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Fabrication of Synthetic Nanopores in Thin Films for Studies of Analytical Applications

Permanent Link: http://ufdc.ufl.edu/UFE0041088/00001

Material Information

Title: Fabrication of Synthetic Nanopores in Thin Films for Studies of Analytical Applications
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Jin, Pu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: asymmetric, electroosmosis, membrane, mica, nanopore, pulse, rectification, resistive, sensing, synthetic
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The goal of this research is to develop nanoporous membrane devices for resistive-pulse sensing or separation applications. The first part of the research involves the fabrication of single conical nanopore in poly(ethylene terephthalate) (PET) membranes. A model analyte poly(styrene sulfonate) (PSS) was driven electrophoretically through the nanopore and the corresponding ion current pulses were observed. The effects of the supporting electrolyte pH, transmembrane potential and concentration of polyelectrolyte on pulse frequency were studied. The second part of the research involves developing a multi cycle chemical etch method for fabrication of a single or multiple pyramidally shaped nanopore in muscovite mica membrane. The effects of the etchant concentration, applied transmembrane potential and number of etch cycles on nanopore opening sizes and geometry were studied in detail. A significant ion current rectification was observed in such a single pyramidally shaped nanopore embedded mica membrane. In the third part, a new mechanism for improving the resistive pulse signal intensity was investigated by using single pyramidal nanopore in mica. The experimental results were the same as what we predicted based on this hypothesized mechanism, the pulse signals were current drops when the particles transported from tip to base and the pulses were current enhancement when the particles transported from base to tip in the nanopore. The effect of the nanopore geometry on resistive pulse detection was studied as a control experiment to test this hypothesized mechanism. In the last part of this work, electroosmosis in pyramidally shaped multi-pore mica membrane was studied. The asymmetric nanopores in these mica membranes demonstrate the ability to rectify the ion current flowing through the nanopore. As a result, the solution resistivity in the pore changes depending on the polarity of the applied electric field. If a constant current was applied to flowing through the nanopore membrane from the opposite directions, a rectified electroosmotic flow was observed. It was found that this rectified electroosmotic flow was the inherent result of ion current rectification in these asymmetric nanopores.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pu Jin.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Martin, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041088:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041088/00001

Material Information

Title: Fabrication of Synthetic Nanopores in Thin Films for Studies of Analytical Applications
Physical Description: 1 online resource (149 p.)
Language: english
Creator: Jin, Pu
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2009

Subjects

Subjects / Keywords: asymmetric, electroosmosis, membrane, mica, nanopore, pulse, rectification, resistive, sensing, synthetic
Chemistry -- Dissertations, Academic -- UF
Genre: Chemistry thesis, Ph.D.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: The goal of this research is to develop nanoporous membrane devices for resistive-pulse sensing or separation applications. The first part of the research involves the fabrication of single conical nanopore in poly(ethylene terephthalate) (PET) membranes. A model analyte poly(styrene sulfonate) (PSS) was driven electrophoretically through the nanopore and the corresponding ion current pulses were observed. The effects of the supporting electrolyte pH, transmembrane potential and concentration of polyelectrolyte on pulse frequency were studied. The second part of the research involves developing a multi cycle chemical etch method for fabrication of a single or multiple pyramidally shaped nanopore in muscovite mica membrane. The effects of the etchant concentration, applied transmembrane potential and number of etch cycles on nanopore opening sizes and geometry were studied in detail. A significant ion current rectification was observed in such a single pyramidally shaped nanopore embedded mica membrane. In the third part, a new mechanism for improving the resistive pulse signal intensity was investigated by using single pyramidal nanopore in mica. The experimental results were the same as what we predicted based on this hypothesized mechanism, the pulse signals were current drops when the particles transported from tip to base and the pulses were current enhancement when the particles transported from base to tip in the nanopore. The effect of the nanopore geometry on resistive pulse detection was studied as a control experiment to test this hypothesized mechanism. In the last part of this work, electroosmosis in pyramidally shaped multi-pore mica membrane was studied. The asymmetric nanopores in these mica membranes demonstrate the ability to rectify the ion current flowing through the nanopore. As a result, the solution resistivity in the pore changes depending on the polarity of the applied electric field. If a constant current was applied to flowing through the nanopore membrane from the opposite directions, a rectified electroosmotic flow was observed. It was found that this rectified electroosmotic flow was the inherent result of ion current rectification in these asymmetric nanopores.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Pu Jin.
Thesis: Thesis (Ph.D.)--University of Florida, 2009.
Local: Adviser: Martin, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2011-12-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2009
System ID: UFE0041088:00001


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1 F A BRICATION OF SYNTHETIC NANOPORES IN THIN FILM S FOR STUDIES OF ANALYTICAL APPLICATIONS By PU JIN A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2009

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2 2009 Pu Jin

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3 To my loved parents

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4 ACKNOWLEDGMENTS In the pas t five years of my graduate study and pursuing the truth of science, I have always been supported, advised and encouraged by many individuals around me. They are behind every progress that I made to achieve more success in my research. First of all, I would like to thank my research advisor, Dr. Charles R. Martin for his great guidance and support, especially his encouragement th r ough my doctorate research. I am grateful to have a n advisor who allow s independent thinking and scientific creativity. His serious attitude about scientific research really impressed me and made me a professional scientist thr oughout my future career. I also acknowledge the entire martin group and every member in the past five years. I would thank Dr. Lane A. Baker, who ga ve me the insight of my first research project and guide d me with his experience. I also thank Dr. Hitomi M ukaibo for teaching me a lot about electrochemical experiments. I would thank my great colleagues, Lloyd Horne, Jiahai Wang, Lindsay Sexton, Gregory Bishop, Peng Guo, Kaan Kececi, Dooho Park, Fan Xu for their supports and sharing their wisdom and experienc e with me. I truly appreciate the chances and time to work with these great people I also give special thanks to the scientist s at Major Analytical Instrumentation Center in Department of Mate rial Science and Engineering at UF. They provided me the great help in instrumentation training and then allowed me to access the instrument by myself. I would appreciate my parents for caring and support ing me in study and personal life. I also thank all my friends in Gainesville and I did have a great pleasing time in this peaceful town.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................8 LIST OF FIGURES .........................................................................................................................9 ABSTRACT ...................................................................................................................................12 CHAPTER 1 I NTRODUCTION AND BACKGROUND ...........................................................................14 Introduction .............................................................................................................................14 Fabrication of Conic al Nanopores in Thin Film Materials .....................................................15 Track Etch Method ..........................................................................................................15 Formation of Latent Ion Tracks .......................................................................................15 Ion Track Chemical Etching ............................................................................................17 Fabric ation of single conical nanopore in poly(ethylene terephthalate) with two step etch method ............................................................................................19 Fabrication of asymmetrically shaped n anopores in other membrane materials .....21 Characterization of Conical Nanopores and Properties ..........................................................23 Electron Microscopy .......................................................................................................23 Electrochemi cal Measurements .......................................................................................24 Ion Current Rectification .................................................................................................25 Electrical Field Focusing .................................................................................................28 ResistivePulse Sensing ..........................................................................................................29 ResistivePulsing with Biological Nanopores .................................................................29 ResistivePulsing Sensing with Synthetic Nanopores .....................................................30 Other Nanopore Based Sensing Strategies .............................................................................31 Mass Transfer in Asymmetric Nanopores ..............................................................................32 Dissertation Overview ............................................................................................................33 2 RESISTIV E P ULSE DETECTION OF A MODEL POLYMER ANALY TE USING A CONICAL NANOPORE IN PET MEMBRANE S ................................................................45 Introduction .............................................................................................................................45 Experimental ...........................................................................................................................46 Materials ..........................................................................................................................46 Fabrication of Single Conical Nanopore in Ion Track PET Membrane ..........................46 Current Voltage Curves Measurement ............................................................................47 Characterization of Sodium Poly(styrene sulfonate) .......................................................48 ResistivePulse Measurement of PSS ..............................................................................48

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6 Results and Discussion ...........................................................................................................49 Preparation and Characterization of Single Conic al Nanopore .......................................49 Characterization of Sodium Poly(styrene sulfonate) .......................................................50 ResistivePulse Measurement of Poly(styrene sulfonate) ...............................................51 Effect of supporting electrolyte solution pH ............................................................51 Detection limit for resistive pulse measuring PSS ...................................................52 Effect of analyte concentration on pulse frequency .................................................52 Effect of transmembrane potential on pulse frequency ............................................53 Translocation time and magnitude of current blockade ...........................................55 Conclusions .............................................................................................................................57 3 FABRICATION OF PYRAMIDALLY SHAPED NANOPORES IN MUSCOVITE MI CA MEMBRANES ............................................................................................................64 Introduction .............................................................................................................................64 Experimental ...........................................................................................................................65 Materials ..........................................................................................................................65 Asymmetric Nanopore Fabrication and Characterizations ..............................................65 Fabrication of asymmetric nanopore in mica ...........................................................65 Scanning electron microscopy .................................................................................67 Equivalent base and tip diameters ............................................................................68 Equivalent base and tip diameters for the singlepore mica membranes .................69 Electroosmotic flow measurement ...........................................................................70 Acid base neutralization in nanopores .....................................................................71 Nanopore Surface Modification ......................................................................................72 Results and Discussion ...........................................................................................................73 Fabrication of Nanopore with Ion Track Method ............................................................73 Nanopore breakthrough in iontrack mica ...............................................................73 Current time traces during anisotropic chemical etch ..............................................74 Electroosmotic flow in nanopores ............................................................................74 Acid and base neutralization in the nanopores .........................................................75 Track etch rate of mica .............................................................................................77 Morphology of Nanopore Openings in Muscovite Mica ................................................78 Asymmetric Nanopore Base and Tip Etch Rate ..............................................................79 Geometry of Asymmetric Nanopores ..............................................................................80 Effect of etchant concentration on nanopore geometry ............................................83 Effect of applied voltage on nanopore geometry .....................................................84 Fabrication of Single Pyramidal Nanopore in Muscovite Mica ......................................85 Conclusions .............................................................................................................................88 4 RESISTIVE PULSE DETECTION OF NANOPARTICLES USING A SINGLE PYRAMIDAL NANOPORE IN MICA MEMBRANE S .....................................................101 Introduction ...........................................................................................................................101 Experimental .........................................................................................................................103 Materials ........................................................................................................................103 Fabrication and Characterization of Single Pyramidal Shaped Nanopore in Mica .......103

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7 Characterization of Poly(styrene) Nanoparticles ...........................................................104 Current Voltage Curves Measurement in the Presence of Coating Reagents ...............104 ResistivePulse Detection of Poly(styrene) Nanoparticles ............................................104 Results and Discussion .........................................................................................................105 Characterization of Poly(styrene) Nanoparticles ...........................................................105 Current Voltage Curves of Nanopore in the Presence of Coating Reagents. ................106 ResistivePulse Detection of Poly(styrene) Nanoparticles ............................................107 Resistivepulse detection of nanoparticles with conventional mechanism ............107 Resistivepulse detection of nanoparticles involving coating reagent ...................107 Validation of the hypothesized resistive pulse detection mechanism involving coating reagent ....................................................................................................108 Current pulse shape ................................................................................................109 Current pulse amplitude .........................................................................................110 Current pulse duration ............................................................................................111 Current pulse frequency .........................................................................................112 Control Experiments for Studies of the Hypothesized Sensing Mechanism .................112 Conclusions ...........................................................................................................................113 5 STUDIES OF ELECTROOSMOTIC FLOW RECTIFICATION IN PYRAMIDAL NANOPORES IN MICA MEMBRANE S ...........................................................................120 Introduction ...........................................................................................................................120 Experimental .........................................................................................................................120 Materials ........................................................................................................................120 Fabrication of Pyramidally Shaped Nanopores in Muscovite Mica ..............................121 Fabrication of Straight Nanopores in Muscovite Mica .................................................121 Nanopores Characterization ..........................................................................................122 Phenol Transport Measurements ...................................................................................123 Results and Discussion .........................................................................................................124 Characterizations of Nanopore Size in Multipore Mica Membrane .............................124 Ion Current Rectification in Asymmetrically Shaped Multipore Mica Membrane .......126 Phenol Transport Measu rements ...................................................................................126 Electroosmotic flow in straight multipore mica membrane ...................................126 Calculation of electroosmotic flow velocity in nanopore membrane .....................128 Electroosmotic flow in asymmetric multipore mica membrane ....................................129 Relationship between the ion current rectification and electroosmotic flow rectification ................................................................................................................131 Conclusions ...........................................................................................................................131 6 CONCLUSIONS ..................................................................................................................138 LIST OF REFERENCES .............................................................................................................141 BIOGRAPHICAL SKETCH .......................................................................................................149

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8 LIST OF TABLES Table page 51 Estimated equivalent diameters of straight nanopore openings. ......................................137 52 Estimated equivalent diameters of asymmetric nanopore base and tip openings. ...........137 53 EOF velocities and ioncurrent and EOF rectification ratios for the membranes studied here. .....................................................................................................................137

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9 LIST OF FIGURES Figure page 11 Schematic of track etch method for the fabrication of nanopores in membranes. ............35 12 Schematic of ion track i n different membrane materials ...................................................35 13 Diagram of the electrochemical cells for nanopore chemical etch and electrochemical measurement. .....................................................................................................................36 14 Schematic of track etch and bulketch of a conical pore. ..................................................36 15 Schematic of anisotropic etch of a single conical nanopore in PET and the resulting current time trace. ..............................................................................................................37 16 Schematic of isotropic etch of a single conical nanopore in PET and resulting current time trace. ..............................................................................................................38 17 Chemical structure of polymers ty pically used for track etching ......................................39 18 SEM images of conical nanopores in different membrane materials. ...............................40 19 FESEM images of replicas of nanopore in different membrane mater ials. .......................41 110 A typical current voltage curve used to determine the single conical nanopore tip opening diameter. ...............................................................................................................42 111 Schematic of ion current rectification model. ....................................................................42 112 Distribution and line profile of the electric field across a conical nanopore membrane. ..........................................................................................................................43 113 Illustration of the resistive pulse sensing method. .............................................................44 114 Illustr Hemolysin protein embedded in a lipid bilayer support. .........................44 21 Current voltage curves of a single conical nanopore in PET membrane. ..........................58 22 Distribution of hydrodynamic diameter of PSS at different concentrations and the relationship between the gyration radius and polyelectrolyte concentration. ....................58 23 Current time transients of resistive pulse measuring PSS at different concentratio ns. .....59 24 Current time transients of resistive pulse measuring PSS at different pH value. ..............60 25 Relationship between the number of resistive pulse events and PSS concentration. ........61

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10 26 Relationship between the number of resistive pulse events and transmembrane potential. .............................................................................................................................61 27 Histogram of normalized current pulse amplitude of resistive pulse measuring PSS translocation. ......................................................................................................................62 28 Histogram .......................................................................62 29 Scatter plot of normalized current........63 31 The relationship between a rhomboidal opening and an equivalent circular opening. ......90 32 Schematic of modification GPTMS on muscovite mica to produce hydroxyl terminated surface groups. .................................................................................................90 33 Current time traces of the anisotropic etching of multitrack muscovite mica membranes. ........................................................................................................................91 34 Concentration of phenol in the permeate solution at different time when it was transported from base to tip in the nanopore. ....................................................................91 35 Current time traces during the HAc and NaOH neutralization and the schematic of eletrolyte concentration profile. .........................................................................................92 36 Current voltage curves of multi track mica membranes that were anisotropically etched for one 10minute cycle. .........................................................................................93 37 FESEM images of the base and tip openings of asymmetric nanopores in muscovite mica membranes at different etching time. ........................................................................94 38 Calibration of equivalent diameters of the base and tip openings of the pyramidally shaped nanopores that were anisotropic etched for different time. ...................................95 39 FESEM images of carbon replica of pyramidal nanopores in muscovite mica that were anisotropic etched for different time. ........................................................................96 310 Current voltage curves for single nanopore in mica membrane prepared by multi cycle and one step methods. ..............................................................................................97 311 FESEM images of carbon replica of pyramidal nanopores in muscovite mica that were anisotropic etched with different concentration of HF.. ...........................................97 312 Schematic of voltage effect on HF concentration profile and FESEM images of carbon replica of asymmetric nanopores. ..........................................................................98 313 Current voltage curves of single pyramidal nanopore in mica membrane in different salt concentration solutions and resulting ion current rectification ratio. ..........................99 314 Current voltage curves of single asymmetric nanopore in PET and mica membrane. ....100

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11 315 Current Voltage curves of a single asymmetric nanopore mica membrane before and after the surface modification with hydroxyl terminated gro ups. ...................................100 41 Schematic of hypothesized mechanism of resistivepulse detection. ..............................115 42 Size distribution of poly(styrene) nanoparticles in the buffer solution. ...........................116 43 Current voltage curves of a single pyramidal nanopore in mica measured in different supporting electrolyte solution. ........................................................................................116 44 Current time transients for conventional resistive pulse detection of nanoparticles using a single pyramidal nanopore in mica. .....................................................................117 45 Current time transients for coating reagent assisted resistive pulse detection of nanoparticles using single pyramidal nanopore in mica.. ................................................117 46 Current time transients for coating reagent assisted resistive pulse detection of nanoparticles using single pyramidal nanopore in mica. .................................................118 47 Expanded views of current pulse for nanoparticles translocation from the opposite directions. .........................................................................................................................118 48 Histogram of current pulse amplitude for nanoparticles translocation from the opposite directions. ..........................................................................................................119 49 Histogram of current pulse duration for nanoparticles translocation from the opposite directions. .........................................................................................................................119 51 FESEM images of asymmetric nanopore and straight nanopore openings in mica membranes used in EOF experiments. .............................................................................133 52 Current voltage curves of multipore mica membrane with straight or pyramidal nanopores etched for different time. ................................................................................134 53 Amount of phenol transported across a straight multipore mica membrane. ..................135 54 Schematic of ion accumulation and ion depletion in asymmetric nanopore and direction of corresponding electroosmotic flow. .............................................................136 55 Amount of phenol transported across an asymmetric multipore mica membrane. ..........136

