CVD. This was necessary to

expose the underlying mica surface for dissolution with HF so that the carbon nanotube can be

revealed.

Asymmetric Diffusion

In some systems, diffusive transport through membranes may in some circumstances pass

more readily in one direction than the other. This phenomenon is known as asymmetric

diffusion, and is known in the context of transport across membranes,87-8 and in the context of

osmosis.90, 91 The above asymmetric phenomena is explained either by the binding of particles at

intra-pore sites or other electrostatic interaction with the pore surface. In this research, we show

that asymmetric diffusion can occur with no binding or electrostatic interaction of particle with

the pore surface. It is demonstrated in Chapter 5 that asymmetric diffusion can take place by

purely geometric constraints. Here, conical nanopores, with tip diameter comparable to that of

the diffusion molecule, are the used for asymmetric diffusion of neutral molecules.









Dissertation Overview

The goal of this research is to develop conical nanopores and nanotube (single and

multipore) in polymers and mica membranes by the track-etch method and extension of this

technology for sensing and other applications, and to investigate the transport properties of these

conical pores. The previous part of Chapter 1 has reviewed background information for this

dissertation including the ion track-etch method, membrane based template synthesis, electroless

metal deposition, chemical vapor deposition, resistive-pulse sensing, plasma based etching and

asymmetric diffusion.

In Chapter 2, an extension of the track-etch method to make conical pores in a reproducible

fashion is demonstrated. We have shown here that we can not only reproducibly prepare track-

etched based conical nanopore sensor elements, but that we can predict from the experimental

parameters used during the second etch, what the diameter of the all-important nanopore tip will

be. For these reasons, we believe that the track-etch method will prove to be the technology of

choice for taking artifieial-nanopore resistive-pulse sensors from the bench top to the practical

prototype-device stage of the R&D effort.

Chapter 3 describes a method to make asymmetric pores in tracked muscovite mica films

using an etch-refi11-etch approach. Tracks in the fi1ms were initially etched away with

hydrofluoric acid to form nanoporous membranes. These nanopores were then refi11ed with

silver nanowires or "metal tracks" using an electroless plating method. One face of the

membrane was then exposed to a solution of hydrofluoric acid and nitric acid, which etched the

bulk material and the nanowires respectively, at two different rates. By controlling the

concentration ratio of hydrofluoric acid to nitric acid, tapered pores with diamond shaped cross-

section were obtained. Replicas of the asymmetric pores were accomplished by carbon vapor










deposition, and scanning electron microscopy was used to give evidence of the resulting

nanotubes. In this study, excellent control over tip size and cone angle was demonstrated.

In Chapter 4, electroless gold plating properties on the surface and pore walls of track-

etched Kapton polyimide nanoporous membranes were studied. Scanning electron microscopy

(SEM), atomic force microscopy (AFM), and ion current measurements were used to

characterize the surfaces and pore dimensions of the membrane. Nanoporous Kapton polyimide

membranes were electroless gold plated over different times and the pore diameter characterized

using SEM. Ion current measurements were used to measure the diameter of very small pores.

AFM images show that after electroless gold plating, the gold surface layers are smooth

compared to a similarly structured polycarbonate membrane. Electroless plating the membranes

for 12 hours produced Au wires in the pores. Removing the membrane by oxygen plasma

revealed that the plating in the walls is also relatively smooth.

Finally, Chapter 5 describes asymmetric diffusional transport of neutral molecular species

through nanoporous polymer membranes. Membranes containing conical nanopores from

polyimide (Kapton HN Dupont) foils were prepared by the track-etching technique, based on

irradiation of the polymer with swift heavy ions and subsequent etching of the latent tracks. The

transport properties (e.g, flux) of these membranes were investigated using UV-Vis

spectroscopy. Transport experiments were performed with bare polymer membranes without

any modification. We report the preferential direction of the flow of molecules through conical

nanopores and spatial or concentration dependence of diffusion coefficient for a particular flow

direction.














Swilt heavy tom


''L'
r:';$ ~5~ ;~


,Latant lon
tackg


I, r ~I
~.'~'. .~


Figure 1-1. Swift heavy ions impinge on a dielectric material creating damaged ion tracks.


\0bitfion


Etlds:Int ; n.-arm r.


Etchi-\to a
solutionn


Elds~ant


Figure 1-2. After irradiation, the materials are subject to chemical etching which preferentially
removes the latent ion track.


DIctric
material













r
T


- lon track


Figure 1-3. Etched pore geometry in a homogeneous isotropic medium to a first approximation,
showing track etch rate, Fr, and bulk at rate, Va~


Figure 1-4. Scanning electron micrographs of Au nanocones replicas of PET conical pores.
Increasing fraction of ethanol in the etch solution results in greater cone angle (A-
C).[Adapted from Scopece, P.; Baker, L. A.; Ugo, P.; Martin, C. R. Nanotechnology
2006, 1 7, 3951-3956.]


V

































Figure 1-5. Scanning electron micrographs of etched particle tracks in single-crystal mica
































Figure 1-6. Scanning electron micrographs of a porous polycarbonate, alumina and mica
membranes used for template synthesis (A-C, respectively).



























Figure 1-7. Schematic diagram of Au electroless plating procedure


,>SnLI Ag

,Sn2


7,SXn4+ Ag
SAg

'>iSn 'Ag
/) Ag


SnCl


Au
`X4+
Au

'Sn *A
Au


+ Ag*


Au' Formaldhdeyd
4 OC










Top View

Membrane Face Pore Gold surface film

Electroless
Plating
O Gold Plating



Au nanotubes lining
SSide View 1 the pores

Electroless

Gold Plating

Membrane Pores


Figure 1-8. Schematic illustration of Au nanotubes obtained from electroless gold deposition
















Tim












Time

$ nlyewt ngtvecag
Figre1-. chmaicilusra ionofrsitv-usse in









Vestibule/cavity\


soA


IlrliM


Constriction***
Lipid bilayer 11


Transmembrane
il barrel


~- 20A


Figure 1-10. Essential features of the staphylococcal a -hemolysin pore shown in a cross-
section based on the crystal structure.[Adapted from Bayley, H.; Martin, C. R.
Chemical Reviews (Washington, D. C.) 2000, 100, 2575-2594.]


100 A









PET membrane


12 prm


Base
opening
(520 nm)


TIp
Opening
(10O to 60 nm)


Conical nanopore


Figure 1-11. Schematic of a conical nanopore sensor element showing the base diameter and
range of tip diameters used in these studies (drawing to scale)









CHAPTER 2
A1VIETHOD FOR REPRODUCIBLY PREPARING SYNTHETIC NANOPORES FOR
RESISTIVE-PULSE BIOSENSORS

Introduction

Resistive-pulse60 SCHSOTS for molecular and macromolecules analytes60-77 USe a nanopore in

a synthetic or biological membrane as the sensor element. This method, which when applied to

such analytes is sometimes called stochastic sensing,60-69 entails mounting the membrane

containing the nanopore between two electrolyte solutions, applying a transmembrane potential

difference, and measuring the resulting ion current flowing through the electrolyte-filled

nanopore. In simplest terms, when the analyte enters and translocates the nanopore, it transiently

blocks the ion current, resulting in a downward current pulse. The frequency of such analyte-

induced current pulses is proportional to the concentration of the analyte, and the identity of the

analyte is encoded in the magnitude and duration of the current pulse.60-77

The maj ority of such resistive-pulse biosensing data has been obtained using a biological

nanopore, a-hemolysin (a-HL), embedded in a supported lipid-bilayer membrane as the sensor

element.60-6 A key advantage of this biological-nanopore sensor element is that it can be

reproducibly prepared from the commercially available a-HL protein. This is of great

importance if practical, real-world, sensing devices are ultimately to be derived from this

technology.' There is, however, a key impediment to developing practical sensors based on the

biological nanopore. This problem concerns the fragility of the supported bilayer membrane that

houses the nanopore. Such membranes typically survive for periods of only hours before

rupture, much too short of a time to make a practical sensing device.69

One approach for solving this problem is to replace the biological nanopore, and bilayer

membrane, with an artificial nanopore embedded in a mechanically and chemically robust

synthetic membrane.69-77, 79, 80 Such artificial nanopores are often prepared by microlithographic









methods, using for example a focused ion" or electron72 beam to bore the nanopore into a silicon

or Si3N3 membrane. We and others are exploring an alternative technology, called the track-etch

method,16, 19, 81, 82 for preparing nanopores for resistive-pulse sensorS.70, 75-77, 79, 80 Analytes

detected with prototype track-etched nanopore sensors include small molecules,70 DNA,75, 76

proteins" and nanoparticles.79 Furthermore, there are older reports of developing virus sensors

based on track-etched nanopores.so

This field of artificial-nanopore resistive-pulse sensing is currently in its infancy. A key

question that must be addressed before practical sensors can be developed is can the nanopore

sensor element be prepared reproducibly, as the biological nanopore can? We address this

critically important issue here.

The sensor elements we evaluated were conically shaped nanoporeS70, 75-77, 79, 81 prepared

by the track-etch method in polyethylene terephthalate (PET) membranes. Such conical

nanopores have two openings the large-diameter (base) opening at one face of the membrane

and the small-diameter (tip) opening at the opposite face (Figure 2-la). We have found that the

diameters of both of these opening can be controlled with good reproducibility using a new two-

step pore-etching procedure. Furthermore, we have developed a simple mathematical model that

allows us to predict the diameter of the tip opening from the parameters used during pore

etching. Good agreement was obtained between the predicted and experimentally measured tip

diameters.