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12 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 SYNTHETIC NANOPORES IN THIN FILM S FOR STUDIES OF ANALYTICAL APPLICATIONS By Pu Jin December 2009 Chair: Charles R. Martin Major: Chemistry The goal of this research is to develop nanoporous membrane device s for resistive pulse sensing or separation applications The fir st part of the research involves the fabrication of single conical nanopore in poly(ethylene terephthalate) ( PET) membrane s A model analyte poly( styrene sulfonate) (PSS) was driven electroph ore tically through the nanopore and the corresponding ion current pulses were observed The effect s of the supporting electrolyte pH, transmembrane potential and concentration of polyelectro lyte on pulse frequency were studied. The second part of the research involve s developing a multi cycle chemical etch method for fabrication of a single or multiple pyramidally shaped nanopore in muscovite mi ca membrane. The effect s of the etchant concentration, applied transmembrane potential and number of etch cycle s on nanopore opening size s and geometry were studied in detail. A significant ion current rectificatio n was observed in such a sin gle pyramidal ly shaped nanopore embedded mica membrane. In the third part, a new mechanism for improving the resi stive pulse signal intensi ty was investigated by using single pyramidal nanopore in mica. The experimental results were the same a s what we predicted based on this hypothe sized mechanism, the pulse signals were current drops when the particle s transported from tip to base and the pulse s were current enhancement

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13 when the particle s transported from base to tip in the nanopore The effect of the nanopore geometry on resistive pulse detection was studied as a control experiment to test this hypothesized mechanism. In the last part of this work, electroosmosis in pyramidal ly shaped multi pore mica membrane was studied. The asymmetric nanopores in these mica membranes demonstrate the ability to rectify the ion current flowing thr ough the nanopore. As a result, th e solution resistivity in the pore changes depending on the polarity of the applied electric field. If a constant current was applied to flowing through the nanopore membrane from the opposite direction s a rectified electroosmotic flow was observ ed. It was found that this rectified e lectroosmotic flow was the inherent result of ion current rectif ication in these asymmetric nanopore s

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14 CHAPTER 1 INTRODUCTION AND BACKGROUND Introduction At the nanometer scale, the physical pro perties of the macroscale world no longer hold.1 Due to this phenomenon, the physical and chemical properties of a nanoscale material may be different from the macroscale material composed of the same substance. These materials usuall y have special properties which have potential applications in a wide variety of sectors, including electronics, energy, biotechnology and e nvironmental monitoring et. al.1 There is an increasing interest in the research of the nanostructure s in thin film in recent years. Nanoporous membrane can be used for divers e nanotechnology applications, includi ng template synthesis2 8, bio separation3, 9 13 and bio sensing paradigm1417. These membranes are attractive platform s for nanotechnology due to the s imple, yet effective method s in which they can be used. Membranes offer a convenient way to handle and manipulate nanostructure s or nanomaterials without the use of a specialized instrument. Furthermore, homogenous pores ensure homogeneous nanomaterials, a characteristic that is not easily achieved at these small scales. Appropriate membranes can be commercially available, or can be fabricated and relatively simple technique s can be used to chemically or physically modify the membrane properties. There is a great deal of interest in developing the synthetic nanopore s as analogues of biological protein channel s for biosensing applications.1820 Such artificial nanopores have been fabricated in various materials by using different techniques.21 26 In the Martin research group, we have been exploring fabrication of conically shaped nanopore s polymeric and inorganic membranes by using the track etch method.2729 These membrane s have been used to study mass

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15 transfer properties,28, 3033 biosensing applications10, 3437 and as template for two dimensional nanomaterials synthesis.2, 3840 This research focused on the fabrication of asymmetric ally shaped nanopores in muscovite mica membrane. It was shown that the p yramidal shaped nanopore s in such material highly rectifi ed the ion current flowing through the nanopore. Correspondingly, an interesting electro kinetic phenomenon, rectified electroosmotic flow was also observed in these nanopores. Based on the strong ion current rectification in these pyramidally shaped nanopores, a new resistive pulse detection mechanism was proposed to enhance the current pulse signal intensity. F abrication of Conical Nanopores in Thin Film Materials Track Etch M ethod The track etch method entails t he chemical etch of the latent damag e track s that formed during the irradiation of th in film materials with a high energy, heavy ion beam (Figure 1) This method allows uniform pore siz e with adjustable pore diameter. The nanopore orientation could also be controlled by the heavy ion trajectory. In industry, this method has been utilized to prepare various types of nanoporous polymer membrane s for filtration applications The membrane materials containing nanopore are also good template s for two dimensional st ructure synthe sis. A variety of dielectric membrane materials (crystals, glasses, and polymers) are compatible with this technique, yielding diverse pores with different s hapes .41 Polymer membrane1, such as porous poly(carbonate) (PC)42, 43, poly(ethylene terephthalate) (PET)44, 45 and poly(imide) (Kapton)46 48 membranes are all commo nly used to prepare nanopores via this technique Formation of Latent Ion Tracks Damage tracks are usually prepared by bombarding the membrane materials with high energy particles or ions.49, 50 A very convenient way to obtain energetic heavy particles of about

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16 1 MeV/nucleon specific energy is spontaneous fission from artificial nuclides.41 Such high energy particles can usually leave damage tracks in the solid s Nuclear reactors provide improved beam intensity and collimat ion so that larger scale application like the commercial production of nuclear track filters with track density at 1010 per cm2 becomes realistic.41 The third, rather exclusive way to irradiate track with excellent control is to use a heavy ion accelerator. Such an accelerator can provide highly parallel ion beams leading to damage trails in the form of laten t tracks.41 For crystal materials, the penetrating ion creates a positive ion cloud around its path, whic h explodes by electrostatic repulsion. This leads to atomic displacements (defects), a highly disordered zone, with a core of decreased density surrounded by a sheath of increased density (Figure 1 2 A) .41, 49 In organic polymer materials, the track core consists of a zone of drastically reduced molecular weight, corresponding to broken molecular bonds close to the track (Figure 1 2B).41, 49, 51 Latent tracks consist of metastable or permanently changed zones with increased chemical reactivity. The etching transforms the latent track into an inerasable structure by supplying the required amount of ener gy for the enlargement process.41 Gesellschaft fuer Schwerionenforschung (GSI, Darmstadt, Germany) has developed a techniqu e to control that there is only one single track prepared in each membrane.52 Single heavy ion irradiation is achieved by placing a shutter between the ion beam and the membrane and an ion detector behind the membrane. The shutter is closed when the ion detector senses that a single ion traverse the membrane to prevent further exposure of the membrane to ion beam. Such technique provides promising application s for these nanopore membranes t hat were

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17 fabricated from the ion tracked films, especial ly those with only single pore as a paradigm for sensing devices.16, 42, 48 Ion Track Chemical Etching After irradiation, the latent damage tracks can then be chemical etched to create pores. A variety of membrane materials were fabricated with nanopores by using the ion track etch m ethod.16, 42, 48, 53 D epends on the chemical structure and composition of each material, the chemical reagents and etch condition vary in detail, especially for accurate controlling the nanopore dimensions and geometry. In the case of prepar ing the nanopore membrane s that were used as f ilters cylindrical shaped nanopores are preferentially fabricated.10, 13, 54, 55 For example, the commercial available nanoporous membranes, the ion tracked membranes are simply immersed into the etching solution for a certain time and the damage tracks are etched from both sides of the membrane s This leaves straight, usually cylindrical shaped nanopore through the membrane s The nanopore opening size s could be controlled by carefully choosing the type s of etchant, etchant concentratio n and et ch time Conically shaped multipore membrane s were also prepared when the high flux is preferred and the narrowest part of the nanopore s t ill need to be smal l .28 There is an increasing interest in developing c onica lly shaped nanopore in polymer membrane s because of their novel characteristics56, 57 and the potential application as a paradigm for sensing.16, 58 To fabricate asymmetric n anopore an iontrack membrane is mounted between two halve of electrochemical cell as shown in F igure 13.53 An etching solution that reacts with the polymer along the ion tracks is added to one side of the membrane. A stopping solution that does not react with the membrane is placed to the other side. The latent damage track s are preferentially etc hed along the direction of the tracks from the surface of membrane where in contact with the etchant.

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18 When the etchant breakthrough the latent damage tracks, it is neutralized or reacted with the stopping solution. This results in asymmetrically shaped nanopore s with larger opening (base) facing the etch ing solution and small opening (tip) facing the stopping solution.53 The reaction between the etchant and stopping solution is t he key for fabrication of asymmetrically shaped nanopore and this effect is referred as chemical stopping. Another important effect favorite the fabrication of conically s haped nanopore is electrical stopping.56 Dur ing the chemical etching a Pt electrode is placed in the solution on either side of the membrane and a constant transmembrane potential is applied. After the nanopore breakthrough, i n the case of essential etch species is charged, the electrical field drives the charged species cl ose to or fa r away from the membrane to affect the nanopore etch For example, PET membranes with only single conical nanopore were successfully prepared by Apel et al.47 After the PET membrane s were irradiated with heav y ions and latent track s were left in the center of the membrane. An etch solution of 9 M NaOH was placed on one side of the membrane and a stopping solution of 1 M formic acid with 1 M KCl was on the other side. A Pt electrode was placed in each solution and a transmembrane potential of 1 V was applied with the anode on the etchant side. The s e fabricated nanopores have large openings (base) facing the NaOH and small openings (tip) facing the formic acid After the nanopore broke thro ugh, the electrical field drove the OHetchant electrophoretical moving far away from the nanopore small openings At the sa me time, formic acid neutralized the OHetchant and decreased the chemical etch rate at the tip region. This usually result ed in conical nanopores with small tip diameter (<10nm) The etchant etch the polymer in both the direction s parallel (track etch) and perpendicular ( radial etch) to the damage tracks (Figure 14) Usually the track etch rate vT is much faster than

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19 the bulk materials etch rate (bulk etch, vB) Both of the vT and vB determine the shape of track e tch pores as well. The geometry of the conical nanopore is usually described by using the cone half angle (Figure 14 ) defined as the ratio of vB/ vT at high track etch case. The track etch ing rate is influenced by the sensitivity of the materials to tracking, UV irradiation condition59, post irradiation storage and etching conditions.5962 For materi als with a very high track bulk etch ratio, the etched pore s usually have straight shape with constant cross section .63 To explore the potential application as a resistive pulse sensing device, the reproducibility of the signals that were obtained from different membrane s is important Therefore the reproducibility of the conical nanopore tip base and corresponding geometry become the key issue s of these nanopore fabrications The method which Apel et cl had developed could control the nanopore base openings pretty well, however, has less control about the nanopore tip size. The Martin group has modified this method based on their approach and developed a two step chemical etch method to fine control both base and tip of the conical nanopore with excellent reproducibility.53 Fabrication of single conical nanopore in poly(ethylene terephthalate) with two step etch method The two step chemical etch method involves an anisotropic etch step for control ling the nanopore base opening size and an isotropi c etch step for precise controlling the nanopore tip opening si ze. The first step anisotropic etch is to control the conic al nanopore base opening size while still maintain a small tip. The membrane that is used for etch need to be irradiated under UV at fully sensitize the track etching process.59, 64 It is important to ens ure the nanopore breakthrough so that both ends of the nanopore are open when chemical etch is terminated at the predetermined amount of time The first step etch is quite similar to the procedure that was developed by Apel .47 The PET membrane is etched with 9

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20 M NaOH and stopped with 1 M formic acid solution at room temperature and a transmembrane potential of 1 V is applied with anode in the NaOH solution (Figure 15A) The NaOH hydrolyze the ester bonds in PET resulting in the formation of carboxylate groups and hydroxyl groups on the surface of nanopore wall and membrane face.65 Before the etchant breaks th rough the membrane the ion current flowing though the membrane is essential zero A sudden current increase is usually observed when the nanopore breaks through as shown in Figure 1 5B Such a current jump only indicates the na nopore formation; however, t he nanopore may not have the dimension or geometry desired for resistive pulse sensing. The membrane is usually etched for the predetermined time, about two hours in most of the cases. It was determined that the nanopore base openings etched under this condition have a average diameter of 52045 nm.53 To explore the reproducibility of nanopore base openings several multitrack membranes were etched a nd the above average nanopore size was determined by using scanning electron microscopy ( SEM ) But it has been found that the tip size of single conical nanopore s with similar size of base opening vary from membrane to membrane The complicated acid base neutralization make it hard to control the chemical etch at the tip region. It is also difficult to monitor the current accurately so that anisotropic etch cannot be stopped in time before the tip becom es too large However, a second isotropic etching step was developed to fine tune the nanopore tip opening.53 The second step isotropic etch is similar to the anisotropic etch. The only difference is that both halves of the chem ical cell are filled with the etch solution of 1 M NaOH (Figure 1 6 A) As known, t he conductance of nanopore increases when the pore size increases and the rate of such an increase dep ends on the bulk etching rate.51, 66

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21 There is an assumption that is the etch rate is the same at every position along the axis of the nanopore during the isotropic etch in other words, both the nanopore tip and base increase the same amount of value during this step etch.53 The observed ion current increasing demonstrates the nanopore is increasing at the same time. Instead of being stopped at a predetermined time, the isotropic etch step is ended at a predetermined current value As shown in Figure 1 6B, the isotropic etch es of three ind ividual membrane were terminate d when the ion current value reached ~40 nA. The real time current monitoring allows the termination o f isotropic etch in time. Besides a low concentration of etchant etches th e nanopore very slowly and it can be easily neutralized to terminate the isotropic etch. Therefore, highly reproducible tips with accurately controlled diameter were obtained by stopping the isotropic etch process at a pre determined current value, Fabrication of asymmetri c ally shape d nanopores in other membrane materials A v ariety of membrane materials were utilized for track etch technique. Polymer membrane s have the greatest use due to their chemical and mechanical robustness. A number of polymer materials are suitable for preparing ion track etched conical nanopores, including poly(carbonate) (PC),42 poly(ethylene terephthalate) (PET),47 Poly(imide) ( Kapton),47 poly(propylene) (PP) .67 The chemical structures of the three commonly used poly mer membranes (PC, PET and Kapton) are shown in Figure 17. Beside s, asymmetrically shaped nanopores were also fabricated in inorganic materials, such as glass.41, 68 The Martin research lab recently started exploring the fabrication of asymmetric pores in crystal materials, such as muscovite mic a.69 According to the materials composition, the etchant, stopetching solution, etch temperature differ from one to another ; correspondingly, nanopore opening dimensions and geometries vary a lot.

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22 The fabr ication of conical nanopore in poly(carbonate) is similar to that of PET. A 9 M NaOH i s used as etchant and a solution containing 1 M formic acid and 1 M KCl is the stopping solution. Chemical etch is performed at room temperature w ith a transmembrane potential of 1 V applied on the anode in the NaOH solution .62 It is also demons trated that the cone angle increase by using higher transmembrane potential.62 The ion tr acks in poly(imide) (Kapton) are etched by using a NaOCl solution with a n a ctive chlorine content of 13%. A 1 M KI solution is used as the stopping reagent .70 The chemical etch is usually performed at 50C to mak e the nanopore breakthrough quickly. When the etchant NaOCl breakthrough to the other s ide of the membrane, the iodide ions reduc e the hypochlorite ions to produce chloride ions as a result, the chemical etch is stopped Etching of Kapton results in the formation of carboxylate groups via hydrolysis of imide bonds.46 Conical shaped nanopore s w ere fabricated not only in polymeric membrane, but also in inorganic thin films, such as glass.41 Ion tracks in glass ar e usually etched with hydrofluoric acid. These cheap material offers a low but highly reproducible etch ratio.41 Another promising candidate for a technological application is the very homogeneous and isotropic phosphate glass. For highly ionizing heavy ions, phosphate glass has an etch ratio vT/ vB of the order of ~10. This allows prolonger etch breakthrough time and the tracks can be easily etched to spherical sections m) .41 Compare with glass, it is a great challenge to fabrication asymmetrically shape d nanopore in crystal materials, such as muscovite mi ca. M ica has a very high relative track bulk etch ratio in hydrofluoric acid (VT/VB>> 103).71 Because of such a high track etch rate, etchant HF breakthrough to the other side too fast to etch in the radial direction larger enough. Unlike using polymer membrane, it is not easy to fabricate asymmetric nanopore in mica.

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23 Martin group start ed to explore the fabr ication of asymmetric pore in mica.69 One approach is to first etch straight nanopore s, followed by replacing the pore with a material that is more controllably etched, for instanc e metal nanowire s Then a mixture of HF and HNO3 was used with HNO3 etch ed the nanowire s and HF etch ed the bulk mica around the metal. Asymmetric nanopores were prepared this w ay, but it has less control over the etch process so that it is difficult to maintain the nanopore tip small enough for resistive pulse sensing. Characterization of Conical Nanopores and Properties The dimension and geometry of nanopore s are critical important regarding their application s in resistive pulse sensing53 and separation9. S canning electron microscopy was used for imaging and measuring both the base and tip openings of nanopore s in multi pore membrane. However, it is challenging to obtain such images for nanopore in single pore membranes. This is especially true of the tip opening because they are so small and there is only one per sample. An alternative method, electrochemical measurement, is u sed for measure the conductance of nanopore filled with electrolyte solution and calculate nanopore tip opening. Electron M icroscopy Both the tip and base openings on multi pore membrane surface were measured with field emission scanning electron microscopy (FESEM). The base diameter, dbase, of conical nanopore in PET membrane s obtained after the anisotropic etch was used to determine the bulk etch rate vB of PET in 9 M NaOH at room temperature. SEM images were obtained from different individual membrane s and an average etch rate was obtained. The PET bulk etch rate vB using 9 m NaOH is ~2.17 nm min1.72 Therefore, it is possible to calculate the nanopore base size as a function of etch time if the membrane is etched under the same condition. This is especi ally useful to estimate the nanopore base size for single pore membrane. For example, the nanopore base is

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24 about 520 nm in diameter after 120 minutes anisotropic etch with 9 M NaOH .53 Figure 18 shows the FESEM images o f conical nanopore etched in P C73, PET, Kapton69, glass41 thin films. Although the SEM images o f nanopore base and tip opening clearly demonstrate the asymmetric shape of nanopore, but the exact geometry of nanopore is still unknown. To investigate this issue, an alternative method was developed to image pore geometry in multipore membrane entails depo siting a material such as metal62 or carbon29 within the pores. Fo r most of the nanopore polymer membrane s (PC and PET) a well established elec troless gold plating produced is used to deposit gold inside the empty nanopore.16, 62 This also leaves a gold layer on both sides of the membrane. After removing t he surface gold layer, the p art remains is the gold replica of conical nanopore s For inorganic materials, such as mica, a chemical vapor method was used to de posit carbon inside the nanopore channel.29 Both these methods create replicas of the pores, which can be liberated by dissolution of the t emplate membranes and imaged by using FESEM Figure 19 s hows the exampl e of gold replica from conical nanopore in PET and carbon replica from straight nanopore in mica, respectively. Electrochemical Measurements Because it is difficult to measure the base and ti p opening of single conical nanopore in membrane by using SEM, a current voltage curve measurement is helpful to determine the nanopore tip size (dtip) based on a few assumptions. This measurement entails mounting a membrane containing a single pore betwee n the two half cells (Figure 1 3) An electrolyte solution with known ionic conductivity is introduced into the cell s on both sides of the membrane with a Ag/AgCl electrode on each side A curre nt voltage curve is obtained via a linear scan ning of transmembrane potential and measuring the resulting ion current flowing through the nanopore. The slope of the current voltage curve represents the ionic conductance (G) of electrolyte filled nanopore. Sometimes the ion current -

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25 voltage curve is not com plete linear in the entire range of scanned voltage s only the most linear part of the current voltage curve (usually the part between 200 mV and +200 mV) is used to calculate the nanopore tip diameter.53 Equation 1 1 describe s the ionic conductance of the nanopor e filled electrolyte solution as, = 4 (1 1) (equal to the thickness of membrane), dtip and dbase are the diameters of tip and base opening, respectively. Base opening diameter dbase is usually calculat ed from the etch time and bulketch rate. Tip opening diameter dtip can be calculated using Equation 11 if pore conduce G can be measured experimentally. However, t here are two assumptions that have to be satisfie d if Equation 11 can be used to calculate single pore tip size First, it is assumed that the bulk etch of single track membrane is the same as that of multi track membrane, which is also ~ 2.17 nm min1 in 9 M NaOH. The second assumption is that the nanopore is in a perfect cone shape or cylindr ical shape It need to be mentioned that the increase of b considered in the case of the second step et ch. The diameter of base opening dbase after the isotropic etch need s to be correct ed with the original size and its increase Ion Current R ectification It has been known that the currentvoltage curve of single conical nanopore is not always linear, especially when the nanopore is filled with low ionic strength solution.57 It is usually observed as the ion current is higher at one electrode polarity and it is lower at the same absolute voltage but the opposite polarity ( as shown in Figure 110) There are several models have been developed to explain ion current rectification in artificial nanopore or nano capillary system.2 4, 57,

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26 7476 Among thes e models, ratchet model76 and i on accumulation and depletion model74 are widely accepted by researcher s The chemical etching of the ion tracks in PET and Kapton membrane s leaves carboxylate groups on the membrane surface.47 When the pH is above the polymers isoelect ric point ( ~ 3 for PET, Kapton), the carboxylate groups deprotonated, which results in negative charges on the surface. It is proposed that the membrane becomes cations permselective when the nanopore size is comparable to the thickness of electrical doubl e layer (EDL) on the pore wall which means the nanopore will preferentially transport cations and reject anions .30 T herefore in these theories only cations transport are considered for the surface negatively charge d pore In the model proposed by Siwy et. al., cations is electrostatic trapped in the nanopore where is close to the tip region at positive potential (anode at base side of the membrane). The electrostatic trap effectively inhibits the movement of cations and decreases the current flowing throug h the conical nanopore. However, at the negative potential (anode at the tip side of the membrane), the electrostatic trap is eliminated and larger current is observed.57, 76 In the case of positively charged surface, this r atchet model will be reversed. There are three requirements for the ion current rectification base on ratchet mode. Such requirements are (i) pore su rface is negatively or positive ly charged, (ii) pore has an asymmetric shape, (iii) the dimension of tip is comparable to the thickness of electrical double layer on the pore wall. It is also demonstrated that a cylindrical nanopore with equal size opening s on both sides of the membrane does not rectified the ion current.76 It is reported that the nanopore at certain size will rectify the ion current more strongly in lower ionic strength s upporting electrolyte solution.57

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27 Another well accepted model, introduced by Cervera et. al. also has the requirements as pore wall surface charge and asymmetric pore shape. As shown in Figure 111A, when the anode is on the tip side of the nanopore, cations ar e transported from the tip (anode) to base (cathode). Anions are driven from the external s olution on the base sid e into the nanopore by the electric field. However, they cannot pass through the nanopore tip because of the electrostatic repulsion from the negatively charged pore wall at tip As a result, anions accumulate in the nanopor e. To maintain electrostatic neu trality more cations are needed in this tip region to balance the extra anions. As a result, the local salt concentration in the nanopore increases and the resistance of membrane decreases Therefore, a higher ion current is obtained when a transmembrane pot ential is applied at such electrode polarity We ref er th is situation to the on state (Figure 1 11A). On the contrary, when the anode is placed on the base side of the nanopore, cations are transported from base to tip in the nanopore (Figure 111B). Anions inside the nanopore are vacated from the nanopore and into the bulk solution at the base side by electric field. However, the anions on the tip side are less likely to enter the nanopore because of the strong electrostatic repulsion from the anionic pore wall at the tip. As a result, the local salt concentratio n in the nanopore decreases, and t he resistance of membrane increases. When a transmembrane potential is applied at such electrode polarity a lower ion current is obtained. We re fer th is situa tion to the off state (Figure 11 B) In both of S iwy or Cerveras model, one critical assumption or requirement is that the nanopore tip is cation or anion perm selective. In general, for surface negative ly charged materials, the transference number of the cations is larger than of the anions, in other words, nanopore preferentially transport s cations but reject anions This might raises a problem for

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28 application s of these membranes in separation or biosensing, if the analyte is electrostatic repelled by the charge s on pore wall Electrical Field F ocusing A very important feature of the conical nanopore is that the voltage drop caused by the ion current flowing through the nanopore is focused at the nanopore tip.16, 77 Lee et. al. had simulated the electrical fiel d distribution across a single conical nanopore embedded in a polymer membrane.77 They demonstrated that the majority of the potential drop across the membrane occurs in proximity of the small opening of the pore. In this investigation, even the electrical potential applied across the 6 thick membrane is only 1 V, the elect r ic al field strength a t the nanopore tip is enormously huge (~ 1 106 V/m) (Figure 1 12) .77 In the nanopore, t he electrical f ield strength decreases dramatically at the position with larger cross section. The consequence of electrical field focusing at the tip is that the steady state current is extremely sensitive to any resistance c hange close to the tip. If an analyte passes through the field strength focused region and results a resistance change, such resistance change can be reflected by the ion current change. It is know n that the electrical field strength is not uniformly distributed along the nanopor e (Figure 1 12B). The electric field decays to ~1% of its value at the pore base opening, equivalent to ~1104 V/m. Thus, an appreciable electric field exists far from the pore tip opening. It is only the part in close to the tip is sensitive to the resistance change and that region i s usually called the sensing zone. If we arbitrary setup a criterion that the effective length of sensing zone in the pore is that the length over which 80% of the voltage is dropped, then the sensing zone is only about 50 nm 72 Such short sensing zone provides some advantages of conical nanopore than straight nanopore as a

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29 paradigm sensing devices. For example, a short sensing zone would greatly decrease the possibility that multi analyte enter the zone at the same ti me, therefore make single analyte molecule signaling possible. More important ly such simulations show that t he effective length of the sensing zo ne can be controlled by varying the cone angle of the nanopore. This demonstrates the significance of tailoring the nanopore size and geometry to allow analyte species to be detected. Resist ive P ulse S ensing The resis tive pulse sensing method14, also referred as stochastic sensing, is based on an electrochemical cell in which a small aperture separates two electrolyte solutio ns. A n ionic current is passed through the aperture. When an analyte with dimensions comparable to the diameter of this aperture is driven through the pore by electrical field, the analyte partially occludes the pathway for ionic conduction and the ionic current flowing through t he aperture decrease (Figure 113) Depending on the device, such change in ion current can be used to size identify and determine the concentration of the analyte species. Current research in the field of resistive pulse sensing is aimed at the detection and characterizati on of DNAs, proteins, ions, small organic molecules et.al The developments of nanopore with molecular size in both biological and synthetic membrane make such detection s possible. In particular, a great number of resistive pulse sensing research had been accomplished by using biological nanopores.14, 17, 78 89 It is also important to develop the synthetic nanopore because of their more adjustable size and g eometry and surface properties. ResistiveP ulsing with Biological Nanopore s hemolysin,78 a type of protein nanopore embedded in a lipid bilayer support (Figure 114) .90 The nanopore is formed by either w ild -

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30 type78 or engineered protein .81, 86, 87 The extramembraneous domain contains a large cavity which hosts the transmembrane domain during the assembly pr ocess. Biological nanopore have been used to detect DNA s ,83, 88 metal ions ,80 anions ,82 organic molecules,84, 85 proteins ,89 polymers79 and enantiomers of drug molecules .91 There are two advantages that make biological nanopore very useful. The first one is that the excellent reproducibility of nanopore from sample to sample. The second advantage is that the powerful bioengineering technique allow s such hemolysine pores to be modified through genetic engineering and chemical reaction s introducing a highly selective sensor platform. The large amount of work that has hemolysine has a n important influence on the developm ent of resistive pulse sensing. However, t he planar lipid bilayers cannot endure a wide range of pHs, temperatures, applied transmembrane potentials, or solvents, and they are sensitive to vibrations .78 An alternative way is to using more stable bilayer with enhanced solid support92 or artificial membrane Another limitation of such protein channel s is that their dimensions of cavity allow the translocation of small analytes only at a couple of nanometer scale. Therefore, a significant a mount of research focused on the developing of synthetic nanopores as analogues of biological channels to be resistive pulse sensing device s ResistiveP ulsing S ensing with Synthetic Nanopore s Many techniques have been explored to fabricate single nanopore in solid state materials.93, 94 One widely used method entails using electro n beam21 or ion beam23 to drill a single nanopore in silicon nitride and silicon oxide membranes. While monitoring the fabrication process using transmission electron micro scopy (TEM), the size of such a nanopore could be controlled down to 1 nm. Th ese type s of nanopore s have been used to study mainly DNA,95103 also some protein sensing and analyte interaction s with nanopore s .104 The microfluidic channel fabricated using

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31 soft lithograph has been used to detect cell,105, 106 DNA,107 the binding of antigens to primary antibody coated colloidal particle s,108, 109 and quantitatively distinguish colloidal particles at different size.110 A single carbon nanotube embedded in an epoxy membrane have been used as sensing device for detection of nanoparticles111 113 and DNA translocation through such a device w as observed using fluorescence microscopy.11 4 A femtosecondpulse laser based technique has been used to create single pore in glass and such nanopore is used to examine immune complexes.115117 A feedback electrochemical etching method has been used to fabricate nanopore in silicon.118 In the Martin group, polymer membranes contains single conically shaped nanopore were widely used for resistive pulse detection of different analyte s .16, 42, 45, 48, 78 Other Nanopore Based S ensing Strategies Other sensing strategies have also been used with single conical nanopores fabricated in polymer membranes. A single conical Au nanotube in a PET membrane was used to design a new type of protein biosensor.35 The nanotube was modified with various biochemical molecular recognition agents (MRAs) to detect analytes in soluti on with an on/off response. Similar to the resistive pulse sensing method, this sensing protocol also involves passing an ion current through the single nanotube membrane However, current pulse translocation events were not observed in this case. Instead, as the an alyte bound to the MRAs that had been attached on the pore surface, the current flowing through the nanopore was permanently shut off. Blockage of the ion current occurred because the diameter of the anal yte was of comparable dimension to that of the nanotube tip. This sensor has bee n shown to be a highly sensitive and selective type of biosensor, and it should be possible to modify the Au nanotube surface with a wide range of MRAs to selectively detect a wide variety of analytes. Conical nanopore sensors that use the ion current rect ification phenom enon have also been developed.58, 119 There devices consist ed of a single conical nanopore in polymer membrane and

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32 demonstrate d ion current rectification when nanopore is filled with low concentration electrolyte solution. When the nanopore m embrane was exposed to the analyte solution, the analyte bound on the nanopore surface and changed the local charge condition. An ion current rectification change was observed corresponding to the analyte binding. The quantitatively correlation between the analyte concentration and the ion current rectification ratio was observed.119 Mass Transfer in Asymmetric Nanopores Single asymmetric nanopore in membrane materials were widely studied as a potential sensin g device. The membrane contains multiple nanopore have been studied for filtration and separation applications T here is a increasing interest in the mass transfer in the asymmetric nanopore .32, 120 A widely studied phenomenon is the rectified ion transport in the nanopore32, 67 or nanochannel121123 system These studies demonstrated interesting transport phenomenon for the charged species under the effect of electrical field. It is observed that a larger current flowing through the membrane from one direction and a s maller curren t flowing through the membrane from the opposite direction. This means there is a higher transport rate of charged species from one direc tion and lower transport rate from the other direction. It was also observed that the nanopore geometry had a effect on th e diffusion.69, 120 It has been demonstrated that the diffusion rate of analyte entered from the lar ge opening of the membrane is higher than that of ana lyte entered from the small openings when the size of the analyte is comparable to the size of t he nanopore small opening. Electroosmotic flow (EOF) is an electro kinetic phenomenon that occurs when an ionic current is passed through a channel or porous materials that contain excess surface charge.124132 EOF is used to pump fluids through microfluidic126 and capillary electrophoresis127 columns. In addition, we and others have been exploring EOF through charged membranes that contai n

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33 straight nanopores. We are wondering whether the nanopore geometry has an effect on the electroosmosis in the nanopore. In other words, is that possible to have a rectified electroosmotic flow phenomenon in the asymmetric nanopor es. Dissertation Overview The goal of this research is to explore the fabrication of asymmetric nanopore s in thin film materials, including polymer and crystal materials and the corresponding potential applications in analytical chemistry. In particular, the research has been foc used on the fabrication of pyramidally shaped nanopore s in muscovite mica membrane and the relative studies of resistivepulsing sensing by using this type of nanopore platform. Chapter 1 has reviewed necessary background information for this disse rtation including, track etch method, ion current rectification in asymmetric nanopore resistive pulse sensing by using biological nanopores and artificial nanopores. In Chapter 2, w e prepared a single conical nanopore in PET membrane with good reproducibility. A model polymer analyte, sodium poly(styrene sulfonate) (PSS) was used for resistive pulse method studies. The translocation of PSS through the nanopore were detected and quantitatively measured. The relationship between the transmembrane potential and the resistive pulse frequency was studied. In Chapters 3, the fabrication of asymmetric nanopore s in a crystal mate rial, muscovite mica, was discussed in detail. The effects of etch time, transmembrane potential on nanopore sizes and geomet ry were studied. A chemical vapor deposition method was used to deposit carbon in the nanopore s to produce the nanopore replica s for geometry studies. A highly ion current rectification was observed in these pyramid al shaped nanopore in mica. We think thi s highly ion current rectification is because of the high charge density on mica surface.

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34 In Chapter 4, a new mechanism was proposed to improve the resistive pulse detection signal intensity based on the nanopore ion current rectification characteri stic. A coating molecule was introduced to modulate the nanopore local charge condition. The current pulses were studied based on the interaction of model analyte nanoparticles and such coating reagent. In Chapter 5, w e have shown for the first time that the asymmetric nanopore in mica membrane rectifies EOF. We studied the phenol transport through the membrane from different direction of the membrane. When a constant current were applied during the transport, a higher transport ra te was observed from the base side to the tip side and a lower t ransport rate was observed from the opposite direction. We demonstrated that such rectified EOF is the direct consequence of the ion current rectification in asymmetric nanopore s

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35 Figure 11. Schematic of track etch method for the fabrication of nanopore s in membrane s. A) Irradiation of a thin film with swift, heavy metal ions. B) Formation of latent damage tracks along the path of ions. C) Selectively chemical etch of damage track resulting pore openings across the thin film. Figure 12. Schematic of ion track in different membrane materials A) I n crystal. B) I n polymer.

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36 Figure 13. Diagram of the electrochemical cells for nanopore chemical etch and electrochemical measurem ent. Figure 14. Schematic of track etch and bulketch of a conical pore Bulk etch rate vB, track etch rate, vT

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37 Figure 15. Schematic of anisotropic etch of a single conical nanopore in PET and the resulting c urrenttime trac e. A) Schematic of anisotropic etch. B) Current time trace of single pore anisotropic et ch for 120 minutes. 0 20 40 60 80 100 120 0.0 0.2 0.4 0.6 BCurrent (nA)Time (minute)

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38 Figure 16. Schematic of isotropic etch of a single conical nanopore in PET and resulting current time trace. A) Schematic of isotropic etch. B) Currenttime trace of single pore isotropic etch until final current at 40 nA 0 20 40 60 80 100 120 140 0 10 20 30 40 B Current (nA)Time (minute)

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39 Figure 17. Chemical structure of polymer s typically used for track etching. A) P oly(carbonate) (PC). B) P ol y(ethylene terephthalate) (PET). C) P oly(imide) (Kapton).

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40 Figure 18. SEM images of conical nanopore s in different membrane materials. A) PC73. B) PET. C) Kapton72. D) Glass41.

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41 Figure 19. FESEM images of replicas of nanopore in different membrane materials. A) Gold replica of conical nanopore in PET membrane B) Carbon replica of straight nanopore in mica membrane.