Experimental

First Etch Step

The tracked PET membrane (from GSI) was mounted in cell shown in Figure 2-1b, and the

etch solution (9 M NaOH) was placed in one half cell and the stop solution (lM formic acid plus

1 M KC1) in the other. A platinum wire electrode was placed in each solution and a Keithley









6487 was used to apply a transmembrane potential difference of IV during etching, with polarity

such that the anode was in the etch solution. The electrochemical reactions occurring at the

anode and cathode are discussed in the Supplementary Materials. Etching was terminated after

two hours by replacing the contents of the etch half-cell with stop-etch solution. The membrane

was then rinsed with purified water (Barnstead D4641, E-pure filters).

Determination of the Diameter of the Base Opening

Multi-track membranes (106 CA-12) were subj ected to the same first etch step as used for the

single-track membranes, and the base openings were imaged via FESEM (JEOL JSM-633 5F).

The average base diameter obtained (520+45 nm) is associated with measurements of 50 pores in

five different multi-track membrane samples.

Electrochemical Measurement of the Tip Diameter

The membrane was mounted in the cell, and the half cells were filled with 100 mM

phosphate-buffered saline, pH = 7.0 that was also 1 M in KC1. The specific conductivity of this

solution was measured using a conductometer (YSI 3200) at 250 C; a conductivity of 0. 107 S

cm-l was obtained. A Ag/AgCl electrode immersed into each solution was used in conjunction

with the Kiethly 6487 to obtain the current-voltage curve for the nanopore (Figure 2-2). We

have validated this electrochemical method by comparing diameters obtained via this method

with diameters for the same pores measured by electron microscopy.92, 93

Second Etch Step

The etch solution in this case was 1 M NaOH, and its conductivity was measured at 0. 160

S cml (250 C). The membrane was mounted in the cell, and the half cells were filled with this

etch solution. A platinum electrode was immersed into each half-cell solution, and the Keithley

6487 was used to apply a transmembrane potential difference of IV and measure the nanopore

ion current as a function of time. Etching was terminated at specified current values (Figure 2-3,









Figure 2-4) by replacing the etch solution in both half cells with the stop-etch solution. The

membrane remained in the stop etch for at least 30 min and was then rinsed with purified water.

Bovine Serum Albumin (BSA) Resistive-Pulse Sensing

Conical nanopores sensors having two different tip diameters, 58 nm and 44 nm, were

prepared; the base diameter for both sensors was 520 nm. After etching the pore walls were

coated with gold nanotubes using the electroless plating method described previously.28 After

electroless plating the tip diameters of the resulting gold nanotubes were measured using the

electrochemical method discussed above; tip diameters of 32 nm and 23 nm were obtained. The

Au-coated nanopore walls were then functionalized with a thiolated PEG (MW 5000 Da, Nektar

Therapeutics) to prevent non-specific protein adsorption.94 This was accomplished by

immersing the nanotube-containing membrane in a 0. 1 mM solution of the PEG at 4 oC for ~15

hours. The membrane was then immersed in purified water for 1.5 hours to remove any unbound

PEG. The tip diameters were then re-measured; values of 27 nm and 17 nm were obtained.

The BSA (Sigma) was dissolved in 10 mM phosphate-buffered saline that was also 100

mM in KCl (pH 7.4). The concentration of BSA was 100 nM, and the BSA solution was placed

on the tip side of the membrane. Buffer was placed on the base side and a transmembrane

potential of 1000 mV was used to drive the protein through the nanopore (tip to base) by

electrophoresis.

Results and Discussion

Conical Shaped Nanopores are Ideal Resistive-Pulse Sensor Elements

Resistive-pulse sensing entails mounting the nanopore membrane between the two halves

of an electrolyte-filled cell (Figure 2-1 (b)) and passing an ionic current through the electrolyte-

flooded nanopore. In a conically shaped nanopore the voltage drop caused by this ion current is

focused to the electrolyte solution in the tip opening of the nanopore.79 Indeed, the field strength









in the solution within the nanopore tip can be greater than 106 V m- when the total voltage drop

across the nanopore membrane is only 1 V.79 A consequence of this focusing effect is that the

nanopore ion current is extremely sensitive to analyte species present in the nanopore tip. That

is, there is an analyte "sensing zone" just inside the tip, which makes conically shaped nanopores

ideally suited for the resistive-pulse sensing application. This has been demonstrated with

prototype conical-nanopore sensors for analyte species ranging in size from small molecules, to

proteins, to nanoparticleS.70, 75-77, 79

The Core Technology: The Track-Etch Method

The track-etch method has been practiced commercially for decades to make polymeric

nanopore membranes for filtration applications.16, 19, 81, 82 It entails passing high energy particles

through the membrane, to create damage tracks, followed by chemical etching to convert these

damage tracks into pores. While the commercial process yields membranes that contain high

pore densities, a method for preparing single-damage-track membranes was developed at the

Gellsellschaft fur Schwerionenforschung (GSI).19 We purchased such single-track PET

membranes from GSI for these studies. This is an important point 0I ithr regard to the overall

sensor-fabrication technology the key precursor material, the tracked membrane, can be

obtained commercially.

The First Etch Step

Both etch steps used the cell shown in Figure 2-1b. Step 1 entails placing a solution that

etches the damage track on one side of the membrane and a solution that neutralizes this etchant

on the other side.8 For PET the etchant is NaOH, and the etch-stop is formic acid. This yields a

conically shaped pore (Figure 2-la) with the base opening facing the etch solution and the tip

opening facing the etch-stop solution. To determine when the etchant has broken through to the

etch-stop solution, and a contiguous pore has been obtained, an electrode is placed in each









solution, and a potential difference is applied across the membrane. Before breakthrough, the

transmembrane ion current is zero, and breakthrough is signaled by a sudden rise in the current.8

We previously showed that the diameter of the base opening could be controlled by

varying the potential applied across the membrane during this first etch step.92 An applied

transmembrane potential of 1.0 V was used in the first etch step for all of the nanopores

investigated here. To obtain a measure of the reproducibility of the base diameter obtained after

the first etch step, we subj ected multi-track membranes (106 tracks cm-2) to the same first etch as

used for the single-track membranes, and imaged the base openings using field emission

scanning electron microscopy (FESEM, Figure 2-4). We used multi-track membranes for this

study because it is difficult to locate the base opening in electron micrographs of a single-

nanopore membrane. We have previously shown that the pore diameter obtained for tracked-

etch membranes is ind pendent of track densi y.95 A base diameter of 520+45 nm was obtained,

indicating good reproducibility in base diameter after the first etch step.

However, the tip diameter varied between 1 and 7 nm and could not be reproduced from

etch to etch. We reasoned that this is an inherent feature of this "anisotropic" etch process. This

is because the etch and etch-stop solutions are mixing (and neutralizing each other) in the

nascent tip, which makes it difficult to control the etch rate in this critically important region of

the nanopore.

Measuring the Diameter of the Tip Opening after the First Etch

Because the tips after the first etch are so small, they are very difficult to find and image

via electron microscopy. Therefore, an electrochemical method described previously70, 76, 81, 92

was used to measure the diameter of the tip. This entailed mounting the membrane sample in the

cell (Figure 2-1b), filling both half cells with an electrolyte solution of known ionic conductivity,









and obtaining a current-voltage (I-V) curve associated with ion-transport through the nanopore

(Figure 2-2).

The experimental slope of this linear I-V curve is the ionic conductance, G1, (in Siemens,

S) of the nanopore, which is given bys

G1 = (GKal x db dt) / 4 L (2-1)

where GKal is the experimentally measured conductivity of the KCl-based electrolyte used (S cm-

1), L is the length of the nanopore (membrane thickness), db is the experimentally measured

diameter of the base opening, and dt is the diameter of the tip opening. Because all of the other

parameters in Equation 1 are known, dt can be calculated.

The Second Etch Step

In the second step NaOH etch solution is placed on both sides of the membrane. Again, a

transmembrane potential is applied, and the ion current flowing through the nanopore is

measured as a function of time during this etch. Our key innovation is that the second etch is

stopped at a prescribed value of this nanopore ion current rather than at some prescribed time

after starting the second etch (e.g., Figure 2-3). We adopted this approach because of the

variability in tip diameter obtained after the first etch step. The consequence of this variability is

that if we stopped the second etch at a prescribed time, we would obtain a corresponding

variability in the tip diameters obtained after the second etch step. In contrast, as we will see

below, there is an exact mathematically relationship between the ion current flowing through the

nanopore when the second etch is stopped (If) and the diameter of tip opening.

To prove that the pores obtained after the second etch truly are conically shaped, we used

an electroless plating method28 to deposit correspondingly conically shaped gold nanotubes

within the pores of the multipore membranes described above. The PET membrane was then









dissolved and the conical nanopores collected by filtration and imaged by FESEM.l These

images show that a nearly ideal conically shaped pore is obtained (Figure 2-5).

Measuring the Diameter of the Tip Opening after the Second Etch

The same electrochemical method was used, but the mathematics is slightly different. We

define the diameters of the base and tip openings after the first etch step as db1 and dtl and the

diameters after the second etch as dbf and dtf. These diameters are related via

dbf = (db1 + Ax) (2-2)

dtf = (dr y + Ax) (2-3)

where Ax is the change in diameter during the second etch. Substituting Equations 2-2 and 3-3

for db and dt in Equation 2-1 yields

G2 (Kal n (db1 + Ax)(dr y + Ax)) / 4 L (2-4)

where G2 is the slope of the current-voltage curve used to determine the tip diameter after the

second etch, db1 and dol are the experimentally determined base and tip diameters, respectively,

after the first etch, GKal is the experimentally measured conductivity of the electrolyte used (100

mM phosphate-buffered saline, pH 7, that was also 1 M in KCl; a = 0.107 S cm- ), L is the

membrane thickness, and Ax is the change in diameter between the first and second etch steps.