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42 Figure 110. A typical current voltage curve used to determine the single conical nanopore tip opening diameter. Figure 111. Schematic of ion current rectification model Model proposed by Cervera et. al .74 (drawing of pore not to scale). -1.0 -0.5 0.0 0.5 1.0 -10 -5 0 5 10 Current (nA)Voltage (V)

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43 Figure 112. Distribution and line profile of the electric field across a conical nanopore membrane A) Distribution of electric field across a conical shaped pore with base nd membrane thickness 77 B) Line profiles of the electric field strength across the membrane corresponding to the centerline axis of the pore.77

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44 Figure 113. Illu stration of the resistive pulse sensing method. A) S teady state current in the absence of analyte. B) R esistive pul se event in the presence of analyte. Figure 114. Illustration of Hemolysin protein embedded in a lipid bilayer support It formed a nanopore across the support lipid bilayer .90

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45 CHAPTER 2 RE SISTIVE PULSE DETECTION OF A MODEL POLYMER ANALY TE USING A CONICAL NANOPORE IN PET MEMBRANE S Introduction There is increasing interest in using nanopores in synthetic or biological membranes for resistive pulse studies of molecular14, 78, 80, 82 and macromolecular35, 42, 83, 88, 96, 97, 101, 102, 108, 114, 133, 134 analytes The resistive pulse method entails mounting a membrane containing a nanopore that is filled with electrolyte solution and measuring the ion current flowing through the nanopore when applying a transmembrane potential. If a charged analyte is moving into the pore and blocking the na nopore, a transient current pulse signal is usually observed. The current pulse frequency is usually related the analyte concentration and the identity of the analyte is encoded in the currentpulse signature, usually define d as the current pulse amplit ude and current pulse duration.14, 16 A single conically shaped nanopore in PET membrane was fabricated through a two s tep etch method and used for resistive pulse detection studies. The two st ep chemical etch method includes a n anisot r opic etch (the first step) where nanopore large opening (base) size is well controlled and a n isotropic etch (the second step) where the nanopore tip opening size is accurately adjusted. The model analyte used in this study, sodium poly(styrene sulfonate) is a negatively charged linear polyelectrolyte which is s table at different pH solution. We studied the pulse frequency varied with polyelectrolyte concentration. We also investigated the effect of transmembrane potential on pulse frequency and explore a method to lower the limit of detection.

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46 Experimental Materials Poly(ethylene terephthalate) membranes (~3 been irradiated with a heavy ion of 2.2 GeV kinetic energy to create a single damaged track through the film) were obtained from GSI Darmstadt, Germany. Sodium polystyrene sulfonate standard (Mw = 126,700 by gel permeation chromatography and molecular distribution Mw/Mn = 1.17) was obtained from Scientific Polymer Product INC. (Ontario, NY). All other chemic als were reagent grade and used as received. Purified water, obtained by passing house distilled water through a Barnstead, E pure water purification system, was used to prepare all solutions. Fabrication of Single Conical Nanopore in Ion Track PET M embran e The iontracked PET membrane with a single damage track was irradiated under the UV light at wavelength ~320 nm for 15 hours before etch. To modulate the conically shaped nanopore tip size more accurately, we modified the etching pr ocedure described in literature .56 We refer to our method as the two step chemical etch met hod.53 The first step is an anisotropic chemical step and is used to control the size of the conically shaped nanopore large openings (base). The second step is an isotropic chemical etch step which is used to tailor the conical nanopore tip size with accuracy at the nanometer scale. The first step is the same as described by Apel previously.56 A single tracked PET membrane was mounted between two half conductive cell s. A ~3 mL 9 M NaOH (etching solution) was added to one half cell and 1 M formic acid (HCOOH) and 1 M KCl (stopping solution) was added to the other. A Pt wire electrode was placed in each condu ctive cell. A transmembrane potential + 1 V was applied on the electrode placed in the etching solution during the first etch step by using Keithley 6487 picoammeter voltage source. The ion current during the e tch ing was monitored at real time to allow us to collect the information about etch. The

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47 anisotropic chemical etch was terminated at 120 minutes and solutions on both sides of the membrane were replaced with a fresh stopping solution (1 M HCOOH and 1 M KCl). The membrane was then rinsed with d eionized water and immersed in the stopping solution for 15 minutes aft er rinsing The membrane was untouched and kept in the cells and immersed in deionized water for storage. A second step chemical etch was used for fine control the single conical nanopore tip size. At the second step chemical etch, the same etchant 1 M NaO H was placed on both sides of the membrane. A Pt wire electrode was placed in each conductive cell and a potential + 1 V was applied on the anode that was placed in the etching solution. The ion current was monitored at real time and chemical etch was terminated when the desired ion current was achieved. In these studies, the isotropic etch was stopped when the ion current increased to 5.0 nA. The etchant 1 M NaOH was replaced with mixture of 1 M HCOOH and 1 M KCl and kept for 15 minutes. The membrane was then rinsed and immersed in deionized water for 1 hour. Current V oltage Curves M easurement A membrane sample contained only single nanopore was mounted between the two half cells and a 100 mM phosphate buffer solution (pH 7.0) that was also 1 M KCl was placed on both sides of the membrane. A Ag/AgCl electrode was placed in each solution. A current voltage was scanned from 1 V to +1 V with working electrod e at the base side of the membrane The conical nanopore tip size c an be calculated via Equation 2 1, = 4 (2 1) where G is the conductance of nanopore filled with electrolyte solution. The conductivity of supporting e lectrolyt cm1, l is the length of nanopore, dbase and dtip are the diameter of conical nano pore base and tip, respectively.

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48 Characterization of Sodium Poly(styrene sulfonate) Sodium poly(styrene sulfonate) (PSS) standard (Mw = 126,700 and Mw/Mn = 1.17 by gel permeation chromatography) were obtained from Scientific Polymer Products Inc (Ontario, NY). To deter mine the dimensions of PSS in the solution used for resistive pulse measurements, dynamic light scattering (DLS) measuremen ts under identical conditions were performed by using a ZetaPlus particle size analyzer (Brookhaven Ins of of PSS using the same buffer solution. DLS measurements were ta ken for 5 minutes for samples at each concentration. Data were analyzed using ZetaPlus particle sizing software, assuming PSS adopts a wormlike coil under the conditions employed for DLS measurements Resistive P ulse Measurement of PSS A bare single nanopore PET membrane was mounted between the two half electrochemical cell s and both cell s were filled with 100 mM phosphate buffer s olution (100 mM KCl pH.7.0). A constant transmembrane potential was appli ed on the Ag/AgCl electrodes that were inserted in each solution The ionic current flowing thr ough the single nanopore filled with electrolyte was measured with an Axopatch 200B current amplifier (Molecular Devices Corporation, Union City, CA) in the voltage c lamp mode with a low pass B essel filter at 2 kHz bandwidth. The signal was digitized using a Digidata 1233A analogto digital converter (Molecular Devices Corporation). Data were recorded and analyzed with pClamp 9.0 software (Molecular Devices Corporation ). The ion At least 5 minutes data were recorded for each experimental condition. During the analyte translocation experiment, the anode was placed in the solution facing the base open ing of the conical nanopore and the cathode on the other side of the membrane was grounded. The steady state ion current of the buffer solution without analyte was measured for half an hour before

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49 being replaced with pre diluted sodium polystyrene sulfonat e solution that had the same ionic strength as the buffer solution. Results and Discussion Preparation and Characterization of Single Conical Nanopore The first requirement for this research is to prepare a single conical nanop ore with good reproducibility. A two step etch procedure, including anisotropic and isotropic etch, was performed for tailoring the shape of nanopore.53 The single ion tracked PET membrane was anisotropic etched for 120 mins. Because it is hard to locate a single nanoscale pore on the sample of 0.78 cm2 under SEM, so we took the image s of multi pore mem brane and use the mean base diameter for cal culating the si ze of the single pore base opening. Also according to the literature, we use the PET bulk etching rate of 2.170.19 nm min1 t o calculate the base size as a function of etching time.53 The tip size was calculated via Equation 2 1 after measuring the characteristic current voltage (I V) curve (blue curve in Figure 21) in the pH 7.0 100 mM phosphate buffer solution with 1 M KCl. After the anisotropic etching, a conical ly shaped nanopore was obtained with nanopore dbase ~ 521 nm and dtip ~ 0.9 nm. In most of these resistive pulse sensing measurements we would like to fabricate the nanopore with a tip size comparable to the size of the analyte. In this stud y of PSS, a nanopore tip size of 10~ 15 nm was needed. Therefore, isotropic etching was performed and the etching process was monitored via the ion current. The currentvoltage curve (red straight line in Figure 21) after the second step etch (isotropic etch) showed that conical nanopore dtip ~ 13.5 nm with assumed dbase ~ 533 nm. We notice d that the nanopore s show current rectification when the tip size is small, however, the ion current rectification disappear when the tip size gets larger, especially when the tip size is larger than 15 nm.76

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50 Characterization of Sodium Pol y(styrene sulfonate) The hydrodynamic diameter of PSS in 100 mM pH 7.0 phosphate buffer with 100 mM KCl was measured by using dynamic light scattering (DLS) Because there is a requirement for sample volume fraction in DLS measurement, we only measured PSS resistive pulse measurement (shown in Figure 22A). It is reported that the gyration radius of polyelectrolyte changes with the polymer concentration and ionic strength of the solution.135, 136 We assume that the gyration radius follow s the relationship shown in Equation 22 which is applied in semi dilute region.135 We can predict the approximate dimensio n of PSS in our experiment. 1 8 (2 2) where Rg is the radius of gyration and C is the co nc entration of polyelectrolyte. In Figure 2 2B, w e plot the radius of gyration vs. the PSS concentration C1/8, a relationship between these two parameter is obtained. From this linear fit relation, the gyration radius at very low PSS concentration could als o be calculated, for example, the Rg of PSS at 10 nM i s about 21.2 nm. Even though the average gyration radius of PSS is about twice of the nanopore tip size (13.3 nm) the polymer can still adopt different conformations in the solution to enter the nanopore under the effect of electrical field. By combing the light scattering, zeta potential measurement s we can approximate the mobility of these polyelectrolytes in salt solution. The mobility and zeta potential are also obtained from the light scatte 2.09104 2 V1s1), and the negative sign means that the PSS molecules are negative ly charged.

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51 ResistiveP ulse Measurement of P oly(styrene sulfonate) In the resistive pulse measurement of poly( st yrene sulfonate) we choose the nanopore tip size of 14 nm. The electrochemical cell s were setup in similar to the etching setup and Ag/AgCl electrodes were used. Before measuring the PSS transport, the buffer solution in the absence of PSS (100 mM phospha te pH 7.0 with 100 mM KCl) was added to both sides of the membrane and the background ste ady state current was obtained. Figure 23A shows t he background current a smooth current in absence of any resistive pulse events. I n the follow ing resistive pulse measurement, we always applied positive potential on the base side of the membrane (permeation side) to electrophoretically drive n egative charged PSS from tip to base in the nan opore. It was clearly observed that current pulses occurred when the buffer so lution at the cathode side was replaced with the solution containing 1 nM or 10 nM PSS (Figure 2 3BC). It is also demonstrated here that the pulse frequenc y increas ed when PSS concentration was higher in the solution To confirm tha t the resistive pulse ev ents were caused by the migration of negative ly charged PSS, we switched the polarity of transmembr ane potential. When the anode was on the permeation side, we observe d the pulse events. We then hypothesize d that i f the anode was on the feed side, the nega tively charged PSS would migrate far away from the nanopore tip and no pulse events w ould be observed. The experiment results supported our hypothesis (Figure 2 3D). Effect of supporting electrolyte solution pH We also investigated th e effect of solution pH value on the detection of PSS. In Figure 24, we demonstrate the currenttime trace when PSS passed through the nanopore with solution pH at 3.0 and pH 7.0, respectively. It was easy for us to observe regular pulse events in the solution at pH 7.0. However irregular events were observed and pulses overlapped and baseline current fluctuated when the solution was at pH 3.0.

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52 These results suggest that the pol yelectrolyte might interact with or adsorb on the nanopore when the surface carboxylate group i s protonated and the possible adsorption cause the longer events duration time and baseline current fluctuation. On the contrary, the PSS polymer might have less interaction with the pore wall and migrate faster because the electrostatic repulsion between the negative charged surface and PSS reduce the adsorption. So we think that a supporting electrolyte solution at pH 7.0 is preferred for resistive pulse measurement of PSS if considering optimizing the signal to noise ratio. Detection limit for resistive pulse measuring PSS Different from the definition of detection limit in spectrometry, we nee d to define our criteria in the resistive pulse measurement. There is no loss of s ignal/ noise ratio even at the low concentration as long as the analyte size is com parable to the nanopore tip size. Theoretical, it is supposed to have single molecule detection capability for resistive pulse system if we can wait for long enough time to get the resistive pulse events corresponding to that single molecule translocation. Practical ly however, we define the detection limit at which at least one resistive pulse current blockade can be observed in one minute time window This time window scale combines the needs for low concentration analyte detection, time efficiency, and t he co st. We studied the PSS resistivepulse measuring in a large dy namic range, which is PSS concentration changed from 10 pM to 10 nM. Effect of analyte concentration on pulse frequency From Figure 23, we can easily know that the number of resistive puls e events should increase if we raise the conce ntration of PSS. As Equation 23 shown, the electrophoretical flux Ji(x) is proportional to the concentration of analyte (Ci),137

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53 ( ) = ( ) (2 3) where F is faradic constant (96485.3 C mol1) R is the gas constant, Di is the diffusion coefficien t of species i, T is the tempe rature and (x)/ x is the electrical field strength at point x. If we multiply the Avogadros number N0 (6.021023) and area of nanopore tip and time scale we could know the relationship between the number of events (Npulse) in certain time window (t) with analyte concentration, = ( ) 0 2 (2 4) where Rtip is the radius of conical nanopore tip opening. Figure 25 shows that the resistive pulse frequency increased with PSS concentration. At lower concentration range in our experiments (10 pM to 1 nM), the pulse frequency was almost proportional to the PSS concentration, but at the 10 nM concentra tion of PSS, the relationship was not linear. We think such a nonlinear relationship might be because the increased analyte interactions outside of the tip at high polyelectrolyte concentration affect the detection. It seems that we could predict the number of resistiv e pulse events from Equation 24, but it is really hard to make any prediction accurately because of the uncertainty of several parameters in the equation, such as the effective charge, real electric field strength at the tip. Effect of transmembrane potential on pulse frequency Besides the concentration dependen ce, we also investigated the effect of transmembrane potential on resistive pulse event frequency. The PSS polyelectrolyte is supposed to be negatively charged in the pH 7.0 buffer solution so that the polyanions should migrate in diffe rent direction s if t r ansmembrane potential polarity is switched. If the anode was placed at the permeate solution side, the polyanions were supposed to migrate toward s the nanopore membrane and generat e resistive pulse events. On the contrary,

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54 the polyanions were driven far a way from the nanopore and no events would be observed if the anode was placed in the feed solution. As predicted from Equation 24, we should observe higher resistive pulse events frequency when the transmembrane potential increases. However, we d id not observe a linear relationship between the pulse frequency and electrical field stren gth as described in Equation 2 4. Instead, we found that t he resistive pulse frequency had an exponential growth relationship with transmembrane potential (as shown in Figure 26). Actually, the frequency of current blockade might not be proportional to the electric field strength. Because the hydrodynamic dia meter of PSS in dilute solution was larger than the tip size of nanop ore, the polymer chains need activation energy to overcome an activation barrier In other words, they must pay an entropy penalty to reptate into the nanopore. It was suggested that the DNA threads through the pore rather than going though as a random coil.134 There was literature repo rted that the elongation of these polyions conformation occurs under the electric field.138 so that it is possible for PSS to change conformations and reptate into the nanopore. Similar to the tra nsport of DNA into an nanopore, the current blockade rate R is described by Vant hoff Arrhenius or transition state relation.134 = exp [ ( ) / ] (2 5) where is a probability fac is the activa tion energy or barrier height. is the reduction in the energy barrier due to the applied transmembrane potential, k is the molar gas constant and T is the absolute temperature. The higher transmembrane potential leads to higher energy, = | | (2 6) where z is the effective total number of elemen tary charges on the polymer and e is the

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55 magnitude of the elementary charge. The rate of blockade in a vanishing applied potential (E) is 0= exp ( / ) (2 7) Then it gives the equation for blockade rate at different transmembrane potential = 0 exp ( | | / ) (2 8) and R0 is independent of E It is expected to observe the exponential growth of current blockade rate with the transmembrane potential. We can expect to have more resistive pulse events at th e same concentration of analyte if applying a high potential. W e think w e might be able to find a way to improve the detection limit further. The higher electric field strength which speeds up the mass transfer can lead to the higher migration flux and increase the detection efficiency as long as th e mass transfer is fast enou gh.81 In summary, the polarity and magnitude of transmem brane potential have important effect s on the PSS translocation through the nanopore. The polarity c ould change the direction of polyelectrolyte migration The magnitude of el ectric field strength will affect different transport flux and provide different energy for polymers to translocate through the nanopore. Thus it might be a way to improve the detection limit by applying higher transmembrane potential, which is a n important advantage of using synthetic membrane th an biological nanopore system. Transloc ation time and magnitude of current blockade In the resistive pulsing, the translocation events duration time and amplitude of current blockade are used to identi fy the analyte DNA .42 We also studied these two parameters in the experimen t related with polyelectrolyte electromigrat ion. In this study, as an analogy to data analysis in spectrometry, we defined a current pulse as one detection event only when the am plitude of that current pulse was twice of the baseline noise. For example, the background noise of 14 nm tip conical nanopore filled w ith 100 mM

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56 phosphate buffer and 100 mM KCl at applied potential 1 V was about 30 pA. In this case, only t he current pulse amplitude equal to or larger than 60 pA was counted by the software as a pulse event. As we know, the background current decrease if the applied potential decrease. In su ch case, the signals were only counted as events when the signal/noise ratio at 2:1, but not base on the absolute value of 60 pA. It is also known the values of ion current background and am plitude of curren t pulse depend on the tip size of nanopore.53 So it is hard to compare the amplitude of pulse me asured from the nanopore at different size s In this study, the normalized current pulse amplitude ( I%) and average background current value (I0), as shown, % = 0 100% (2 9) Figure 27 shows the histogram of normalized PSS current pulse amplitude measured with 14 nm tip size conical nanopore membrane. It was observed that most of the current pulse amplitude is less than 15% of the background current value. Compared t o the 90% current block when a single strand DNA passed throug Hemolysin channel, the amplitude of current pulse in this synthetic conical nanopore embedded in PET membrane is really small. The other parameter, current pulse duration, is also importa nt because it might include the information about the sp eed of analyte migrate in the pore, interactions between of analyte and pore wall or binding affinity. It seems that the duration of PSS translocate the nanopore sensing zone is really short (Fig ure 28). Most of the events are shorter than 40 ms, which might mean the PSS has little interaction with nanopore and pass through the sensing zone quickly. This might be an advantage for detection of ana lyte at high concentration. It is possible that pulse si gnal will overlap at high pulse frequency if duration is too long. But the duration of PSS at

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57 molecu le weight Mw = 127,000 still had a broad distribution, from several milliseconds to one hundred milliseconds. Scatter plots of current pulse amplitude ( I) versus current pulse duration ( ) were usually used to summarize resistive pulse data It was shown that the current pulses had large distribution for both pulse amplitude and duration. The variability of the current pulse s might be be cause the polyelectro lyte can adopt different conformations and thus different pulse signals. Conclusions We prepare d a single conical nanopore in PET memb rane with good reproducibility. PET membranes contained conical nanopore at ~ 530 nm base and 14 nm tip was used for the resistive pulse studies of polyelectrolyte translocation. Experimental conditions for resistive pulse se nsing of a prototype linear polymer analyte were investigated Resistive pulse events of PS S translocation were observed at a large ran ge of PSS concent ration s It was observed that the current pulse frequency increased with polyelectr olyte concentration in this range. The lowest detection limit for a one minute measurement was 1 0 pM of polyelectrolyte in the solution with transmembrane potential at 1 V. It might be able to improve the limit of detection if apply much higher voltage as long as the nanopore membrane is stable. However, the current pulse signature of this polyelectrolyte model analyte had a very broad distribution for both the pulse amplitude and pulse duration. It is very difficult to distinguish the same polyelectrolyte but at different average molecule weight. It is also important to improve the selectivity of such experiment in the future instigation.