We define the parameter M as


M = KCl (2-5)
4L

which allows us to write, after some simple algebraic manipulation,


u2= dbldtl + (db1 + dtl)Ax + Ax2 (2-6)


Substituting (dtf dtl) for Ax (where dtf is the diameter of the tip opening after the second etch),

and again applying some simple algebraic manipulations, yield










d- + (db1 + d,,)dv = 0 (2-7)

This is a quadratic equation in dtf for which the solution is


-(b d,,)+ (db1 ,-d,)z)+4G', /M
d,. = (2-8)

Note that the quadratic formula has two roots; i. e., there should be a + instead of a + between the

two terms in the numerator of Equation 2-8. However, the root that results when subtraction is

used yields a negative value for the tip diameter.

Because all of the parameters on the RHS of Equation 2-8 are known, dte can be calculated.

Furthermore, because the base diameter at the start of the second etch is large (520 nm), the

change in base diameter during the second etch (Ax in Equation 2-2) is negligibly small for all

but the very largest tip investigated here (60 nm, Figure 2-6).

Reproducibly Varying the Tip Diameter

Figure 2-6 shows a plot of nanopore tip diameter, measured after the second etch step (Eq.

2-5), vs. the nanopore ion current at which this etch was stopped (If). We see that the tip

diameter can be reproducibly varied over the range from 10 to 60 nm (data points in Figure 2-6).

This is important because this is exactly the range in tip diameters we used in our prototype

protein," DNA,76 and nanoparticles79 Sensors. The lower limit (10 nm) is determined by the tip

diameter obtained after the first etch which, again, was in the range of 1 to 7 nm. However, we

have shown that the walls of such nanopores can be lined with gold nanotubes," and that the

diameter of these tubes can be controlled at will down to 1 nm.96 Hence, if tip diameters smaller

than 10 nm are needed, a pore with a 10 nm tip can be gold plated to reduce the tip to any desired

value. Furthermore, tips larger than the 60 nm maximum shown in Figure 2-6 can be easily

prepared by simply stopping the second etch at larger values of If.









The Mathematical Model

We begin by defining a new conductance, Getch, which is the ion current at which the

second etch is stopped (If) divided by the transmembrane potential applied during the second

etch (Eap). With this definition, Equation 4 can be rewritten as

If = Eap (etch n: (db1 + Ax)(dtl + Ax)) / 4 L (2-9)

where oetch is the experimentally measured conductivity of the NaOH solution used in the second

etch. Equation 2-9 is again a quadratic Equation in dtf for which the solution is


-(db1 -d,,)f + j(db1- d,,) + 4I /
der (2-10)


where K = Eap oetch n/4L (see Derivation of Equation 2-5, below). Equation 2-10 allows us to

calculate the value of the tip diameter after the second etch step (dtf) for any value of If at which

the second etch was stopped.

We noted above that the base diameter before and after the second etch is essentially the

same for all but the largest tip in Figure 2-6. This allows us to simplify Equation 2-9 to

If = Eap oetch n: db1 dtf / 4 L (2-11)

which can be rearranged to

dtf = If 4 L/ (Eap Getch n: dbl ) (2-12)

This obviously provides a much simpler relationship between dtf and If.

Plots of dtf vs. If calculated using the simplified equation (Equation 2-12) and the exact

equation (Equation 2-10) are shown as the two solid lines in Figure 2-6. The tip diameters

calculated by these two equations are identical for tips below ~20 nm. Furthermore, the

agreement between the experimentally measured (Equation 2-8) and theoretically calculated

(Equation 2-10) tip diameters is good, especially considering that there are no adjustable









parameters in the calculations. For example, at If = 20 nA the experimental and calculated tip

diameters differ by less than 10%, and at 40 nA they are identical.

The calculated tip-diameter values are, in general, slightly smaller than the experimental

values. This results from an interesting feature of the transport properties of conical nanopores -

if there is charge on the pore wall and if the tip opening is small, such nanopores act as ion

current rectifiers.97 The consequence of this rectifieation phenomenon is that the ionic

conductivity of an electrolyte solution within the tip of the nanopore can be lower than the value

measured for a bulk sample of the same electrolyte. Since we used the bulk-solution

conductivity in our calculations, the calculated values are in general low. Excellent agreement is

obtained between the experimental and calculated diameters for the largest tip (Figure 2-6)

because large-tip pores do not rectify the ion current.98 We believe that procedures to obviate the

small disagreement between the experimental and calculated tip diameters can be developed, and

we are currently pursuing this issue.

To illustrate the importance of controlling the tip diameter in resistive-pulse sensing, we

obtained current-pulse data for a prototype protein analyte, bovine serum albumin (BSA), with

nanopore sensors having two different tip diameters. The sensors in this case were conical PET

nanopores that had been lined with gold nanotubeS28, 77 and then coated with a poly(ethylene

glycol thiol) (PEG) to prevent nonspecific protein adsorption.94 The tip diameters, 17 nm and 27

nm, were measured after PEG functionalization.

Figure 2-7 shows current-pulse data obtained for BSA with these two different sensors.

The current-pulse signature can be defined by the average duration and magnitude (AI) of the

current pulses. The magnitude of the current pulse is important because if AI is not larger than

the peak-to-peak noise in the background current, the current pulse will be undetectable. We









found that AI is larger for the nanopore sensor with the smaller tip opening (AI = 80+20 pA) than

for the sensor with the larger tip opening (AI = 35+9 pA). This is because the roughly 4 nm x 4

nm x 14 nm94 BSA molecule more effectively blocks the ion current as it translocates the

smaller, 17 nm, tip.

Electrochemical Details

Pt electrodes were used to apply the transmembrane potential difference in both of the etch

steps, and the applied potential was 1.0 V in both cases. The half reaction occurring at the Pt

cathode was the reduction of the dissolved 02 in the solution.

02 + 4H+ + 4 e- + 2H20 (2-13)

The low (nA-level) currents, and the fact that the solutions were exposed to air during

etching, insured that the Oz WAS not depleted. The half reaction occurring at the Pt anode was the

reverse of Equation 2-15.

Conclusions

In his review of nanowire-based chemical and biosensors, Lieber stresses the importance

of being able to reproducibly prepare the nanowire sensing element.'" The same is true for

artificial nanopores to be used as resistive-pulse sensor elements. We have shown here that we

can not only reproducibly prepare track-etched based conical nanopore sensor elements, but that

we can predict from the experimental parameters used during the second etch, what the diameter

of the all-important nanopore tip will be. For these reasons, we believe that the track-etch

method will prove to be the technology of choice for taking artificial-nanopore resistive-pulse

sensors from the bench top to the practical prototype-device stage of the R&D effort.










PET membrane


12 pm


Base


(520 nm)


Conical
nanopore


Electrodes Pressure Clamping
J plate screw




IC1.0 cm3.cm


Solution
chamber\


/ 1
Membrane Poly(chlorotrifluoroethylene) cell


Figure 2-1. Schematic of a conical nanopore sensor element and etching cell. (A) The base
diameter and range of tip diameters used in these studies (drawing not to scale). (B)
Cell used to do the etching and to make all electrochemical measurements.


Tip
opening
(10 to 60 nm)

















Q



-.2-0.1 0D 0.1 0.2

o -1-


2 -2-
Applied transmembrane potential (V)

Figure 2-2. A typical current-voltage curve used to measure the tip diameter of the conical
nanopore.













40-

S35-




F 20-

E 15-

o 10-

z5,

0 20 40 60 80 100 120 140

Etch time (minutes)

Figure 2-3. Current-time transients obtained during the second etch step for three membranes
that were subjected to the same first etch. The second etch was stopped in each case
when a final nanopore ion current (If) of 40 nA was obtained.































Figure 2-4. Scanning electron micrograph of the base openings of two conical nanopores in a
multi-track PET membrane that had been etched (first etch step) as per the single-
track membranes used in these studies.




































Figure 2-5. Scanning electron micrograph of conical gold nanotubes deposited in a conical
nanopore membrane.












80

70



50

f 40~




I I I



0 10 20 30 40
Final nanopore ion current during second etch, I,(nA)

Figure 2-6. Plot of tip diameter measured after the second etch step vs. the final nanopore ion
current (If) at which the second etch was stopped. The points are the experimentally
measured tip diameters. The error bars are measurements on three different
membrane samples prepared identically. The solid curves were calculated using the
simplified equation (Equation 2-12, red curve) and the exact equation (Equation 2-10,
blue curve).









.r"r" nl- nr rr71-~nrr~lrl-~r~n 'Imn~nr~rrmrr


.~LLL 1.


100 s


20 pA [


1 1 I


1 (


Tll"r~ n I


Figure 2-7. Current-pulse data obtained for a prototype protein analyte, bovine serum albumin
(BSA) using PEG-modified conical nanotube sensors. (A) Tip diameters of 17 nm
and (B) Tip diameters of 27 nm. BSA concentration = 100 nM. Applied
transmembrane = 1000 mV.









CHAPTER 3
ETCH-FILL-ETCH METHOD FOR PREPARING TAPERED PORES IN ION TRACKED
MICA FILMS

Introduction

The Martin research group and others have been investigating a general method for

preparing nanomaterials known as template synthesis.23, 96 This method entails the synthesis or

deposition of a desired material within the pores of a nanopore membrane that serves as a

template. These template membranes contain monodisperse pores that are typically cylindrical

in geometry, and the pore diameter can be varied at will from tens of nanometers to tens of

microns. Since typical pore geometries are cylindrical, correspondingly cylindrical

nanostructures are usually synthesized via the template method; depending on the membrane and

synthetic method used, these may be solid nanowires or hollow nanotubes. 99

Recently, our research team and others have become interested in nanopores that have a

conical pore shape and the correspondingly conical nanostructures synthesized via the template

method within these pores. A number of applications can potentially benefit from conical pore

geometry. For instance, it has been shown that such conically shaped nanopores can be used as

the sensing element for new types of small molecule, 70 DNA, 75, 98, 100, 101 prOtein 77and

particle79 Sensors. Conically shaped gold nanotubes deposited within such pores can also act as

mimics of voltage gated ion channelS. 102 Membranes used for separations might also benefit

from a highly asymmetric pore structure. Finally, in addition to sensing and separations

platforms, conical nanostructures prepared by more conventional methods have been proposed

for use as cathodes in field-emission displayS. 103 To date, the vast majority of conical pores

have been fabricated using tracked polymers. 81, 89, 93, 95, 97, 104, 105 However, we are interested in

exploring materials other than polymers to use as conical pore templates because their different

properties may prove potentially superior for certain applications, and other applications might









be realized that are not possible with polymers. One such material of interest is tracked

muscovite mica.

Because mica is an inorganic crystalline material, it possesses some properties not present

in polymers that might make it superior in some aspects for certain applications. For example,

the surface of mica is molecularly flat, 106 and is a good candidate for platform for AFM imaging

of DNA, 106 and support layer for lipid bilayers. 107 Also, mica is very chemically resistant and

has high thermal and mechanical stability. 22, 108 Mica conical pores might prove much more

stable for resistive pulse sensors than polymers. Furthermore, these properties make it possible

for template synthesis of materials that require high temperatures. Additionally, nanostructures

that demand special geometry that is difficult to obtain by conventional methods may be

realized. For instance, tapered-shape carbon structure can provide mechanical stability yet

provide very sharp tips, which may be useful for enhanced electron field emission. 109

We and other have shown that the well-known track-etch method 16 can be used as a

starting place for preparing such conical nanopores. 81, 89, 93, 95, 97, 104, 105 This method entails

bombarding a thin film (5-20 Cpm) film of the material with a collimated beam of high-energy

particles to create parallel damaged tracks through the film. To make cylindrical pores, the

tracked membrane is simply immersed into a chemical etch bath, where preferential etching

along the damaged track converts each track into a cylindrical pore. To make conical pores, the

tracked membrane is mounted in an etching cell with an etch solution on one side of the tracked

membrane and a stop solution on the other side.8 This is shown schematically in Figure 3-1.

Since the damaged track is etched at a longer duration time and faster at the face of the

membrane exposed to the etch solution than at the face of the membrane exposed to the stop

solution, conically shaped nanopores are obtained.









While this stop-etch approach has been successful in making conical nanopores in a variety

of polymer materials, the method fails for the preparation of conical pores in mica membranes.

The key impediment is due to the ratio of the track to bulk etching rate. The track etch rate is

much faster than the bulk etch rate in mica (about 3000 time faster), los so that the etch solution

traverses the entire membrane before any significant bulk etching takes place. This means that

all parts of the membrane start etching almost at the same time in an isotropic fashion giving rise

to uniform pores instead of asymmetrical pore shape. One approach to solve this problem is to

replace the tracks of the mica films with a material that is more controllably etched, for instance

metal nanowires. In this study, we developed a method to independently control the solution

etch rate traversing the membrane, and also the etch rate of the surrounding bulk material, to give

asymmetric pores. We prove this by using carbon vapor deposition to replicate the pores,

dissolving the membrane to expose the tapered tubes, and imaging the nanotubes using scanning

electron microscopy. The results of these investigations are reported here.

Experimental

Materials

Muscovite mica wafers (1.181X 0.0004 inches) were purchased from Spruce Pine Co.

USA. Then these bare wafers were irradiated by swift heavy U25+ ions of 2.2GeV kinetic energy

with fluence of 104 tol07 cm-12 (GSI Darmstadt, Germany), which produced damage tracks

through the mica membranes. Hydrofluoric acid (HF, 48~51% from ACROS), for etching and

dissolving mica membranes, was used as received. Anhydrous tin (II) chloride 98% (Aldrich)

and hydrochloric acid (ACROS) were used as received to sensitize the mica membranes for

electroless plating. Ammonium hydroxide (Fisher), silver nitrate (Mallinckrodt), potassium

sodium tartrate tetrahydrade (Aldrich), and magnesium sulfate (Fisher) were used as received to

prepare silver plating solution. Ethylene (30% balanced with Helium, from Praxair) was used as









the CVD carbon precursor gas. Purified water was prepared by passing house-distilled water

through a Millipore Milli-Q water purification system.

Initial Etching of Mica Tracks to Prepare Very Small Pores

Mica wafers, containing damaged tracks, were exposed to low concentration HF solution

on both faces to create pores of about 10nm in diameter. The wafers were sandwiched between

two half cells of a conductivity cell (Figure 3-2.) and 2% HF at 250 C were placed in each half

cell for a period of 10 minutes. The etch process was terminated by quickly removing the etch

solution and replacing it with water for 2 minutes. Fresh water was replaced several times for

two minute intervals. Finally water was allowed to sit in the cell for another two hours.

Preparation of Tin Sensitizing Solution

Tin (II) chloride was used to sensitize the wafers so that electroless plating of silver can

take place on the surfaces. Tin (II) chloride crystals (0.5 g) were placed in 100 mL water and

stirred to give a cloudy appearance. 2 mL of 10 % hydrochloric acid was added using a pipette

causing the mixture to become a clear tin (II) chloride sensitizing solution.

Preparation of Silver Plating Solution

Filling the pores in the wafer with silver wire was done to make a more controllably etched

material. A two part electroless silver plating solution was made, where solution A contains the

silver ions and solution B contains a reducing agent. Solution A was made by dissolving 45.4 g

of silver nitrate to 450 mL of water and then adding ammonium hydroxide drop wise using a

pipette until the solution goes from clear to dark brown and returns to clear. Solution B was

made by dissolving 159 g of potassium sodium tartrate tetrahydrate and 1 1.4 g magnesium

sulfate to 364 mL of water. Both solutions were stored away from light.









Filling the Pores with Silver Wires

The purpose of silver plating the nanopores in the mica membrane was to control the rate

at which etch solution traverses the membrane relative to the lateral or bulk etch rate. First, the

intended stop side of the membrane was exposed to tin by filling one half cell with the solution.

The other half cell was left empty so that one face of the membrane was exposed to air. Tin

solution remains in the cell for 45 minutes giving it time to properly wet and sensitize the inner

walls of the pores. The membrane was then rinsed with water several times then was left to sit in

water for at least one hour. After removal of water from the both half cells, the membrane was

now ready for silver electroless plating. A dilute solution A (0.5 mL Solution A in 45 mL water)

was cooled to 40C and then 0.5 mL of solution B was added. This mixture was then placed in the

half cell that was not exposed to tin solution (side to be etched). The conductivity cell containing

the membrane was placed in a refrigerator, which was set at a temperature of 40C, for one hour.

Following electroless plating of silver, the remaining solution in the cell was removed and the

cell was thoroughly rinsed with water. Figure 3-3 shows the schematic outline for this etch-fill-

etch method.

Etching Silver Filled Mica to form Tapered Pores

The silver wire-containing mica membrane was exposed to HF/HNO3 etch solutions on

one face. This etch solution of HF (variable concentration) and HNO3 (10%) etched mica and

silver wire respectively at two different rates. After 3 hours the etch solution was removed and

rinsed with water several times. The membrane was then left to sit in 10% HNO3 for at least 3

hours to get rid of any residual silver. Finally, the membrane was rinsed in water several times

followed by soaking for at least 3 hours.









Making Replicas of the Tapered Pores

Tapered carbon tubes were obtained using a chemical vapor deposition (CVD) method

described in detail previously. 38, 110, 111 A piece of a porous mica membrane (preparation

methods were described in the previous section) was placed vertically into a quartz tube

(diameter: 4.5 cm, length: 48 cm). This tube was then inserted into a high-temperature tube

furnace (Thermolyne 21100) and the furnace was heated to 670oC under Ar flow. Once the

temperature stabilized, the Ar gas was replaced with an ethylene gas (20 sccm), which thermally

decomposed into carbon on the inner-wall and both faces of the mica template. After a desired

deposition time, the heating was terminated, the ethylene gas was replaced by Ar flow, and the

furnace was cooled down to room temperature. Unlike our previous CVD procedure with

alumina templates, 11 heat pretreatment of the template in this experiment was skipped since the

mica membranes we used can withstand temperatures above 900oC without any physical

deformation. The yielded carbon thickness can be controlled by varying the duration of

deposition.

Preparation of the Carbon Tube Replicas for SEM Imaging

First, CVD carbon/mica membrane was put into 48~51% HF solution for 16 hr to dissolve

away the mica template. Next, HF was then removed by pipette, leaving the liberated carbon

nanoboxes (connected together by the carbon surface film) which were rinsed with methanol and

suspended in methanol. The next preparation procedure entailed the removal of the carbon

surface layer on one face of the CVD-treated mica membrane to expose the carbon tubes. This

was accomplished by using an oxygen plasma etch procedure. A 1 cm x 1 cm hole was pre-

made in one piece of aluminum foil. This hole defines the area of the membrane that is exposed

to the oxygen plasma. The entire assembly, with the hole-containing Al foil facing up, was then

placed in the center of vacuum chamber of a plasma reactive-ion etching system (Samco, model









RIE-1C). The following etch conditions were used: power = 100 W, Oz preSsure = 300 Pa, Oz

flow rate = 30 sccm. After etching away the carbon surface film (determined by measuring the

conductivity of the membrane surface), the membrane was immersed into~- 48-5 1 wt % HF

solution to dissolve the mica template. This step does not result in free carbon tubes because

they are held together by carbon surface film that was not exposed to oxygen plasma. Finally,

the sample was rinsed with distilled water and air dried overnight. Sample imaging was

conducted using JEOL 6335F field emission scanning electron microscope (FESEM). Prior to

FESEM imaging, all samples were sputtered with Au/Pd using the Desk II Cold Sputter

instrument (Denton Vacuum, LLC). The sputter current = 45 mA, Ar pressure = 75 mTorr,

sputtering time = 60sec. The resulting Au/Pd film was ~ 16 nm.