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58 Figure 21. Current voltage curves of a single conical nanopore in PET membrane (B lue) IV curve after the first step etch. (R ed) IV curve after the second step etch. Measured in pH 7.0 100 mM phosphate buffer with 1 M KCl. Figure 22. Distribution of hydrodynamic diameter of PSS at different concentration s and the relationship between the gyration radius and polyelectrolyte concentration It was measured using dynamic light scattering. A) Distribution of PSS at different concentrations. The curves are Gaussian fit data. B) Relation ship between gyra tion radiuses vs. PSS concentration. -1.0 -0.5 0.0 0.5 1.0 -4 -2 0 2 4 Current (nA)Voltage (V) 0 5 10 15 20 0 20 40 60 80 100 ANumberDiameter (nm) 3.0 3.5 4.0 4.5 5.0 5.5 6.0 2.5 5.0 7.5 10.0 B C-1/8 (M-1/8)Radius of gyration Rg(nm)

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59 Figure 23. Current time transients of resistive pulse measuri ng PSS at different concentration s A) Steady stat e current of buffer solution (100 mM KCl in 100 mM PBS pH 7.0). B ) Resistivepulse events w ith PSS concentration at 1 nM. C) Resistive pulse events with PSS concentration at 10 nM. D ) Steady state current of 10 nM PSS solution when anode is on the feed side facing tip opening of the nanopore

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60 Figure 24. Current time transients of resistive pulse measuring PSS at different pH value A) pH 3.0. B) pH 7.0. PSS concentration if 10 nM.

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61 Figure 25. Relationship between the number of resistive pul se eve nts and PSS concentration. It was measured with transmembrane potential at 1 000 m V. Figure 26. Relationship between the number of resistive pulse events an d transmembrane potential. It was measured using P SS concentration of 10 nM feed solution. 1 10 100 1000 10000 0 100 200 300 400 500 600 551.6 101 10 1.2Number of Events / minuteConcentration of PSS (pM) 0 200 400 600 800 1000 0 100 200 300 400 500 600 700 Number of Events / MinutePotential (mV)

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62 Figure 27. Histogr am of normalized current pulse amplitude of resistive pulse measuring PSS translocation. Nano pore tip diameter = 14 nm. PSS concentration = 10 nM. Applied transmembrane potential = 1000 mV. Figure 28. Histogram Nanopore tip diameter = 14 nm. PSS concentration = 10 nM. Applied transmembrane potential = 1000 mV. 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0 100 200 300 400 500 600 700 Number of EventsMagnitude (normalized) 0 20 40 60 80 0 500 1000 1500 2000 2500 Number of Events

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63 Figure 29. Scatter plot of normalized current pulse amplitude Nanopore tip diameter = 14 nm. PSS concentration = 10 nM. Applied transmembrane potential = 1000 mV. 0 50 100 150 200 250 0.00 0.05 0.10 0.15 Magnitude (normalized)

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64 CHAPTER 3 FABRICATION OF PYRAM IDALLY SHAPED NANOPORES IN MUSCOVITE MICA MEMBRANE S Introduction There is an increasing interest in developing asymmetrical ly shaped nanopores16, 28, 32, 45, 47, 50, 53, 57, 61, 69, 70, 119, 139, 140 in synthetic membrane s using the track etch method for resistive pulse48, 133 or ion current rectification studies. However, most of these asymmet rically shaped nanopores were fabricated in polymer membrane s .53, 58, 62, 67, 119 There is also one example to prepare such conically shaped nanopore s in inorganic materials, but it is in amorphous silica thin films.24, 92The Martin group has started exploring the fabrication of asymmetric nanopore in crystal line thin film muscovite mica.69 However, the fabrication proc ess still has less control over the nanopore size and geometry, making nanopores developed in mica membranes less suitable for resistive pulse sensing studies. In this work, we developed a multi cycle anisotropic chemical etch method to prepare asymmet ric ally shaped nanopores in iontrack muscovite mica membranes. The effect of etchant concentration, transmembrane potential and etching cycle number on nanopore size and geometry were investigated in detail The nanopore dimensions and geometry in multipore mica were confirmed by SEM images of bare nanopore membrane s and carbon replica s of the nanopores. The electrochemical measurement and calculation of the size of a single nanopore in mica were discussed as well. The ion transport properties through a single asymmetric nanopore in mica membrane were also studied The ionic current was highly rectified in low ionic strength electrolyte solution. We think this very strong ion current rectification is due to the highly negatively charged surface. To decrease the surface charg e density, the nanopore wall can be modified with an uncha rged

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65 surface functional group. This leads to much less ion current rectification in the asymmetric nanopore. Experimental Materials Muscovite mica (KAl2(AlSi3)O10( F, OH)2) membranes, ~10.5 m thick and ~3 cm in diameter, were obtained from Spruce Pine Co (Spruce Pine, NC). The linear accelerator at GSI (Darmstadt, Germany) was used to ion track the mica; Pb2+ ions of ~8.6 MeV kinetic energy were used. Samples with a single d amage track, and with 105 and 107 tracks per cm2, were prepared. Hydrofluoric acid (~50%) was obtained from Acros Organics USA (Morris Plains, NJ). Glycidoxypropyltrimethoxysilane (GPTMS) was purchased from Gelest (Morrisville, PA). Toluene (anhydrous, 99. 8%), ethanol (anhydrous, 99.5%), mercaptoethanol ( 99.0%) and phenol were obtained from Sigma Aldrich (St. Louis, MO). The ethylene/helium (30% ethylene) mixture was purchased from Praxair (Danbury, CT). All other chemic als were reagent grade and used as received. Purified water, obtained by passing house distilled water through a Barnstead, E pure water purification system, was used to prepare all solutions. Asymmetric Nanopore F abrication and Characterizations Fabrication of asymmetric nanopore in mica A caliper was used to accurately measure the diameter of the membranes, and the membranes were then weighed with an analytical balance. The thickness of each membrane was calculated from the mass and the density of the mica (2.831 g cm3). An average value of A multicycle chemical etching method was developed to fabricate pyramidally shaped nanopores in the ion tracked muscovite mica membranes. A ~1 cm diameter hole was cut into a ~4x4 cm piece of parafilm (Alcan Inc. Neenah, WI), and the mica membrane was sandwiched

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66 between two such films. The sandwiched membrane was placed between the two halves of the etching cell53 described previ ously. To check that there were no leaks in the parafilm seals, the half cells were filled with 1 M KCl that was 100 mM in phosphate buffer, pH = 7.0. A Ag/AgCl reference electrode was placed in to each half cell solution, and a Keithley 6487 picoammeter/voltage source (Keithley Instruments, Inc., Cleveland, Ohio) was used to apply 1 V across the membrane and measure the resulting trans membrane current. If there were no leaks a negligible curren t (<10 pA) was obtained. This is because the ionic conductivity of the unetched damage track is negligibly small. After the leak check, the half cells were emptied, rinsed, and ~3 mL of 10 M NaOH solution was pipetted into one of the cells. The membrane was exposed to this solution for 5 minutes, a Pt wire electrode was inserted into each half cell, and ~3 mL of 10 M HF was then added to the other half cell. The Kei thley was used to apply a trans membrane voltage of 10 V (with the anode in the HF solution) and measure the resulting ionic current through the membrane during etching. Because the ionic conductivity of the unetched track is essentially zero, no current flowed through the membrane a t short time s However, HF etched the mica along the damage tracks t o create the pores, and ultimately the pores broke through to the NaOH solution on the opposite side o f the membrane. This c aused a rapid rise in the trans membrane current, with the current being carried by the nascent electrolyte filled pores. Because NaO H does not etch mica at room temperature, but instead neutralizes the HF etchant that makes its way through t he pore, a n asymmetrically shaped pore was obtained with the large diameter (base) opening facing the HF solution and the small diameter (tip) open ing facing the NaOH solution.

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67 After 10 minutes the etch was terminated by placing 1 M NaOH on both sides of the membrane and applying 10 V for 5 minutes, to ensure complete neutralization of the HF in the pores. The membrane was then rinsed with water, and water was placed in both half cells for 30 minutes to wash the NaOH from the pores. We call this procedure a 10 minute etch cycle. Membranes were etched using between one and eight of such 10minute etch cycles. Scanning electron microscopy For the multitrack mica membranes, field emission scanning electron microscopy (FE SEM, JEOL 6335F) was used to measure the dimensions of the base and tip openings of the asymmetric pores. To obtain images of the tips, the mica membrane was applied with the base side d own to a piece of double side copper tape, which was then attached to a SEM sample stub. Images of the base openings were obtained by mounting the sample with the tip side down. Another method we have developed to image pores in multipore membrane entails depositing a material such as metal62 or carbon29 within the pores. This creates replicas of the pores, which can be liberated by dissolution of the template membrane and imaged. The replicas in this case were composed of graphitic carbon, deposited within the pores by a CVD method .29 The etched mica membrane was placed ver tically into a quartz tube (diameter ~4.5 cm, length ~48 cm) with the nanopore base opening facing the gas flow. The quartz tube was then inserted into a hightemperature tube furnace (Thermolyne 21100, Aldrich) and heated to 670 C under argon flow. When the temperature stabilized, the argon gas was replaced with a 30% ethylene/helium mixture at a flow rate of 20.0 sccm (standard cubic cm per min). After 5.5 hours, the flow was changed back to argon, the furnace heater was turned off, and the tube was allo wed to cool to room temperature. The asymmetric shaped carbon replicas deposited within the pores were then exposed by dissolution of the mica template. This was accomplished by first using a methanol wetted cotton

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68 swab to remove the carbon surface film co vering the tip side of the mica. The base side was then attached to a piece of double side copper tape, which was attached to the bottom of a small (3 cm) plastic Petri dish. Several drops of 50% HF solution were pipetted onto the center portion of the tip side surface. The Petri dish was then covered with parafilm and allowed to sit for 8 hours to dissolve the portion of the mica that was covered with the HF solution. The parafilm was then removed and the HF solution allowed to evaporating to dryness. Seve ral drops of deionized water were then applied to the same spot on the surface, and this was allowed to evaporate. The sample was then mounted on a SEM stem and sputtered with Au/Pd using a cold sputter instrument (Desk II, Denton Vacuum). Equivalent base and tip diameters Both the base and tip openings in tracketched mica are rhomboidal in shape.141 As a result the lengths of both the long (al) and short (as) axi s of the rhomboid are required to describe the size of these openings (Figure 3 1). To simpl if y this situation, we define here an equivalent circular opening141 which is a circle with t he same area as the rhomboidal opening. The area of a rhomboidal opening (Arhomboid) and of a circular opening (Acircle) can be calculated using Equation 3 1 and Equation 3 2, respectively. =1 2 (3 1) =1 4 2 (3 2) To obtain the circular area equivalent to the rhomboidal area, we set Equation 3 1 equal to Equation 32 and solve for the diamet er of the equivalent circle, dEq. = 2 (3 3)

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69 This equation can be simplified by noting that the minor angle of a rhomboidal pore in mica is approximately 60.71 Simple geometry shows that for such a pore the al is equal to 1.732 as. This allows us to write = 0 .606 (3 4) The al values were ob tained from the electron micrographs of the multipore mica membranes, and Equation 34 was used to obtain the corresponding equivalent diameters. Both of the equivalent base diameter, dEq b, and the equivalent tip diameter, dEq t, were determined this way for the multipore mica. Equivalent base and tip diameters for the single pore mica membranes While the base and tip openings of nanopores in the multipore mica membranes could be easily imaged with FESEM, it is much more difficult to obtain such images for single nanopore membranes. This is especially true of the tip openings because they are so small and there is only one per sample. As will be discussed below, for the multipore mica membranes we have obtained experimental plots of equivalent base diameter dEq b, vs. etch time. The slope of such plots provides the etch rate. As per our prior work,53 dEq b for the single pore was calculated from the etch time and such an experimental etch rate. The equivalent diameter of the tip opening, dEq t, was then measured using an electrochemical method described in detail previously.32, 53 Briefly, the mica membrane containing a single asymmetric nanopore was mounted between the two half cells, and an electrolyte solution of measured conductivity was placed on either side of the membrane. In this case, the electrolyte solution was 1 M KCl that was also 100 mM in phosphate buffer at pH 7.0; the measured conductivity of this solution was ~11.8 Sm1. A

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70 Ag/AgCl electrode was placed into ea ch half cell solution, and a current voltage curve was obtained by scanning the potential from 1.05 V to +1.05 V at a scan rate of 1 V min1. For single asymmetrically shaped nanopore such current voltage curves are typically nonlinear.57 However, the portion between about +200 mV can be approximated by a straight line, and the slope of this line provides the ionic conduc tance of the electrolytefilled pore, G. G is related to dEq t, dEq b,, and the membrane thickness, L, via = 4 (3 5) The dEq t, values for the single nanopores were calculated from Equation 35. Electroosmotic flow mea surement As will be discussed below, both the nanopore base and tip size increased with etch time We wonder whether there is electroosmotic flow (EOF) in the nanopore to carry the etchant HF molecule towards the tip region during the chemical etch, an EOF experiment was performed under the similar ionic strength conditions as we etched the nanopore EOF was investigated by measuring the flux of a probe molecule (phenol) across the membrane. A mica membrane containing asymmetric nanopores (pore density ~108 cm2) w as mounted between the two half cells. A 3 mL permeate solution that was 1 M phosphoric acid was added to the cell at the tip side of the membrane. A 3 mL feed solution that was 1 M phosphoric acid containing 10 mM phenol was added to the other cel l. A Pt wire electrode was placed into each half cell. A Solartron SI 1287 electrochemical interface (Farnborough, UK) was used to apply a constant current (5.0 mA, 10.0 mA or 15.0 mA) flowing through the multipore m ica membrane. Such current is responsib le for the EOF in the nanopores. The Pt wire in the feed solution will be used as working electrode and the Pt wire in the permeate solution will be used as reference and

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71 counter electrode. The faradic reactions occurring at the Pt electrodes that support the ionic current in the nanopore were mainly the reduction and oxidation of water. After 20 minutes, the applied current was terminated and both the permeate solution and feed solution were pipetted out of the half cells. An Agilent 8453 UV visible spectr ometry system (Waldbronn, Germany) was used to measure the UV absorbance of phenol in the permeate solution. The concentration of phenol in permeate solution was calculated from the calibration curve measured at wavelength 2 70 nm.142 After the UV visible spectrum measurement, the permeate solution was placed back into the same half cell for next 20 minute cycle translocation. A new feed solution with the same concentratio n of phenol was placed at the feed side for next cycle translocation. The total translocation time is 120 minutes and UV visible spectrum of permeate solution was measured after each 20 minute translocation cycle. The diffusion of phenol across the membrane without applying current was also measured at the same time interval s Acid base neutralization in nanopores As will be discussed below, to study the ionic current decay we observed d uring the mica etch w e designed an acidbase neutralization experiment to mimic the process that might occur the same way during mica etches In this study, to keep the HF from continuing etching and increasing the nanopore size, we replaced the HF with another weak acid acetic acid (HAc). A multipore (pore density ~105 cm2) mica membrane that etched for one 10minute cycle was used a nd mounted between the two half cells. In this experiment, a Pt wire electrode was placed on each half cell. A NaOH solution was first put into the cell facing the tip side of the membrane and a HAc solution was added into the other cell The Kei thley was used to apply a trans membrane voltage of 10 V (with the anode in the HAc solution) and measure the resulting ion current flowing through the membrane for 10 minutes. Different concentration (1 M, 2.5 M,

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72 5 M, 10 M) of NaOH were used in a series of experiments and the corresponding ionic current was recorded. Nanopore Surface Modification Nonlinear current voltage curves are typically observed for conical ly shaped nanopore s that have tip diameter s in one nm to tens of nm range.31, 57 This nonlinearity is an ion current rectification phenomenon, and has been discussed in detail in the literature .24, 56, 57, 74, 143 In order to see rectification, the pore wall must have excess su rface charge, and the extent of rectification is affected by the ionic strength of the solution and the size of the nanopore tip.57 A s trong in current rectification is observed when the nanopore surface charge density is high, the tip diameter is small, and the ionic strength is low. As will be discussed below, the asymmetric mica nanopores investigated here show greater ioncurrent rectification than the conical pores in polymeric mem branes investigated by us and others To confirm that the excess surface charge present on the muscovite mica141 is responsible for rectification, we reacted silanol sites on the mica surface144 with a neutral siloxane reagent to decrease the surface charge (Figure 3 2). The chemistry is analogous to that used to att ach reagents to silicon surfaces.145, 146 The single asymmetric nanopore mica membrane was first cleaned by immersion in a highly acidic piranha solution (3:1 v/v of H2SO4/H2O2) for 1 hour. The mica membrane was then rinsed thoroughly with deionized water and stored in water for 1 hour followed by drying under a stream of nitrogen. The mica was then immersed for 30 minutes into 10% (v/v) glycidoxypropyltrimethoxysilane (GPTMS) dissolved in an hydrous toluene. This was done in a polyarylic box under nitrogen flow to limit GPTMS hydrolysis by atmospheric water. The membrane was then immersed into three portions of anhydrous toluene for five minutes for each

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73 portion. It was then immersed into thre e portions of anhydrous ethanol for five minutes for each portion, and dried in air for 12 hours. The surface epoxide terminal groups of the attached GPTMS were then converted to hydroxyl groups by r mercaptoethanol (Figure 3 2) .146 This was accomplished by mercaptoethanol dissolved in pH 7.4 phosphate buffered silane for 3 hours. The mica was then rinsed with deionized water and dried under nitrogen. Current voltage curves were obtained for these hydroxyl modified mica membranes using the same conductivity cell and Ag/AgCl electrodes as described above. However, a lower ion ic strength electrolyte was used 10 mM phosphate buffer (pH 7.0) that was also 10 mM in KCl. Results and Discussion Fabrication of Nanopore with Ion Track Method Nanopore breakthrough in ion track mica We began our studies with multi track mica membranes (105 tracks cm2) because it is easier to image the resulting pores in such multi track membranes. Figure 33 shows plots of ion current flowing through the membrane during the first 10minute etch cycle as a function of etch time for three identical mica membranes. Because the ionic conductivity of the unetched track is essentially zero, no current flowed through the membrane a t short time s However, HF etched the mica along the track, and ulti mately the resulting pores broke through to the NaOH stopping solution on the opposite of the membrane. This was signaled by the sudden jump in current observed at about 1 minute. When a lower concentration of etchant HF was used for etching, it took a longer time for nanopore to breakthrough.

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74 Current time traces during anisotropic chemical etch As shown in Figure 33, after the current jump signaling nanopore breakthrough, the current initially decayed and then slowly increased with fur ther etching. We think that such current change was related with the acid base neutralization during the etch process. We hypothesize that this acid base n eutralization will cause a trans membrane acid concentration gradient, which is cr itical for fabrication of asymmetrically shaped nanopore in mica. To investigate these questions, we designed two e xperiments to help us explain what is happening during the anisotropic chemical etching. The first one was to study whether there was EOF in the nanopore in the presence of applied current. The second one was to use HAc (weak acid) and NaOH (strong base) and their neutralization to mimic the reaction process between HF and NaOH in the nanopore during etch. Base d on the results from these two experiments, we hope to develop a model of HF transport a nd its effect on nanopore etching Electroosmotic flow in nanopores As we will discuss below, it is observed that the asymmetric nan opore tip opening size increased with the number of etch cycles. So we believe that the HF was always transported to the tip region during the whole process even though the concentration of HF might be very low. We wonder if there is any other flow, such as electroosmotic flow (EOF) to c arry the neutral molecule HF towards the nanopore tip during etch. In order to investigate whether there is electroosmotic flow (EOF) to carry the etchant HF molecule towards the tip region during the chemical etch, an EOF experiment was performed under the similar conditions. EOF was investigated by measuring the flux of a probe molecule (phenol, pKa =9.99) across the membrane (Miller and Martin 2002) in acid solution. The buffer used in such an investigation was 1 M phosphoric acid at pH ~1.08. Therefore, phenol was

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75 neutral at this pH value and its translocation through the nanopore was only carried by diffusion and EOF. During the che mical etch of mica, the anode was at the base side of the membrane and the cathode was at the tip side of the membrane. So HF was carried from base (anode) side towar ds the tip (cathode) if there was EOF during the chemical etch. In the EOF study, the electrolyte solution was 1 M phosphoric acid. Although it was different from the solution we used during nanopore etching (10 M HF). However the calculation shows that cation and anion concentration are almost similar to those in the 10 M HF. In 10 M HF, the cation H+ and anion Fconcentration are about 0.084 M (Ka = 7.1104 molL1). In 1 M H3PO4, the cation H+ and anion H2PO4 are about 0.083 M (Ka1 = 6.9103 molL1). Therefore, t he pH of these two solutions were around 1.08 so that the nanopore surface charge condition were similar in both cases. Therefor e, if we can prove that there was EOF from base to tip in the phenol experiment, then we might be able to conclude that there was also EOF carrying HF in the nanopore when we etched the mica. Figure 34 shows the concentration of phenol in the permeate solution under different transport conditions. It was observed that the phe nol transport rate is much higher in the presence of constant current than it is with no applied cu rrent. We think that the enhanced phenol flux was because the elec troosmotic flow pumps the solvent and phenol to the permeate solution. Therefore, it is rea sonable for us to assume that there was also EOF that pump ed the HF towards nanopore tip even at a low flow rate, resu lting in a constant etch rate at the nanopore tip. Acid and base neutralization in the nanopores We designed another experiment to mimic the acid base neutralization during nanopore chemical etch. In this investigation, to av oid the nanopore size increasing because of HF etching, HF was replaced with another weak acid acetic acid (HAc). We assumed that the mass transfer and neutralization of HAc with NaOH are similar to that of HF. Figure 3 5A shows plots of ion

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76 current flowing through the membrane during acidbase neutral ization as a function of time for different NaOH concentrations. It was observed that the c urrent time traces show a simi lar trend to that we observe d during HF etching mica, which was that the currents start ed to decrease after the acid and base contact. It is clear that the ion current flowing through the membrane is larger when the concentration of NaOH is higher. This is because high concentration NaOH consumes HF faster so that HF dissociates faster to p rovide ions to support the ion current. Since we know that t here was electroosmosis pump ing the HF from base to tip and etch ing the tip all the time, so there was HF always in the nanopore during the chemical etch. In this similar experiment, it is assumed that electroosmosis will pump HAc to the tip and HAc will f ill the nanopore. Because NaOH and HAc do not coexist, so we can assume that NaOH did not actually enter the na nopore since HAc always existed even though the concentration is lower than that of the bulk solution. Driv en by the electric field, OHmoved fr om the bulk towards nanopore tip and HAc was pumped out of the nanopore to the external solution on the tip side. We hypothesize that there was a region right outside the nano pore tip where NaOH and HAc contact ed. In this region, the base OHand acid HAc neutralized each other. We refer this region as a neutralization regi on. As shown in Figure 35B, OHwas driven to this region and react ed with HAc. The concentration of both OHand HAc dropped to zero somewhere in the neutralization region. This caused the concentration of HAc at the tip also dropped to a certain value which was higher than zero but lower than the concentration of HAc on the base side. As a result, a concentration gradient of HAc was established in the nanopore. The concentration of HAc was lower at the tip and high at the base. Analogously, we can assume there was also a concentration gradient of HF in the nanopore during anisotropic etch ing of mica The concentration of HF was lo wer at the tip and it was higher at the base. As a resul t of the HF

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77 transmembrane concent ration gradient, the etch rate of mica was lower at the tip opening and higher at the base opening. Therefore, asymmetric ally shape d nanopores were obtained in muscovite mica membrane. Track etch rate of mica The current time transient observed after nanopore breakthrough for the multi track mica membrane show ed a sudden increase If the b reakthrough of ion tracks occurred one after another, ion current would increase gradually, which was usually observed when multi track PET membranes w ere etched in aqueous solution. But if all the ion tracks break through at the similar time, then a very large current would rise rapidly because there were lots of pores that could support the current at the same moment. It was observed tha t the ion current flowing though the mica membrane was nearly zero in the first minute and dramatically increased to a peak value within a few seconds (Figure 33). We believe that this rapid rise in current was due to all the ion tra cks on the membrane be ing etched at a similar track etching rate and breaking through at almost the same time. It was also observed that it took a comparable amount of time (55 to 60 seconds) for ion tracks to break through in different mica membrane s of the same thickness tha t etched in the same way. In other words, good tracketching rate reproducibility was observed from different mica membranes and the average track etching rate is about 18110 nm sec1 using 10 M HF. It was observed that in the experiment of NaOH and HAc, the ion current approached a constant value at the end of neutralization. This is because the mass transfer and the acid base neutralization achieved an equilibrium state. However, in the mica etch process, the ion current stopped decreasing and start ed to increase at abo ut two to three minutes. This was because HF kept etching mica and increasing the size of nanopore tip and base openings in this case. The

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78 resistance of the multipore membrane decreased so that the total ion current inc reased at the end (Fi gure 3 3). Figure 36 shows that the current voltage curves obtained from three identical mica membranes after the initial 10 minute anisotropic etch cycle. The similar conductance observed here showed that nanopores on the different membranes might have c omparable size after one etch cycle if they were assumed to have the same number of p ores from membrane to membrane. However, the nanopore si ze in multipore membrane was not calc ulated from the conductance. Taking a scanning electron microscopy image is th e common method to characterize these nanopores. Morphology of Nanopore Openings in Muscovite M ica Figure 37ABCD shows the FESEM images of the base openings of nanopores in mica membranes that were etched under same condition but for a different number of total etch cycles. As per our prior work, the rhomboidal shape opening was characterized by the length of their long and short diagonals.29 value.141 Larger pores were obtained when the mica membranes were etched for more cycles. The length of diagonal of rhomboidal opening was almost proportional to the etch time under the same etch con dition and the long diagonal reach ed ~1 m after 8 cycles of 10 minute etch (shown in Figure 37D). There was only one side of the mica membrane was exposed to the 10 M HF etchant, and HF was neutralized by 10 M NaOH on the other side. It was presumed to have a HF transmembrane concentration gradient where the concentration was lower at the tip and higher at the base. We p redicted that the nanopore openings facing the NaOH solution were smaller and the openings facing the HF were larger. Figure 37EFGH shows the FESEM images of the tip openings of nanopores on mica membranes that were etched for a different number of total

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79 cycles. Compare images of the base and tip openings in the mica membrane at the same etch cycle, it was found that the base openings were always much larger than those at the tip. It is clearly demonstrated that th e nanopores fabricated via such a multicycle anisotropic etch method had an asymmetric shape. S ince we found that there was EOF in the pore to carry HF to the tip, it is rea sonable that we observe that the nanopore tip opening size increased with etching time. Asymmetric Nanopore B ase and Tip Etch R ate The lengths of the nanopore base and tip opening long diagonal s after different etch cycles were obtained from FESEM images and converted to equivalent diameter of the mica nanopore via Eq uation 34. Figure 38A shows the plot of nanopore base opening equivalent diameter (dEq b) vs. etch time. It indicates that the base opening equivalent diameter increased almost linearly with etch time. The values are arithmetic averages with standard deviations calculated from three independently prepared mica membranes. Because the bulk etching rates are anisotropic for different cleavage planes of mica, the radial etching rate of equivalent circular opening w as used; in this case, it was about 4.0 nm min1 for mica etched with 10 M HF. As long as we know the etching rate, it is easier to precalculate how many 10minute etch cycles are needed to make asymmetric nanopores of desired base size. Therefore, we can obtai n asymmetric nanopores with different base size s by varying the etching time. Figure 3 8B shows that the equivalent diameter of the nanopore tip also increased with etching time. This is reasonable because tip was still etched by a low concentration of HF pumped by EOF in the nanopore. The radial etching rate of ti p equivalent circular opening was about 0.57 nm min1.

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80 Ge ometry of Asymmetric N anopores The geometry of these nanopores was investigated by chemical vapor deposition of car bon into the nanopores and subsequent removal the mica template to expose ca rbon replicas of the nanopores.29 Figure 39ABCD shows the FESEM images of nanopore replicas which reflect ed the pyramidal shape of the nanopores etched for different cycles. The length of the nanocones was about 10 m, as same as the thickness of mica membrane. The width of the nanopore replica base was almost equal to the length of the long diagonal of rhomboidal base opening. These images also proved that the base opening of nanopores increased with longer etch time. The surface morphology of these carbon replicas also indicates that t he ent ire inner surface of the nanopore was extremely smooth. Instead of using the one step method similar to the one we used to fabricate conical nanopores in iontracked polymer membrane s we would like to use this multic ycle procedure to etch pyramidal ly shaped nanopores in mica membrane s In order to demonstrate that the multicycle anisotropic etch method is more effective in preparing asymmetrically shaped nanopores in iontracked mica membrane, nanopores were fabricated with the multicycle etch (8 10 minutes) and the one step etch (180 minutes). The equivalent diameters of base and tip opening of those nanopores obtained from the one step 80minutes etch were around 55410 nm and 19542 nm, respectively. While the equivalent diameters of base and t ip opening of those nanopores fabricated via multi cycle etch were around 62338 nm and 8916 nm, respectively. The base size of the nanopore fabricated by the differen t etch procedures were not very different, but the tip size of the nanopores obtained vi a the multicycle etch was much smaller than that of the nanopore made via the one step etch. All the carbon replicas of these nanopores obtained from the multi cycle etch showed uniform pyramidal geometry spanning the entire length (Figure 39ABCD). Howev er, the carbon

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81 replica of the nanopore obtained from the one step e tch did not show obvious asymmetric shape (shown in Figure 39E). As we hypothesized, the NaOHHF neutralization region was right outside of the tip and HF was pumped to this region by EOF in the nanopore. At longer etch time s this region might become less efficient at neutralizing the HF. As a result, the concentration of HF at tip increased and larger nanopore tips were obtained even though there was NaOH to stop the etching. Another reas on might be that the temperature increased at the tip region so that the etch rate increase d as well. We think there might be two factors that contribute to the temperature increase at the tip region. The first reason might be the resistive heat generated at tip because we know there was a very high electric field strength fo cused around the tip in the asymmetric nanopore .62 The other reason was the heat generated from acid base neutralization reaction. In the one step 80minute etch, the heat accumulated at the tip and continued incre asing the temperature of local environment. As a result, the nanopore tips etch rate increased dramatically when the temperature increased. This temperature effect was extremely significant when the multiple track mica membrane was etched, especially if the track density is very high. It was found that the solution temperature increased when we etched the mica membrane with track density at ~108 per cm2. During the multi cycle etch method, both etchant and stop ping solution were replaced. T he temperature of solution remained at around at 25 C during each 10minute etch cycle. This prevented the temperature increasing dramatically so that the etch process became more controllable. Besides multip ore membranes, the sin gle asymmetric nanopore membranes were also prepared by using multicycle anisotropic etch method. In this fabrication process, mica membranes with only one track were used for chemical etch. Because the nanopore size was

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82 small and there was only one in me mbrane, it was extremely hard to characterize the nanopore size by using electron microscopy. Instead, an electrochemical measurement was used as described previously.53 Figure 310 shows the current volta ge curves of such single asymmetric nanopore fabricated by using the multi cycle method and one step method, respectively. The overall etch time fo r both mica membranes were the same (80 minutes) so that we could assume the same e quivalent diameter of the base openings for both membranes (dEq b 640 nm). From the slope of the current voltage curves, we could calculate the equivalent diameter of tip opening using Eq uation 35. The calculations showed that the multicycle etch method made an asymmetric nanopore with a tip equivalent diameter of 62 nm. The one step method gave a much larger tip equivalent diameter of 155 nm if we assumed that each has the same base equivalent diameter of 640 nm. Both the SEM images of multipore membrane s and current voltage curves of single pore membrane s indicated that a multicycle approach is more useful for preparing asymmetric nanopores in iontracked mica membrane. In this study, we investigate d the multi cycle anisotropic chemical etch with each cycle at 10 minutes. It was shown that the multi cycle etch proce ss offers better co ntrol for fabrication of asymmetric nanopore in mica than the one step method. We wonder whether a n etch cycle with shorter time window will offer even better control over nanopore geometry. However, because the nanopore etching, stopping and rinsing process is rather cumbersome, a short cycle time but with more number of cycles is impractical. Shorter cycle duration definitely requires a higher cycle count to make the nanopore base opening large enough. Considering this, the 10 minute et ch cycle is desirable because both

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83 efficiency and control of the process are optimized. In all other research about changing the parameters for mica etch, we always used 10minute time window for one etch cycle. Beside the duration for each etch cycle, sev eral other parameters were investigated to optimize the etch conditions, including etchant concentration and trans membrane potential. Effect of etchant concentration on nanopore geometry To reduce the time requi red for fabricating these asymmetric nanopor es in iontracked mica membrane, a lar ge bulketching rate is desirable. It is estimated that a higher concentration of etch ant would produce a higher bulketching rate, which results in a larger base opening at the same total etching time. Different concentrations of etching solution (5 M, 10 M, 25 M) HF were evaluated when keeping the same concentration of stopping solution 10 M NaOH. As anticipated, base opening long diagonal of 48710 nm and 102962 nm were observed after eight 10 minute cycles etched v ia 5 M HF and 10 M HF, respectively. In both cases, small nanopore tips were obtained. The SEM images of nanopore replicas demonstrate that a uniform pyramidal geometry spanned the entire replica from tip to base (Figure 3 11AB). Thus, when using 5 M and 10 M HF as etchant, 10 M NaOH could effectively stop the HF etching of mica. However, increasing the etchant concentration even higher to 25 M HF greatly reduced the effectiveness of the 10 M NaOH stopping. It was found that carbon replica with ~1.2 m base long diagonal and ~200 nm tip long diagonal was obtained from a nanopore chemically etched in 25 M HF for only two 10minute cycles. It was observed that the asymmetric geometry only existed at the region a few micrometers from the tip end. The remaining nanopore appeared to have a more constant crosssectional area (Figure 3 11C). So it is estimated that much larger tip and base will be obtained if the mica membrane is etched for eight 10 minute cycles with 25 M HF. In conclusion, at least 1:1 ratio of stopping solution to etchant concentration is required to make the nanopore have a uniform asymmetric geometry.