Results and Discussion

During chemical etching of ion-tracked membranes, the damaged zone of the latent track is

transformed into nanopores. 16 The simplest description of the etching process defines two

parameters: the bulk etch rate (Va) and the track etch rate (VT). VB depends on the material,

etchant composition and temperature. VT depends on additional parameters, such as sensitivity

of the material to tracking, post-irradiation conditions and etching conditions. 14 When the

tracked film is exposed to an etchant on one face as described above, the results give conical

pores. In most ion-tracked materials, without exposure to extreme conditions (like exposure to

high illumination) the VB/VT ratio defines the cone angle that is formed. 14 However, in mica VT

is 3000 times faster than VB, 108 thus producing cone angles (0.020 los that is almost zero, giving

essentially pores with almost identical cross-sections along the membrane thickness. To improve

the cone angle in polymeric materials, the VB/VT ratio is increased by a number of methods

including increasing the etchant concentration, 1os applying a high transmembrane potential, 92









and modifying the etch solution composition. IsHowever, none of the above methods can work

with mica since VT is orders of magnitude greater than Va. 10s To get around this problem, the

etched ion tracks in mica was replaced with silver metal that can be independently etched with

nitric acid to a wide variety of etch rates. Figure 3-4a is an SEM image of a membrane that was

exposed on one face to 20% HF andl0% nitric acid solution at 250 C for 3 hrs. Here, HF and

HNO3 Solutions etch the bulk mica membrane and the metal track respectively. The base side

clearly shows the tapered "cone" shape that resulted from this etch. The decreasing pores size is

made quite evident from the progressive mica layers going down into the cone. The opposite

face of the membrane (Figure 3-4b) shows the tip to be on the order of a magnitude smaller than

the base.

The Martin group has been using conical pores for resistive pulse sensing of molecules.

One important feature that make conical pores ideal for sensing is, that most of the resistance is

focus in a short distant of the tip. As shown by Lee et al,79 the electric field in conical pores is

focused at the tip. The greater the half cone opening angle, the smaller the focus and hence a

sensing zone for molecular translocation of the pore for resistive pulse sensing. Half cone

opening angle depends on the VL/VT rtio, and we changed the concentration ratio of the etchants

to achieve this. Because the concentration ratio of HF (the lateral etchant) to HNO3 (the track

etchant) is greater than used previously, the lateral to track etch rate increases VL/VT TOSulting in

greater half cone opening angle (~ 210). Figure 3-5 is an SEM image of a tapered mica pore that

was etched with a higher percentage of HF solution than previously in Figure 3-4. We

demonstrate here that the cone angle can be controlled at will, because we can independently

control the solution etch rate traversing the membrane, and also the lateral etch rate, to give

asymmetric pores. This implies that the effective pore length (the part of the pore where









resistance is focused), and hence the focus of electric field can be varied in these tapered mica

pores. This is an important feature for resistive-pulse sensing that is likely responsible for pulse

duration.

To indirectly capture the entire geometry of the tapered mica pores in the membrane, a

replica was done using CVD method. Figure 3-6 clearly shows SEM images of carbon tapered

nanotubes replicas of the mica pores. The angles are well defined and surface of the tube

appears rather smooth at the magnifieation shown.

The half cone opening angle was calculated to be ~ 60. This is a relatively large cone

angle when compared to those of polymers that are etched without any promoters (Lane) or

applied high potential (chad). The half cone opening angle can be calculated as

P = arctan((db d,)/2L) (3-1)

where L is the length of the pore, db and dt are the large and small openings of the pore,

respectively. For db >> dt the equation simplifies to:

p = arctan (db /2L) (3 -2)

Figure 3-7 is a low magnifieation image of the carbon replicas of mica tapered pores indicating

that we can indeed reproduce the tapered geometry mica pore uniformly.

Conclusion

This study described a method to make asymmetric pores in tracked muscovite mica films

using an etch-fill-etch approach. Tracks in the films were initially etched away with

hydrofluoric acid to form nanoporous membranes. We demonstrated that by controlling the

concentration ratio of hydrofluoric acid to nitric acid during etching, tapered pores with diamond

shaped cross-section can be obtained. Additionally, we have shown that the cone angle of the

pores can be controlled be changing the concentration ratio of the bulk and metal etch solutions.









Replicas of the asymmetric pores were accomplished by carbon vapor deposition, and scanning

electron microscopy was used to give evidence of the resulting nanotubes. These conical mica

pores make prove more stable for resistive pulse sensing. Because it is so easy to tailor the cone

angle, this might make these mica pores more suitable sensing devises. One potential capability

that might be realized in resistive pulse sensing, is the tuning of the cone angle to control pulse

duration.


Damallged
ion tratck

Etchatnt a a .


Figure 3-1. After irradiation, the materials are subject to chemical etching which preferentially
removes the damaged ion track


Etchi-stop,
solution


Etcha nt Etch-stop,
solution






















Electrodes


Solution
chamber


1ii~ ri ~ 1 I

$1.0 cm 3.5 cm -


I 1
Membrane Poly(chlorotrifluoroethylene) cell

Figure 3-2. Schematic of cell used to do the etching and to make all electrochemical
measurements.


Pressure Clamping
plate screw








Mica membrane



Tracks


Electroless plating
of Silver


HF


Silver


Pore


Figure 3-3. Schematic diagram of etch-fill-etch method


HF/ HNO3




























Figure 3-4. Scanning electron micrographs of mica membrane that was exposed on one face to
20% HF andl0% nitric acid solution at 250C for 3 hrs. (A) Side exposed to the
etchant (the base). (B) Side exposed to water (the tip).






















Figure 3-5. Scanning electron micrographs of mica membrane that was exposed on one face to
40% HF andl0% nitric acid solution at 250C for 3 hrs. (A) Side exposed to the
etchant (the base). (B) Side exposed to water (the tip).

































Figure 3-6. Scanning electron micrograph of carbon tapered nanotube replica of the mica
tapered pore.


Figure 3-7. Low magnification SEM images of carbon tapered nanotube replicas of the mica
tapered pore.









CHAPTER 4
ELECTROLESS AU PLANTING OF TRACK-ETCHED KAPTON POLYIMIDE
NANOPOROUS 1VE1VBRANES

Introduction

Track-etched polymer membranes have found many applications in industry and research

as filtration and separation materials. 6 Chemical modification of the inner walls of these

membranes has made them more selective and sophisticated separation structures. 27, 28, 31 These

chemical modifications have been made possible by first plating the inner walls of the pores of

these membranes with gold, 23 then attaching a desired thiol terminated functional group. 31 For

reproducible results of any analytical measurements done using the pores of these nanoporous

membranes, it is crucial that the pores remain stable and have a well define internal diameter,

particularly where the pore approaches the size of the analyte molecule. One of the problems

faced when using some polymers such as polycarbonate (PC) and poly (ethylene terephthalate)

(PET) membranes, is that very small pores tend to temporarily block during ion transport

measurement. 112 Another disadvantage discovered when using these membranes to measure ion

current, is that possible dangling alkyl groups 113 render the pores from been well define and

thus gives inconsistent results and lots of noise in the current measurement. 112 The polyimide

Kapton on the other hand does not exhibit these disadvantages. 112

Due to its abilities of maintaining excellent physical, electrical and mechanical properties

at both low and high temperature extremes, Kapton is a very attractive polymer for use as a

particle track-etch membrane for application in separation and filtration in industry and research.

16, 114, 115 It will be advantageous therefore, if like polycarbonate and PET, Kapton can similarly

be electrolessly plated with gold to tailor pore diameter and, further, be modified with desired

thiol terminated functional groups for selective separation.









The objective of this study was to determine if the polyimide Kapton, a very chemically

resistant and stable polymer, can be electrolessly gold plated with similar or better quality

compared to PC porous membranes. Additionally, investigate whether plating can be controlled-

that is, tailor pore diameter of the membrane with plating time. It is interesting to note that to

date; there is no known report of electroless plating of Kapton membranes.

Experimental

Materials

Kapton 50 HN foils (12.5 Cpm thick, 107 tracks per cm2) were obtained from the linear

accelerator laboratory UNILAC at the GSI (Darmstadt, Germany). Boric Acid was obtained

from Fisher and used as received. NaOCl (13% active Cl), KI, SnCl2, AgNO3, NaHCO3, were

obtained from Aldrich and used as received. Trifluoroacetic acid, Na2SO3, NH40H,

formaldehyde and methanol were obtained from Mallinckrodt and used as received. Commercial

gold-plating solution (Oromerse SO Part B) was obtained from Technic Inc. Milli Q water was

used to prepare all solutions and to rinse the membranes.

Chemical Etching

Cylindrical pores were etched in the tracks of the Kapton foils using sodium hypochlorite

solution as describes in detail elsewhere. 116 The shape and size of the pores can be tailored by

the choice of etchant and the etching conditions. 116 To obtain cylindrical pores the etching rate

along the track, the-so called track etch rate yt has to be much faster than the non-specific

etching of the polymer called the bulk etched rate yb. 14, 81 The relation between yt and yb is

explained in figure 4-1. 14, 81 Studies have shown that an efficient etching can be performed in

sodium hypochlorite containing 13% active chlorine content. 116 Furthermore, it has been

demonstrated that the shape of the pore can be regulated with an appropriate choice of pH. 116

When sodium hypochlorite is not buffered its pH ~ 12.6. At these conditions, and elevated









temperature of 50oC, yb of Kapton is high (~ 0.21 (Cpm / h) and the pores become strongly

conical. Buffering the etchant with boric acid to pH ~9 enables one to obtain cylindrical pores.

It is important to note that the etching works only at basic pH when the hydrolysis of imide

bonds by OH- is possible. The membrane was immersed in a Teflon container containing 400 ml

of NaOCl solution at pH 9.8 and temperature of 50oC. First, the container with the etchant

solution is brought to 50oC by placing it in a water bath controled by a water heater. The size of

the pores increases with etching time. After etching, the membrane was rinsed with D. I. water

and left to soak for two hours. The membrane was allowed to dry in air overnight.

Electroless Plating of Kapton

In order to better control the size and surface chemistry of Kapton, the membranes can be

plated electrolessly with gold. 10 Tailoring of the pore size in polymer membranes by the time of

performing the electroless plating with gold has been demonstrated previously. 28' 51 Since

Kapton membranes possess carboxylate groups made available via imide hydrolysis by the

etchant, it is expected that these would act as active sites for the bonding of tin (H). Tin can then

reduce silver ion which later acts as a nucleation site for the reduction of gold. The procedure

followed the recipe for the electroless plating of polycarbonate. 28' 51

Pore Diameter Measurement

Cylindrical pores were characterized by taking scanning electron microscopy (SEM)

images of the membrane surfaces. Also, we have chosen to use an electrochemical technique

based on measuring ion current to measure pore diameter. This entailed mounting the membrane

sample in the cell (Figure 4-2), filling both half cells with an electrolyte solution of known ionic

conductivity, and obtaining a current-voltage (I-V) curve associated with ion-transport through

the nanopore. The experimental slope of this linear I-V curve is the ionic conductance, G, (in

Siemens, S) of the nanopore, which is given by










G = (NA GKal x d2) / 4 L (4-1)

where N is the pore density, A is the membrane area, GKal is the experimentally measured

conductivity of the KCl-based electrolyte used (S cm )~, L is the length of the nanopore

(membrane thickness), d is the pore diameter. Because all of the other parameters in Equation 4-

1 are known, d can be calculated.

Results and Discussion

SEM and lon Current Measurements

Figure 4-3 shows SEM images of Kapton porous membranes before and after electroless

gold plating. Here we see that the pore diameter decreases with plating time. Figure 4-4 shows a

plot of pore diameter as a function of Au plating time from 0 to 12 hours. The two methods of

measuring pore diameter gave very similar results. This is important because only resistance

measurements can be used for small pore diameter that cannot be resolved by SEM. This data

indicate that one can tailor the pore diameter with plating time down to the nanometer scale.

This capability is important for different transport studies and in sensor research where the size

of the pore with respect to the analyte is important. 10, 28, 77, 96

Atomic Force Microscope Images

Figure 4-5 shows the atomic force microscope images of the Kapton membranes before

and after electroless plating. The membranes remain relatively smooth after gold plating

compared to a similarly porous structured polycarbonate membrane after plating with gold.

Conclusion

Electroless gold plating properties on the surface and pore walls of track-etched Kapton

polyimide nanoporous membranes were studied. SEM, AFM, and ion current measurements

were used to characterize the surfaces and pore dimensions of the membrane. Nanoporous

Kapton polyimide membranes were electroless gold plated over different times and the pore










diameter characterized using SEM. Ion current measurements were used to measure the

diameter of very small pores. AFM images show that after electroless gold plating, the gold

surface layers are smooth compared to a similarly structured polycarbonate membrane.

Electroless plating the membranes for 12 hours produced Au wires in the pores. Etching away

the membrane with sodium hypochlorite to expose gold tube replicas revealed that the plating in

the walls is also smooth.













Latn intrc

Fiue -. eintono bl ec rt V n tak th ae t[Aate ro peP
Radiationl Mesrmns201 4 5-6.





Electrodes


Pressure
plate


0.5 cm
1t 1


Clamping
screw


Solution
chamber


lon track membrane

Poly(chlorotrifluoroethylene) cell


Aluminum frame


Figure 4-2. Schematic of cell used for electrochemical measurements.


3.5 cm --+









Before AU


3 hrs plating


Figure 4-3. Pore diameters for different Au plating times.










220
200
180
160
S140

S120
100
80
o
60
40
20 *


0 1 2 3 4 5 6 7 8 9 10 11 12 13
Time (hrs)

Figure 4-4. Pore diameter as a function of plating time with measurements taken from SEM
image (0-7.5 hrs) and ion current resistance measurements (8-12 hrs).










Before Au

Kapton
\4 membrane




Roughness ~ 3.6nm


After 2.5 hrs Au plating


~ 6.3nm


PC membrane After 2.5 hrs Au plating


Before Au


IRoughness ~ 13.6


SRoughness ~ 5.1


Figure 4-5. Atomic Force micrographs of Kapton and PC membranes before and after
electroless Au plating









CHAPTER 5
ASYMMETRY INT DIFFUSIONAL TRANSPORT OF MOLECULES THROUGH KAPTON
CONICAL NANOPORES

Introduction

Membranes and porous materials have found various applications in filtration and

separation processes. s-10 Modern biotechnology has posed new challenges in the application of

such membranes, and requires pores with diameters similar to those of molecules under study 11

(e.g., as small as several nanometers). The nanometer scale of such pores is necessary in both

achieving optimal control of the flow of biomolecules, as well as in developing sensors for their

detection. The transport properties of such nanometer scale pores are not well understood yet.

The hint that nanopores behave differently from micropores, comes from Mother Nature. 12

Biological channels and pores have diameter of ~ 1 nm and are critical for functioning of living

organisms. Ion channels and pores exhibit transport properties not observed with larger pores,

for example (i) selectivity for ions or molecules, (ii) rectification of ion current" (iii) ion current

for constant voltages applied across the membranell (iv) facilitated transport of molecules (v)

transport of ions and molecules against their electro-chemical potential gradient. 119

It has recently been demonstrated that asymmetric conical nanopores in polymer films can

exhibit transport and rectification properties similar to biochannels. For example, conical

nanopores in PET and Kapton membranes are cation selective and rectify ion current with a

preferential direction of cation flow from the tip to the base of the cone. 81, 88, 105, 120, 121

However, unlike the above cases, in this investigation there is no applied transmembrane

potential. Also, there are no electrostatic or binding interactions of the molecule with the pore

surface. Since the molecule under study is neutral, transport through the membrane is by purely

diffusional and geometrical constraints. Yet we found in our studies that the diffusion of

molecules across these membranes, exhibit a rectification behaviour. It means that there was a









preferential direction of diffusion flow. This effect cannot be predicted by considering classical

diffusion with constant diffusion coefficients. The rectification of conical nanopores is of

tremendous significance for industrial filtration processes in which asymmetric membranes are

often applied. One type of asymmetric membranes used in industry consists of a thin 'skin' of

nanoporous material placed on a low resistance support, which assures high fluxes and good size

separation. 122 Finding an optimal direction of concentration gradient will improve the filtration

process. Asymmetric diffusion has been observed before with multi-membrane systems and

membranes with two skins on two membrane faces of a membrane support. 123

Diffusional Transport Described by Fick's Laws

Diffusional transport is described by Fick' s two diffusion laws. For diffusion in one

dimension, Fick' s first law of diffusion describes the flux (the net number of moles of particles

crossing per unit time, t, through a unit area perpendicular to the x-axis and located at x), stating

that the flux of a molecule with diffusion coefficient, D, is directly proportional to the

concentration gradient.


J(x, t) =-D Scx )(5-1)


D normally is assumed constant, however there are known examples when D depends on

position x, concentration c or even time t. 124, 125 The diffusion coefficient determines the time it

takes a solute to diffuse a given distance in a medium. The diffusion coefficient depends on the

physical characteristics of the solute as well as those of the medium. An approximate bulk

diffusion coefficient (like that of molecules of similar masses) was used here, because, we are

more concerned about emphasizing the preferential flux from one side of the membrane than the

absolute flux values, themselves. The second of Fick' s law expresses how the concentration of









the species depends on time. Here again, the general form of the law in one dimension, taking

into account the possible forms ofD is as follows:

Sc f Sc\
-o (5-2)
at ax \ xc

Hindered Diffusion in Cylindrical Pores

The effective diffusion coeffcient of a solute within a pore of comparable size is lower than that

of the bulk solution value. This phenomenon is called hindered diffusion and results from steric

exclusion of the solute at pore opening and hydrodynamic or wall drag resistance due to the

presence of the pore wall. For steady-state diffusion through a membrane with cylindrical pores

that are comparable to the size of an uncharged solute, the effective diffusion coefficient can be

expressed in terms of the solute-to-pore size ratio Ai (= a/r) by the Renking equation: 126, 127

D / D, = (1 A1) (1- 2. 1044Al + 2.089il + 0.948il) (5-3)

Here, restriction to diffusion due to steric hindrance at the entrance to the pores is given by the

partition coeffcient,

O = (1 A) .(5-4)

As established by Ferry,128 a molecule must pass through the opening without striking the edge.

Therefore the center of the solute particle cannot be located at a radius that exceeds (r-a) (Figure

5-1). The partition coeffcient therefore, is equivalent to the fraction of the cross-sectional area

of the pore that is accessible to the center of the molecule.

The second factor in the Renking equation, called the inverse enhanced drag,127

K = (1- 2. 1044Al+ 2.089il + 0.948il) (5-5)

corrects for hydrodynamic or wall drag resistance, which is the friction between a molecule

moving within a pore and its walls (Figure 5-2).