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84 Effect of applied voltage on nanopore geometry As per our prior work, we have shown that transmembrane potential applied during etching iontra cked polycarbonate membrane had an effect on the size of conical nanopore base opening.62 The effect of transmembrane potential on mica membrane etching was also investigated in this case. The ion tracked mica membranes were etched by 10 M HF with 10 M NaOH as the stopping solution for eight 10minute cycles, but under di fferent potential bias es (0.0 V, 2.5 V, 5.0 V, 10.0 V and 50.0 V). The HF diffused to the tip quickly and HF would keep etching the tip openings if HF was not neutralized by NaOH imm ediately. Without applying electric field, OHcould only diffuse to the neutralization region, which is assumed to be located out side the tip of nanopore. Because only a very limited amount of HF is consumed to react with a few OHthat diffused to the neutralization region, the concentrat ion of HF at tip will not be negligible There fore it is reasonable to obtain very large tip openings even with the presence of NaOH stopping solution However, mass transfer of OHwill be much faster if electrophoretical migration is applied by controlling the transmembrane potential. With la rger transmembrane potential, a large amount of OHwas delivered to the neutralization region and consumed much more HF. As a result, the concentration of HF at tip was greatly decreased resulting a much slower etch rate at the tip. It also influenced the concentration gradient of HF across the membrane and the nanopore etched in various ways will have different geometries. Figure 3 12A is a scheme of showing the effect of applied electric field on the HF concentration gradient in the nanopore. If this is true, the higher potential might benefit the etch and make uniform asymmetrically shaped nanopores. The experimental results proved this hypothesis. Figure 3 12BCDE shows the FESEM images of carbon replica from the nanopore etched at different applied volt ages. The SEM images show that the long diagonal of base and tip opening etched without applying a potential

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85 were 125674 nm and 64931 nm. The tip opening was much larger than that obtained by applying +10 V transmembrane potential during etching. In Figure 3 12B, the carbon replica of nanopore s obtained without applying potential showed corresponding geometry wit h only about 1 m long asymmetrically s haped segment at the tip opening with the rest having a uniform cross section However, the segment of as ymmetric shape became longer when an electric field was applied. When the applied voltage was as high as 5 V, most part of the carbon replica show ed obvious asymmetric shape. T he nanopore replica obtained from 10 V etch ha d an asymmetric shape along the entire length. It seems that the higher the electric field that was applied, the better cone shape that was obtained, at least when the voltage was lower than 10 V. Although we demonstrated that larger base openings could be obtained by using higher potential differences in our prior research with polycarbonate membranes,62 however, it is not alw ays t he case that better asymmetric nanopore shape could be obt ained when higher potentials were employed during the etching process In the case of an applied transmembrane potential of +50 V, the resistive heating generated from high ionic current made the et ching too fast to stop. Thus, the entire mica membrane dissolved during the third 10 minute etching cycle. It is obviously that transmembrane potential needs to be well controlled during anisotropic etching for preparing asymmetrically sha ped nanopores in mica membrane. Fabrication of Single Pyramidal Nanopore in Muscovite M ica Single pyramidally shaped nanopore mica membranes were fabricated under the same experimental condition used for etching multi track mica. After each 10 minutes etch cycle, the curre nt vo ltage curves were measured by filling the nanopore with pH 7.0 100 mM phosphate buffer solution that was 1 M KCl (Figure 313A). The equivalent diameter of base opening could be calculated by using the mica equivalent bulk etching rate and total etchi ng time. The tip

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86 equivalent diameter after each etch cycle could then be calculated from the conductance of th e nanopore according to Equation 3 5. It is observed that the current voltage curves are not linear for these single asymmetric nanopore in mica The nonlinearity ion current rectification phenomenon has been discussed in detail in the literature .24, 32, 57, 74, 75 Since the surface of mica nanopore is negatively charged,141 the electrical double layer on the nanopore could make the na nopore cation permselective and demonstrate a on and off state when a transmembrane voltage is applied. The ionic current rectification also changes with the ionic strength of the supporting electrolyte .32, 57 Current volta ge curves of a single asymmetric nanopore in mica membrane after different etch cycles were measured when nanopore was filled with pH 7.0 10 mM phosphate buffer solution that was 10 mM KCl (Figure 3 13B). It is interesting that a very strong ion current rectification was observed in such single asymmetric nanopore embedded in mica mem brane at low ionic s trength solution. The ionic current value measured when a 1 V potential was applied on the base opening increased with more etching cycles. Interesting ly the ionic curren t was still strongly rectified in low ionic strength solution when a +1 V potential was applied on the base side of the membrane We previously defined the degree of rectification as a ratio of absolute value of ion current recorded at a given negative voltage to the same absolu te value at a positive voltage.57, 119 Figure 313C indicates that the ioncurrent rectifica tion ratio of a single asymmetrically shaped nanopore in mica increase d with longer etching time (larger base opening) in pH 7.0 10 mM phosphate buffer solution that was also 10 mM KCl. It was also observed that the rectification ratio was small in the high ionic strength electrolyte solution (1 M KCl). The ion current

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87 rectificatio n is not significant when the electrical double layer thickness is much smaller than the pore size. It also seems that the ion current rectification phenomenon is closely related with the pore geometry. When the base size was also very small, anion s were also excluded outs ide of the pore so that anions could not accumulate in the tip region. As a result, the ion current was not heavily rectified When pore base size increased, the ion current rectification b ecame more significant as long as the tip was not too large. We also compared the ionic current rectification of asymmetrically shaped single nanopore s in mica and PET membrane s with similar base (~640 nm) and tip (~60 nm) size. Current voltage curves i n Figure 314A indicates that the nanopore in mica membrane and the one in PET membrane ha d the similar tip size if calculated from the conduce of nanopore filled with 1 M KCl, pH 7.0 100 mM phosphate buffer solution. The current vol tage curve of nanopore in PET was linear and the one from mica only showed a little ion current rectification However, a much larger ion current rectification was observed from the nano pore in mica membrane than the one in PET in a low ionic strength solution (10 mM KCl, pH 7.0 10 mM phosphate buffer). The current voltage curve was still almost linear for nanopore in PET even in 10 mM KCl pH 7.0 10 mM phosphate buffer. However, a strong ionic current rectification was observed from the nanopore in mica at such low ionic strength. We hypothesized that the larger ionic current rectification might be due to the fact that the surface charge density of nanopore in mica (~2.1 e nm2)147 is higher than that of a nanopore in PET (~1.0 e nm2)148. To test the above hypothesis and explore the reason why an asymmetrically shaped nanopore in mica has a very high ionic current rectification ratio at low ionic strength solution, surface charges were removed from the mica via modification the substrate with

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88 glycidoxypropyltrimethoxysilane ( GPTMS) mercaptoethanol to produce hydroxyl terminated group.145, 146 Current voltage curves in Figure 315A indi cate that the nanopore size had no significant change prior to and after the surface modification because the nanopore conductance were almost the same. However, the current voltage curve s in Figure 315B show that the ionic current was much less rectified after the surface modified with hydroxyl group. The ionic current rectification decrease was probably because some surface charges wer e removed because the hydroxyl group was neutral and surface charge density decreased after the modification. This result could also be used to explain why the bare nanopore in mica has stronger ionic current rectification than bare nanopore in PET because PET has lower surface charge density. Conclusions In this work, we developed a multi cy cle anisotropic chemical etch method to prepare pyramidally shaped nanopores in iontrack muscovite mica membranes. The effect of etchant concentration, transmembrane potential and etching cycle number on nanopore size and geometry were investigated in detail The nanopore dimensions an d geometry in multipore mica were confirmed by SEM images of bare nanopore membrane s and carbon replica s of nanopores. The electrochemi cal measurement and calculation of single nanopore size in mica were discussed as well. The ion transport properties of single pyramidal nanopore in mica membrane were also studied and ionic current was highly rectified in low ionic strength electrolyte so lution. We think this very strong ion c urrent rectification is because the highly negatively charged surface caused a large difference between the transference number of cations and anions By modifying the nanopore wall with an uncharged surface functiona l group, surface charge density decreased. As a result, the pyramidally shaped nanopore demonstrated much less ion current rectification.

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89 We think this extremely high ionic current rectification properties usually not seen in nanopore on polymer membrane might be promising for exploring application s in resistive pulse sensing or analyte separation.

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90 Figure 31. The relationship between a rhomboidal opening and an equivalent circular opening. Figure 32. Schematic of modification GPTMS on muscovite mica to produce hydroxyl terminated surface groups.

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91 Figure 33. Current time traces of the anis otropic etching of multi track muscovite mica membrane s Etching solution was 10 M HF and stopping solution was 10 M NaOH. Data shown here were taken from the first 10 minute etch cycle, track density 105 tracks per cm2. Figure 34. Concentration of phenol in the permeate solution at different time when it was transported fro m base to tip in the nanopore There were constant current across the membrane with current of (red) 0.5 mA, (green) 1.0 mA, (blue) 1.5 mA and (black) without current. 0 2 4 6 8 10 0 10 20 30 40 50 60 Current (uA)Time (minute) 0 20 40 60 80 100 120 0 50 100 150 200 250 Time (minute) Diffusion Galvanostat 5.0 mA Galvanostat 10.0 mA Galvanostat 15.0 mA

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92 Figure 35. Current time trac es during the HAc a nd N a OH neutralization and the schematic of eletrolyte concentration profile. A) Current t ime traces during H Ac NaOH neutralization in asymmetric multipore (105 cm2) mica membrane that was etched for one 10minute cycle). B) Schematic of acid (HAc) and base (NaOH) concentration profile during the neutralization. 0 2 4 6 8 10 0 2 4 6 8 10 ACurrent (uA)Time (minute) 1 M NaOH 2.5 M NaOH 5.0 M NaOH 10 M NaOH

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93 Figure 36. Current voltage curves of multi track mica membrane s that were anisotropically etched fo r one 10minute cycle Track density was about 105 tracks per cm2 and current voltage curves were measured in 100 mM phospha te buffer at pH 7.0 with 1 M KCl -1.0 -0.5 0.0 0.5 1.0 -30 -20 -10 0 10 20 30 Voltage (V)

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94 Figure 37. FESEM imag es of the base and tip openings of asymmetric nanopore s in muscovite mica membrane s at different etching time. ABCD) Base opening etched for 210, 410, 610, 810 minutes, respectively. EFGH) Tip opening etched for 210, 410, 610, 810 minutes, respectively.

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95 Figure 38. Calibration of equivalent diameters of the base and tip openings of the pyramidally shaped nanopore s that were anisotropic etch ed for different time. A) Base openings. B) Tip openings. Equivalent diameter was calculated from the length of long dia gonal obtained via FESEM im ages. 0 20 40 60 80 0 100 200 300 400 500 600 700 AEquivalent Diameter (nm)Time (minute) 0 20 40 60 80 0 20 40 60 80 100 120 BEquivalent Diameter (nm)Time (minute)

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96 Figure 39. FESEM ima ges of carbon replica of pyramidal nanopores in muscovite mica that were anisotropic etch ed for different time. A) 210 minutes. B) 410 minutes C) 610 minutes D) 810 minutes with applied transmembrane volta ge at 10 V. E) 180 minutes without applied voltage.

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97 Figure 310. Current volta ge curves for single nanopore in mica membrane prepared by multic ycle and one step methods. (Red) 810 m inutes anisotropic etching. (B lue) 180 minutes anisotropic etching. IV curves were m easured in 1 M KCl, pH 7.0 100 mM phosphate buffer solution. Figure 311. FESEM ima ges of carbon replica of pyramidal nanopores i n muscovite mica that were anisotropic etch ed w ith different concentration of HF Transmembrane voltage was 10 V A) 5 M HF etched for eight 10minute cycles B) 10 M HF etche d for eight 10minute cycles. C) 25 M HF etched for two 10minute cycles. -1.0 -0.5 0.0 0.5 1.0 -100 -50 0 50 100 Current (nA)Voltage (V)

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98 Figure 312. Schematic of voltage effect on HF concentration profile and FESEM ima ges of carbon replica of asymmetric nanopores A) Schematic of voltage effect on HF concentration profile. BCDE) FESEM ima ges of carbon replica of asymmetric nanopores fabricated in muscovite mica via anisotropic etching with 10 M HF at different applied voltages B) 0 V. C) 2.5 V D) 5.0 V E) 10.0 V for eight 10minute cycles.

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99 Figure 313. Current volta ge curves of single pyramidal nanopore in mica membrane in different salt concentration solutions and resulting ion current rectification ratio. A) IV curves measured in pH 7.0 100 mM phosphate buffer with 1 M KCl. B) IV curves measured in pH 7.0 10 mM phosphate buffer with 10 mM KCl. C) Ion current rectification of single pyramidal nanopore in mica membrane measured in 1 M KCl, 100 mM phosphate buffer (blue) and 10 mM KCl, 10 mM phosphate buffer (red). -1.0 -0.5 0.0 0.5 1.0 -40 -20 0 20 40 A 0 mins 1 10 mins 2 10 mins 3 10 mins 4 10 mins 5 10 mins 6 10 mins 7 10 mins 8 10 minsCurrent (nA)Voltage (V) -1.0 -0.5 0.0 0.5 1.0 -10 -5 0 5 10 B 0 mins 1 10 mins 2 10 mins 3 10 mins 4 10 mins 5 10 mins 6 10 mins 7 10 mins 8 10 minsCurrent (nA)Voltage (V) 0 10 20 30 40 50 60 70 80 90 0 5 10 15 20 25 30 35 CRectificetion Ratio (I-1V/I+1V)Time (minute)

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100 Figure 314. Current voltage curves of single asymmetric nanopore in PET and mica membrane ( B lue) Nanopore in PET (Red) Nanopore in mica. IV curves were measured in A) 1 M KCl, 1 00 mM phospha te buffer pH 7.0, B) 10 mM KCl, 10 mM phosphate buffer pH 7.0. Figure 315. Current Voltage curves of a single a symmetric nanopore mica membrane before and after the surface modification with hydroxyl terminated group s (B lue) Before and (red) after the surface modification IV curves were measured in A) 1 M KCl, 100 mM phosphate buffer pH 7.0, B) 10 mM KCl, 10 mM phosphate buffer pH 7.0. -1.0 -0.5 0.0 0.5 1.0 -40 -20 0 20 40 ACurrent (nA)Voltage (V) -1.0 -0.5 0.0 0.5 1.0 -10 -5 0 5 10 BCurrent (nA)Voltage (V) -1.0 -0.5 0.0 0.5 1.0 -150 -100 -50 0 50 100 150 ACurrent (nA)Voltage (V) -1.0 -0.5 0.0 0.5 1.0 -15 -10 -5 0 5 10 15 BCurrent (nA)Voltage (V)

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101 CHAPTER 4 RESISTIVE PULSE DETECTION OF N ANOPARTICLES USING A SINGLE PYRAMIDAL NANOPORE IN MICA MEMBRANES I ntroduction Single conically shaped nanopore s embedded in polymer membrane s are usually used for resistive pulse detection and ion current rectification studies. The conventional resistive pulse signal is caused by temporary pore blocking by analyte and re sulting r esistance change Recently, the ion current rectification in asymmetric nanopore has been well investigated and employed for sensing applications.58, 119, 139, 140, 149 This type of sensing pa radigm is based on the changes of the single conical nanopore wall local surface charge150 induced by binding or adsorption of an analyte. The analyte binding was detected as a cha nge of the ion current rectification of single conical nanopores. The quantity of the analyte in the solution can be correlated with the degree of ion current rectification Martin group has employed using single conical nanopore in Kapton membrane detecting drug molecule s119 and other small organic molecules.151 The single conical nanopor e demonstrated a strong ion current rectification phenomenon and ion current rectification ratio is large. After exposure of the anionic charged nanopore to the cationic drug molecules the extent of ion curre nt rectification changed. Such a change was due to the adsorption of cationic drug molecules on the pore wall and changing the surface charge density or even charge polarity.119 It was found that the magnitude of the ion current rectification change was c orrelated with the concentration of drug molecules However, this type of sensing based on the ion current rectification change could only obtain the overall informa tion of the analyte in the solution. It is difficult to measure the individual analyte inte ra cting with the pore wall and passing through nanopore to achieve single analyte detection signal A ne w resistive pulse mechanism is proposed in this research to achieve

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102 single analyte detection A coating molecule is introduced to modulate the ion current rectification in single pyramidal l y shaped nanopore in mica. A model analyte, poly(styrene) (PS) latex nano particle s was driven electrophoretically through the nanopore. It is hypothesized that the nanoparticle s will interact with the coating mole cule resulting in a temporary surface charge inversion and generates resistive pulse events. The local charge inversion affecting the ion current rectification in asymmetric nanopore is a well known phenomenon.150 According to our proposed mechanism, i n the 10 mM KCl and 10 mM phosphate pH 7.0 buffer solution, the current voltage curve will have the shape as the blue curve as shown in Figure 41A. The steady state c urrent flowing through the nanopore with working electrode voltage of +1 V is small, showing a n Off state. However, if the nanopore is exposed to the coating reagent, the positively charged coating reagent will adsorb on the negatively charged mica pore wall and decrease the a nionic charge density or even re verse the charge polarity. Therefore, the current voltage curve will have the shape as the red curve as shown in Figure 41A. The steady state current flowing through the nanopore with working electrod e voltage of +1 V is larger, showing an On state. The negatively charged nanoparticle at the tip side of the membrane will be electrophoretically driven into the nanopore tip. Because of the electrostatic attraction between the surface negative ly charged particle and the positive ly charge coating reagent, the local surface charge might change momentarily. Correspondingly, t he ionic current might temporarily change from the on state to the off state so that a current drop will occur However, the local surface charge will change back to its original condition after the nanoparticle moves out of the tip region. The ionic current will recover to the on state. Thus a complete current pulse event will be observed when a single nanoparticle passes through the nanopore from tip to base.