The total flux, J, through a membrane with cylindrical pores of length, L, and pore density, N ,

can then be given as:

Nz ~r2 OK D, AC
J = (5-6)


where D.,is the free diffusion coefficient of the solute in the bulk solution, AC is the

concentration difference across the membrane, and N x r2 is the membrane porosity, E.

Hindered Diffusion in Conical Pores

Equation 5-6 is for a membrane with cylindrical pores where r is constant. The pores used

for this study are conical, and therefore r increases or decreases from one end of the membrane

to the other, thus varying linearly with position. The porosity of conical pores is different from

that of cylindrical pores, and is obtained by the product of the pore density and the geometric

mean cross-sectional pore area:

Nx ~d, d
E = '(5-7)


where d, and d, are the diameters of the large and small opening of the pores respectively.

There are varying hindrance effects (steric and hydrodynamic) on diffusion over the length of the

membrane. The average inverse enhanced drag, K 1, is ~ 1 for conical pores because most of the

wall drag resistance occurs in the region of the tip, which is a very small faction of the pore. For

cylindrical pores, the partition coefficient determines the rate of entry of the molecules into the

pores, and is equal to the ratio of the solute concentration in the pore to that of the bulk solution

at equilibrium. However, unlike cylindrical pores, the partition coefficient that determines

molecular entry into conical pores at steady state diffusion is not the same as the equilibrium

partition coefficient. The partition coefficient,0(r(x)) varies with r; which is a function of

position, x, and therefore changes along the length of the pores. Hence it is expected that the









equilibrium concentration along a pore length is different. However, the partition coefficient and

also the enhanced inverse drag within the pore, averages to be of negligible contribution to the

flux since only a very small fraction of the pore approaches the size of the solute molecule. The

value of these coefficients therefore approximates to 1. The asymmetry in diffusion, then, may

be caused by the difference in steric hindrance at the entrance of either side of the pore. This

will determine the amount of molecules that enter and leave the pore per unit time. The above

arguments suggest that molecules entering from the large opening of a conical pore will have

greater flux than those that enter from the smaller opening, provided that they can leave at the

same rate. The limit to diffusion will be the partition coefficient at the tip. With these

assumptions, the fluxes can be written for dilute solute concentrations as

E Ob K-' D, AC
Jb =(5-8)


E O, K D, AC
J, =(5-9)


where the subscripts b and t represents the directions of the net flux entering the large and small

opening of the pores respectively, and K-1 is equal to 1.

We can also solve the diffusion problem with a constant D, through a conical nanopore

with opening angle ot, openings dt and db, with solute concentration co at the tip, and zero

concentration of solute at the base:


c(x) =C co d, -cot a <; < (5-10)

valid for dt<
The diffusion problem with solute placed at the base side has the following form:











c:lx)=i co 1 ,dt -cota< x

When we calculate the fluxes (moles/s) in the two above mentioned conditions they are equal to

each other:


j =Dod'x (5-12)


These calculations indicated that 'classical' diffusion with constant D cannot describe our

experiments, unless one takes into account the difference in boundary conditions due to the

difference in the partition coefficient.

It was the purpose of this study to investigate the diffusion of neutral molecules from both

ends of conical pores of the polyimide Kapton. Here the dimension of the tip of the conical

pores approaches that of the molecules. To this end the diffusion of the water soluble neutral

molecule phthalazine was studied.

Experimental

Materials

Kapton 50 HN foils (12.5 Cpm thick, 107 tracks cm-2) were obtained from the linear

accelerator laboratory UNILAC at the GSI (Darmstadt, Germany). Sodium hypochlorite

(NaOC1, 13% active Cl) and potassium iodide (KI), were obtained from Aldrich and used as

received. Phthalazine, sodium chloride, sodium phosphate dibasic, sodium phosphate

monobasic, sodium azide was obtained from Fisher and used as received. Milli Q water was used

to prepare all solutions and to rinse the membranes.










Kapton Polyimide Membrane

We used polyimide foils to prepare nanoporous membranes. Polyimide possesses a unique

combination of properties that are ideal for a variety of applications in many different fields.

The film maintains excellent physical, electrical, and mechanical properties over a wide

temperature range. Polyimide also has very good chemical resistance and does not dissolve in

organic solvents. We used 12.5 Cpm thick commercially available Kapton 50 HN, produced by

DuPont.

Irradiation-Track Formation

For the preparation of membranes we used the-so called track etching technique. It is

based on irradiating a dielectric film with swift heavy ions and subsequent chemical

development (etching) of the damaged ion tracks. A unique feature of heavy ion irradiation is

single-particle recording. That is to say, one swift heavy ion which penetrates the foil produces

one damaged track. Therefore, counting the number of ions used for irradiation enables one to

prepare membranes with tailored number of pores from the range 1 up to 1010 ions/cm2. Kapton

foils, which we used for these experiments, were irradiated with uranium ions of energy of 1 1.4

MeV/u, at the heavy ion accelerator UNILAC at the Institute for Heavy Ions Research, (GSI)

Darmstadt, Germany. We used foils irradiated with the fluencies 107 and 10s ions/cm2. The

range of the ions is in all cases larger than the thickness of the polyimide membranes. The ions

penetrate the membranes at normal incidence, creating damaged tracks, which is then followed

by chemical etching to form pores (Figure 5-3).

Chemical Etching of Membrane Tracks

After irradiation of the Kapton foil with heavy ions, the latent tracks have to be chemically

etched. The shape and size of the pores can be tailored by the choice of etchant and the etching

conditions. To obtain cylindrical pores the etching rate along the track, the-so called track etch









rate Yt has to be much faster than the non-specific etching of the polymer called the bulk etched

rate y,. The relation between yt and yb is explained in Figure 5-4.

To obtain conical pores, one needs to choose etching conditions which assure high vb.

Preparation of conical pores is normally performed in a conductivity cell with etchant placed

only on one side of the membrane. The other side of the membrane is in contact with a stopping

medium, which neutralizes the etchant as soon as the pore is etched through. For example, if

NaOH is used as an etchant, we use an acidic stopping medium. The chemical stopping is

further supported by an electric stopping. The etching is performed under voltage with

electrodes arranged in such a fashion that the anode is on the side of etchant, which retracts ions

active in the etching process (e.g. OH-) out from the pore ( Figure5-5).

Etching with an applied voltage and measuring electric current affords a method to monitor

the process. At the beginning of etching the current is zero, because the two chambers of the

conductivity cell are not connected with one another. When the pore is etched through, the

current value is finite and increases in time, indicating an increase of the pore diameter (Figure

5-6). Kapton is very resistant chemically, therefore, development of latent tracks has to be

performed by a very aggressive etchant and at elevated temperatures. Previous studies have

shown that an efficient etching can be performed in sodium hypochlorite containing 13% active

chlorine content. Furthermore, it has been demonstrated that the shape of the pore can be

regulated with an appropriate choice of pH. When sodium hypochlorite is not buffered its pH ~

12.6. At these conditions, and elevated temperature of 50oC, yb of Kapton is high (~ 0.42 (Cpm /

h) and the pores become strongly conical. Buffering the etchant with boric acid to pH ~9 enables

one to obtain cylindrical pores. It is important to note that the etching works only at basic pH

when the hydrolysis of imide bonds by OH- is possible.










To obtain conical pores in Kapton, the irradiated samples were placed between two

chambers of a conductivity cell and etched from one side in sodium hypochlorite. The other half

of the cell is filled with 1 M potassium iodide (KI) solution as a stopping medium for the OCl-

ions of the etchant. As soon as the etchant completely penetrates the membrane, iodide ions

reduce OCl- to Cl- ions: 112, 130

OCl- + 2H' + 21[- It + Cl- + H20 5-13

Via this reaction, the etching process stops immediately after the breakthrough, allowing the

preparation of extremely narrow pores.

Pore Diameter Measurement

SEM was used to characterize the opening diameters of conical pores, especially the big

opening, which we call the 'base' (Figure 5-7). The small opening of conical pores, called the

'tip' is below resolution of SEM, therefore, we have to use another technique for its size

estimation. We have chosen to use an electrochemical technique based on measuring ion

current. The ionic conductance of a conical pore is related to its diameter by the following

equation:

G = nc trdbd, 5-14
4L
where n is the number of pores, cris the conductivity of electrolyte, L is the length of the pore (or

the equivalent membrane thickness), and db and d, are the diameters at the base and tip of the

cone respectively.

Transport Measurement

Measurements of neutral molecular transport through conical nanopores were performed

using UV-Vis spectrometry (Agilent 8453). A U-cell set-up contained the permeate molecule

solution on one side and a buffer, in our case PBS (in which the permeate was prepared) on the

other side of the cell (Figure 5-8). The membrane was sandwiched between two transparent










tapes that have holes in the center that defined the transport area. This tape-membrane

composite was clamped between the U-cell. The permeate solution was removed at certain time

intervals and the permeate concentration was measured be UV-Vis spectrometry. Feed solution

concentrations were varied in 4 ml PBS (pH 7.2), and the permeate side was only 4 ml PBS. The

solution on both sides of the U-tube was stirred using stir bars and stir-plate set-up.

Viscosity Measurements

The viscosities of phthalazine and dextrose were measured using Cannon-Fenske

viscometer No. 75 (model P200; Cannon Instruments)

Results and Discussion

Membrane Characterization

Figure 5-9 is an SEM image of the base side of a typical porous Kapton membrane used

for the transport study. The average pore density, taken from approximately five hundred pores

in five different locations of the image under low magnification, was 10 /cm2. Figure 5-10 is an

SEM image of the base showing a diameter of 1.68 pm. The size of the tip, which is below the

resolution of SEM, was calculated from ionic conductance measurements (Figure 5-11) using

equation 5-14. The average tip size used in this study was ~ 2 nm. The average diameter of the

molecule for transport was ~ 0.7 nm.

Transport Measurements of Phthalazine

From equation 5-4, the partition coefficients at the base and tip approximate to 1 and 0.42

respectively. These values put the partition coefficient at the base about 2 times that at the tip.

From the proposed equations, Figures 5-8 and 5-9 for base and tip fluxes, it is expected that at

low concentrations the flux from base to tip should be about twice that in the opposite direction.

Indeed, Figures 5-12 and 5-13 show that base fluxes are about twice that of tip fluxes for 1 and 3

mM solute concentrations. However, base fluxes approach those of tip fluxes as the









concentration of the solute increases, and both base and tip fluxes are similar at higher

concentrations (Figures 5-14 and 5-15). Figure 5-16 shows this asymmetric behavior over a

wider rage of concentrations.

The higher flux from base to tip direction can be explained by a lower access resistance of the

solute to the pore. Figure 5-17 shows a comparison between the theoretical flux obtain from

equation 5-9, and the experimental flux from tip to base. We see that these are in good

agreement over the wide range of concentration used. The experimental flux from base to tip, on

the other hand, deviates from theoretical values obtained from equation 5-8, particularly at

higher concentrations. To determine whether the base flux or tip flux behaves in a classical way,

a plot showing increase of flux through base and tip, respectively, with respect to the flux

measured at 0.1 mM was generated (Figure 5-18). The straight line: Flux(c)/Flux(0.1mM) =

c/0.1 obtained for the tip flux indicates that transport through tip behaves according to the Fick's

law with a constant D. Transport through base is hindered for higher concentrations indicating

that diffusion coefficient is concentration dependent.

The flux (mol/s) through an aperture of diameter dt with boundary conditions co is given

by: jlim,,= ited Dco. This value is smaller than the flux as given by eq. 5-12 The flux through

the tip is limited by the value of jlimited, While the flux through the base is not. The value of flux

through a conical nanopore as given by eq. 5-12 can be easily obtained when the solute passes

from base to tip.

The open question however remains, why the ratio of fluxes is concentration dependent. In

order to answer this question, we examined behavior of the flux from base and tip side,

respectively for different concentrations of the solute.









The Influence of Cosolute Concentration on Asymmetry

We investigated whether molecular crowding / jamming had any influence in the behavior

of the base flux at higher concentrations. To do this, we used a constant concentration (5 mM) of

phthalazine plus varying concentrations (0 to 100 mM) of the cosolute dextrose on the feed side

of the cell. We then placed identical total concentrations on the permeate side of the transport

cell using dextrose (Figure 5-19). This procedure was done to ensure that there was no osmotic

influence on transport. Figure 5-20 is a calibration curve for phthalazine, and phthalazine with

dextrose cosolute, generated from UV-visible absorption measurements. The two plots were

placed together to show that the addition of dextrose as a cosolute to phthalazine does not affect

the absorbance to any significant degree. Also, to address the issue of viscosity, kinematic

viscosity measurements were done on phthalazine and dextrose over the concentration range

used in this study. Figure 5-21 indicates that there is negligible change in viscosity over the

range of concentrations (0 to 100 mM) used for this study. Figure 5-22 shows the influence of

varying concentrations of the cosolute dextrose on the flux of phthalazine, which is kept at a

constant concentration of 5 mM. The tip flux is not affected over the entire range of

concentrations. However, the base flux is sharply decreased to that of the tip flux when dextrose

is added to the feed solution. Even though phthalazine concentration was kept constant, base

flux approached the tip flux in a very similar way when phthalazine concentrations were

increased. The increase in total concentration on the base side of the membrane causes base flux

to approach the limit of tip flux. Proper mathematical modeling of this effect is needed, taking

into account dependence of diffusion coefficient on concentration of the solute and position in

the channel. We would like to mention that theoretical studies were reported in which two sizes

gas molecules were placed on the base side of conical pores, and one size molecule was too large

to pass through the tip. In this situation a total j amming was found, causing the diffusion of the










small molecules to be zero over time. As expected, the rate of such j amming increased with

concentration. On the other hand, when the molecules were placed on the tip side, no j amming

occurred from that entrance. 131 We think that in our case, an increase in total concentration of

the solutes might lead to similar partial molecular j amming of diffusion from base to tip.

Decrease of the base flux provides evidence that it is the exit rate of the solute from the pore that

limits the diffusion transport in the direction from base to tip.

Conclusions

Diffusion rates through a membrane can be asymmetric due to molecular binding,

electrostatic interaction, and difference in osmotic potential. In this study we have demonstrated

that asymmetric diffusion can also occur by purely geometric constrains of conical pores on the

diffusing particles, where the tip of the cone is comparable to the dimension of the molecules.

We show that asymmetric behavior of diffusion is concentration dependent and there appear to

be some sort of partial j amming effect related to the increase in concentration of molecules from

the large opening of the pores. To further shed more light on this interesting phenomenon,

proper mathematical modeling of this effect is needed, taking into account dependence of

diffusion coefficient on concentration of the solute and position in the channel.






r~ra





Figure 5-1. The partitioning of a spherical molecule of radius, a, in cylindrical pores of radius,
r.[Adapted from Davidson, M. G.; Deen, W. M. Macromolecules 1988, 21, 3474-
3481.]










h = a/r


Figure 5-2. Spherical molecule of radius, a, moving within a cylindrical pore of radius,
r.[Adapted from Davidson, M. G.; Deen, W. M. Macromolecules 1988, 21, 3474-
3481.]




Dielectric
mate rial
Swift heavy ions


a-Betenion


Figure 5-3. Swift heavy ions impinge on a dielectric solid leading to damaged ion tracks witch
can be chemically etched to form pores(A and B, respectively)


.a- t


' ~a
































Figure 5-4. Definition of bulk etch rate Vb and track etch rate Vt.[Adapted from Apel, P.
Radiation M~easurements 2001, 34, 559-566.]


Pressure
0. cmplate




Solution 10c
chamber I I cm 5 e~m


Clamping
screw


Figure 5-5. Conductivity cell used to prepare conical pores.


lon track membrane

Aluminum frame Poly(chlorotrifluoroethylene) cell
















150-







242 244 246 4 250 252
time (min)


Figure 5-6. Etching curve showing moment of breakthrough with sharp increase in ion
current.[Adapted from Siwy, Z.; Apel, P.; Dobrev, D.; Neumann, R.; Spohr, R.;
Trautmann, C.; Voss, K. Nuclear Instruments & M~ethods in Physics Research,
Section B: Beamn Interactions nI itr Materals and Atoms 2003, 208, 143-148.]


























Figure 5-7. Kapton membran showing large opening (base) of conical pores


Feed side




Phthalazine + *--
PBS (pH 7.2) '


U-cell


pH 7.2)


Stir bar


Conical nanoporous
membrane


Figure 5-8. Experimental set-up for transport measurements


Permeate side




























Figure 5-9. Scanning electron micrograph of base side showing pore density (107 pores/cm2) Of
Kapton membrane.


Figure 5-10. Scanning electron micrograph of the base showing diameter of 1.68 Cpm


























V.VVVY~


I = 0.0077E
R2 = 0.9918


0.00020
0.00015
0.00010
0.00005


-


0.02 -0.015 -0.01.005

-0.00010
-0.00015
-0.00020

E (V)


0.005 0.01 0.015 0.02


Figure 5-11. A typical current-voltage curve used to measure the tip diameter of the conical
nanopores.








100

90


HBase
A Tip


0 100 200 300 400 500 600 700 800 900 1000

Time (min)



Figure 5-12. Base and tip fluxes for 1 mM phthalazine feed solution.




















mBase


0 100 200 300 400

Time (min)


Figure 5-13. Base and tip fluxes for 3 mM phthalazine feed solution.




120


HBase to tlp
| ATlp to base


0 50 100 150 200 250 300 350 400 450
Time (min)



Figure 5-14. Base and tip fluxes for 10 mM phthalazine feed solution.



















E

n 150
Z,


A Tip
aBase


0 50 100 150 200 250
Time (min)


Figure 5-15. Base and tip fluxes for 50 mM phthalazine feed solution.


Concentration (mM)




Figure 5-16. Asymmetric behavior of flux versus concentration.















0.6

S0.5
(V_ Theoretical
S0.4

O 0.3




0. Experimental


0 5 10 15 20 25 30

Co ncentration (mM)



Figure 5-17. Theoretical and experimental tip flux vs concentration







350

300 -

250

200 + from ti p
S150 -I from base

100 +

50 m


0 100 200 300

c [mM]/0.1 [mMV]



Figure 5-18. Increase of flux through base and tip, respectively, with respect to the flux
measured at 0.1 mM.


















U-cell


'BS (pH 7.2)
+ Dextrose


Phthalazine +

Dextrose+


PBS (pH 7.2)


Conical nanopore Si a
membrane


Figure 5-19. Experimental set-up for transport measurements of phthalazine with cosolute
dextrose


y = 0003058x
R2 = 0 998450


y = 0003021x
FF = 0 999811


* Phthalazine
SPhthalazine with 490 mM Dextrose








y = 0003021x
R2 = 0 998406


o 1

0 09

0 08

0 07


0d 05









001


0


-0 0035
S0 003
m 0 0025
z 0 002
S0 0015
S0001
m 0 0005
0


02 04 06 08
CONCENTRATION(m icrom olar)


5 10 15 20 25 30 35
CONCENTRATION(m icrm olar)


Figure 5-20. Calibration curve of phthalazine, and phthalazine with the highest concentration of
dextrose cosolute used