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103 Experimental Materials B isb enzimide ( Hoechst 33258) was obtained from Sigma Aldrich (St. Louis, MO). Sulf ate modified poly(styrene) latex (PS) particles were obtained from Invitrogen (Carlsbad, CA). Fabrication an d Characterization of Single Pyramidal Shaped Nanopore in Mica The fabrication of single pyramidal nanopore in mica is described in chapter 3. Briefly, a single tracked muscovite mica membrane was mounted between two half electrochemical cells as described before.53 A n etching solution that is 10 M HF was placed in one cell, and a stopping solution t hat is 10 M NaOH was added into the other cell. A Pt electrode was placed in both of the cells. A transmembrane voltage of 10 V was applied with the cathode in the stopping solution. The chemical etch was terminated at 10 minutes and the solutions in both cells were replaced with 10 M NaOH stopping solution. The membrane was etched for totally eight 10minute etch cycles. The current voltage curve was measured to calculate the nanopore tip size. An electrolyte solution that was 1 M KCl and 100 mM phosphate buffer at pH 7.0 was added to both half electrochemical cells. The voltage was scanned form 1 V to +1 V at 1 Vmin1 with working electrode at the nanopore large opening si de (base) The length of major axis of base opening was calculated with the etch rat e and equivalent diameter of base was obtained from via = 0 .606 (4 1) The single pore small opening (tip) equivalent diameter (dEq tip) can be calculated from the pore conductance via, = 4 (4 2)

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104 In this research, the pyramidal nanopore base equivalent diameter dEq base is ~ 640 nm and the tip equivalent diameter dEq tip ~ 85 nm. Characterization of Poly(styrene) Nanoparticle s The sulfate modified poly(styrene) latex particles with diameter of 29 nm were characterized in the presence of the coating molecule, Hoechst 33258, before used as model anal yte. Suspensions contai ned 50 nM of PS particles with different concentration of Hoechst 33258, were prepared. Brookhaven Z etaPlus was used to measure the size distribution and the zeta potential of PS particles in the suspensio n. Suspensions contained 50 nM of PS particles with different concentration of HATC were prepared and used for size distribu tion and zeta potential mea surement as well Current Voltage Curves Measurement in the Presence of Coating Reagents A current voltage curve of a single pyramidal nanopore in mica was first measured in the solution of 10 mM KCl and 10 mM phosphate buffer a t pH 7.0 without Hoechst 33258. The current t, Hoechst 33258. ResistivePulse D etection of Poly(styrene) Nanop article s A mica membrane contained a single pyramidal nanopore was mounted between the two ha lf electrochemical cell s A supporting electrolyte solution was added to both side s of the membrane. In these series experiments there w e re two types of supporting electrolyte solution s used. The first type of solution was a phosphate buffer solution at pH 7.0 wit h 10 mM KCl. The second solution was A Ag/AgCl electrode was placed in each solution. The ionic current flowing through the single nanopore full of electr olyte solution was measured with an Axopatch 200B current amplifier (Molecular Devices Corporation, Union City, CA) in the voltage clamp mode with a low pass B essel filter at

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105 2 kHz bandwidth. The signal was digitized using a Digidata 1233A analogto digita l converter (Molecular Devices Corporation). Data were recorded and analyzed with pClamp 9.0 software window was 1 minute. A background steady state current was measured without adding model analyte PS particles. At least 5 minutes of data were recorded f or each experimental condition. During the PS particles translocation experiment, the solution at one side of the membrane was replaced with the same buffer solution but also containing 50 nM PS particles. T he working electrode was placed on t he side facing the base opening and the reference electrode was in the solution facing the nanopore ti p opening. In the case of transporting PS particle s through the nanopore from ti p to base, a +1 V voltage was applied on the working electrode so that the electrode facing the tip was cathode. However, a 1 V voltage was applied on the working electrode when PS particles were transported from base to tip so that the electrode facing t he base was cathod e. In the control experiments, a similar approach was used. The only difference was using symmetric n anopore in mica Results and Discussion A new mechanism was proposed to increase resistive pulse detection sensitivity and signal intensi ty by using a coating reagent Hoechst 33258. Other control e xperiment was performed to further study the hypothesized mechanism, such as transport PS through the nanopore from different direction, changing nanopore geometry, and nanopore materials. Characterization of Poly(styrene) Nanoparticles As we known, the negatively charged PS nanoparticles will interact with the positively charged coating reag ent e.g., Hoechst 33258,119 in the suspension and re sult in aggregation or even precipitation of nanoparticles. Since we are trying to detect the resistive pulse signal of

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106 single nanoparticle translocation t hrough the nanopore, it is extremely important to prove that the PS nanoparticle s are monodispersed i n the suspension with the presence of coati ng reagent. The dynamic li ght scattering measurement showed that the mean diameter of the PS nanoparticle is about 26.7 nm in the presence of Hoechst 33258, which is close to the diameter reported by the manufactu re. The size distribution of PS nanoparticle in this solution is shown in Figure 42. The zeta potential measurement shows that the zeta potential of PS nanoparticles surface is about 52.15 mV, which means that particle surfac e is still negatively c harged in solution even with the presence of The above studies demonstrated that the PS nanoparticles were negatively charged and mono dispersed in the solution used in the resistive pulse det ection. Current Voltage Curves of Nanopore in the Presence of Coating Reagents. The current voltage curve of single pyramidal nanopore in mica shows strong ion current rectification in 10 mM phosphate buffer at pH 7.0 with 10 mM KCl. A larger current was observed when negative voltage is a pplied and a smaller current value was obtained at the positive elect r ode polarity (Figure 4 3) However, much less ion current rectification was observed when the coatin in the presen ce (shown in Figure 43) The current v alue at positive voltage increased and the absolute current value at negative voltage decreased after nanopore was exposed to the solution contained coating reagent Hoechst 33258. In this case, it was observed that the direction of current voltage curve re versed because the absolute current was lower at a negative voltage an d the absolute current was higher at a positive voltage. This phenomenon is well known to be related with the asymmetric nanopore local charge inversion.150 The current voltage curve of single pyramidal nanopore in mica also showed such ion current rectification change phenomenon when positively charged coating reagent was added to the solution (shown in Figure 43).

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107 ResistivePulse Detection of Poly(styrene) Nanoparticles Resistivepulse detection of nanoparticles with conventional mechanism A conventional resistive pulse method was used to detect the translocation of PS nanoparticle s through a single pyramidal nanopore in mica membrane The single pyramidal nanopore ha d a tip equivalent diameter (dEq tip) ~ 85 nm and base equivalent diameter (dEq base) ~ 640 nm The steady state current of the background without the PS nanoparticle s was about 380 pA wh en the nanopore was filled with pH 7.0 phosphate buffer with 10 mM KCl The baseline current was also about 350 pA when the nanoparticle s were present in the feed solution Only a single event was observed and the amplitude of this event was small and comp arable to the noise level (Figure 44) We think that there are two possibilities that might prevent large amplitude pulse events occurred. One possibility is that the negatively charged PS nanoparticle did not enter the also highly negative ly charged mica147 nanopore tip because of electrostatic repulsion. The other reason might be the charges carried by the particle including the charge s on the PS nanoparticle and the counter ions, is alm ost equal to the charges in the solution which is replaced by the particle. Therefore, no current pulse was observed even though the particle s did enter the nanopore ti p. It seems that the conve ntional resistive pulse detection is not very sensitive for detecting the nanoparticle s in th is case. Resistivepulse detection of nanoparticles involving coating reagent The steady state current of the nanopore filled with 10 mM phosphate buffer at pH 7.0 with 1400 pA (Figure 4 5) This steady state background current value was comparable to the current value at +1 V when the currentvoltage curve was measured. Because there was a dynamic equilibrium of Hoechst 33258 adsorption/desorption on the pore wall and the ion current was sensitively affected by this equilibrium process, the background current fluctuate d a little.

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108 When the nanopore was exposed to the sam e buffer but also contained 50 nM PS nanoparticles, the steady state current decreased to about 1000 pA. Such steady state current decrease migh t because the presence of nanoparticle partially decreased the concentration of free Hoechst 33258 in the solution, resulted in a baseline ion current change. More interesting, when 50 nM PS n anoparticles was present, a large amount of current pulses were observ ed. The current pulses amplitude were much larger than those obtained without using coating reagent In most of the conventional resistive pulse experiment, the current pulses are downward signals, which mean the absolute cu rrent value decrease when such pulses occur In the case of t ransporting PS nanoparticles from tip to base, similar downward current pulse signals were also observed. W e hypothesize that such large current pulses were generated because of a newly proposed mechanism, instead of the conventional mechanism. An experiment of transporting PS nanoparticles from base to tip was designed to validate this mechanism. Validation of the hypothesized resistive pulse detection mechanism involving coating reagent As discussed above, the current voltage curves inverse d after the nanopore was exposed to the coating reagent Hoechst 33258. When the negatively charged PS nanoparticle s were placed on the base side of the membrane, a 1 V voltage was applied so that PS nanoparticles were driven electrophoretically from base to tip. As shown in Figure 41B the steady state current is larger when the supporting electrolyte solution does not contain the coating reagent and it is defined as on state However, the steady state current is much lower when nanopore is exposed to the coating reagent and this is defined as off state. When the nanoparticle enter the nanopore from the base, negatively charged PS particle will interact with the positively charged coating molecule ca using a nanopore surface charge change. Therefore, the ion current will temporarily increase from the off state to on state. But the ion current will change back to off state

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109 when the nanoparticle move out of the sensing zone. Thus a current pulse will be observed when a nanoparticle pass es through the nanopore tip. Especially, the current pulse event is a current enhance signal, instead of absolute current value drop as the conventional signal. The experiment data confirmed our prediction about the current pulse direction, as shown in Figure 4 6. The background current was about 250 pA because of the adsorption of Hoechst 33258 on the pore wall When the PS particles were present, the basel ine current increased to 450 pA and a large amount o f current pulses were observed. Interestingl y, these current pulses were current enhancement signal s the same as we predicted according to the proposed mechanism. If the signal were produce d according to the conventional mechanis m, the current pulse signa l would either only increase or decrease from the baseline current no matter which direction the analyte was driven into the nanopore But according to our proposed mechanism, the pulse event current decrease when nanoparticle move d from tip to base a nd the pulse event enhanced the current when nanoparticle moved from base to tip. Importantly, our experiments proved that the hypothesis about the local charge inversion induced current pulses is correct. Current pulse shape According to the proposed mechanis m, the directions of the current pulse are diffe rent when the nanoparticles are transported from the opposite directions. More interestingly, the experimental data demonstrated that the current pulse shapes were quite different in these two cases and we believe the pulse shape s were related with t he nanopore pyramidal geometry. When the PS nanoparticle was driven into the nanopore and moved from tip to base by electrical field, the nanoparticle first enter ed the tip region, where has the narrowest entrance and is most sensitive to the resistance change. Especially, the neg atively charged nanoparticle had the greatest chance to interact with the coating reagent that ads orbs on the pore wall, causing the largest ext ent of lo cal charge inversion. Thus, it was expected a large current value cha nge when

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110 the nanoparticle first enter ed the tip region (as shown Figure 47A). When the nanoparticle entered the pore tip region and start ed to move forward, the impact of partic le on the local surface change decrease d because the cross section of nanopore increase d along t he axis direction. As a result, the nanoparticle ha d less effect o n ion current when the particles were in the pore but far from the tip. Therefore the ion curr ent slowly recover ed to the baseline value when the nanoparticle moved from the tip to bas e with time As a result, the curr ent pulse in this case had a tale at the end of each pulse. However, the current pulses had different shapes when the negatively cha rged PS nanoparticles were driven into the base and moved from base to tip by electrical field. At the beginning, the cross section of nanopore base opening was large so that nanoparticle had no or less effect on the local charge inversi on. When the nanopa rticle move d into the sensing zone that was in close proximate to the tip, it began interact ing with the coat ing reagent on the pore wall because of being in a confined space. This resulted in a larger extent of local charge inversion. Thus a n ion current start ed to change gradually corresponding to the local charge change We think that the greatest ion current change occurred when the nanoparticle was closest to the tip where the cross section of nanopore was smallest. Bu t because the nanoparticle moved in the dir ection in which nanopore narrowed down, there were increasing ch ances that nanoparticle collided w ith the pore wall. These collisions might cause the multitime nanoparticle adsorption/desorption on the pore wall and as a result, the current flu ctuate d as responses. At the end, the current recover ed to the baseline value quickly when the nanoparticle left the nanopore from tip (as shown in Figure 47B). Current pulse amplitude One characterist ic of the resistive pulse event is the current pulse a mplitude. In the conventional resistive pulse detection, the pulse amplitudes a re supposed to be the same when

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111 analyte moves from different directions of the nanopore. The current pulse amplitude depends on how much charges in the supporting electrolyte solutio n were replaced by the analyte. However, in the experiment s involving the coating reagent will depend on how much the local charges on the pore wall were affected. The current pulse amplitudes depend on how large the difference of steady state current valu e at the on and off states. As shown in Figure 43, when the anode (+1 V) was on the base side of the membrane, the absolute current difference between the on and of f state was only about 900 pA at +1 V which determined the maximum current pulse amplitude. However, there was a much larger absolute current difference between the on and off states when the anode was on the tip side of the membrane The maximum difference was about 9000 pA at 1 V, which allow ed large pulse signals occurs It is interes ting that the experimental data was coincident with our prediction. The c urrent pulse amplitude ( 679 179 pA) was larger when the nanoparticle moved from base to tip and the pulse amplitude (302 68 pA) was smaller when they moved from tip to base (as shown in Figur e 4 8). Current pulse duration The other important parameter for the resistive pulse events is the pulse duration, which might be able to reflect the information about the interaction between analyte and nanopor e surface. Generally, the current pulse is longer if the analyte move slower i n the sensing zone of nanopore. In our experiment, it was noticed that the average current pulse duration (789 777 ms) when the nanoparticles move d from base to tip was much longer than the duration (22 20 ms) when t hey move d from tip to base. We think the average pulses duration was shor t because the nanoparticles had fewer chances to interact with the pore w all and stay less time because the nanopore became wider alo ng the axis. However, the nanoparticle might collide with the nanopore for multiple times when they moved from bas e to tip and stay longer time because the

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112 nanopore became narrower along the axis. It is reasonable that the longer pulse duration is because of the multiple times col lision of nanoparticle on the pore wall. Current pulse frequency In these experiments, we studied the translocation of 29 nm diameter PS nanoparticles through the asymmetric nanopore with ~ 85 nm tip and ~ 640 nm base. W hen the feed solution was on the tip side, the sensing zone at tip was exposed to the bulk analyte solution. When the feed solution was on the base si de, however, the sensing zone was far from the base and exposed to the solution with lower analyte concentration than t he bulk because partition problem. This difference might cause the pulse frequency difference when we transport ed analyte nanoparticles from the different directions. We did observe such difference in our resistive pulse measurement. It was obtained about 325 22 puls e events on the average when the nanoparticles were transported from tip to base and only an average 28 8 counts when they were transport ed from the other direction. We think that the collisions between the nanoparticles themselves in the ba se region also might slow down nanoparticle translocation. Control E xperiments for Studies of the Hypothesized Sensing Mechanism We think there is one key criterion for successful detection of the analyte according to our proposed mechanism, which is that the nanopore need to rectify the ion current and the ion current rectification has to change according to the surface charge conditions. We tried to prove such a criterion by using nanopore with different geometries and different salt concentration support ing electrolyte solution. As we known, the ion current rectification is widely observed in asymmetric nanopore filled with electrolyte solution, especially when the ionic strength is low.32, 57 If the sensing experiment was conducted in the solution with high salt conc entration where ion current did not rectify, we are not supposed to observe the ion current rectification switch between the on and

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113 off state aft er adsorption of coating reagent. However, it was found out that the PS nanoparticle s and the coating reagent Hoechst 33258 easily aggregated and precipitate d in 100 mM KCl or high concentration solution without adding surfactant to stabilize the monodispe rsed nanoparticles. Therefore, w e were unable to perform such a experiment to prove the mechanism. W e tested the proposed mechanism by using a single straight nanopore, instead of the asymmetric nanopore in mica but still in a low salt concentration solution (10 mM phosphate buffer at 7.0 with 10 mM KCl). It is well know n that the straight nanopore with uniform surface charge distribution will not rectify the ion current even in very low ionic strength solution. Therefore the ion current rectification switch did not occur even though the local charge inverse d when the coating reagent adsorb ed on the pore wall and interacted with nanoparti cles. Our experiment with a straight nanopore in mica membrane (equivalent diameter ~ 75 nm) showed that no pulse event was observed. This experiment demonstrated that ion current rectification in asymmetric nanopore is important for such sensing mechanis m. However, further inves tigations about the proposed mechanism are still necessary and the exactly process how the coating reagent interacts with the analyte and affect s the current pulse signals are important. Conclusions In this research, a single pyramidal nanopore in mica mem brane was used to study the resistive pulse detection of model analyte. Different from the conventional mechanism, a new signal producing mechanism was proposed. Such mechanism involves the use of a cationic coating reagent to modulate the nanopore surface local charge. The analyt e interacted with the coating reagent and temporarily affected the nanopore tip surface charge condition and c urrent pulses with large amplitude were observed. It was observed that there were difference s in the current pulse shape, pulse amplitude, pulse duration and pulse frequency when the nanoparticle model analyte was transported from different direction of the nanopore We believe that the

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114 proposed mechanism is a first step to an alternative means of improving the detection sensitivity and increasing the signal intensity. However, further investigations are necessary to explore and prove the mechanism to improve the sensing effect.

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115 Figure 41. Sche matic of hypothesized mechanism of resistive pulse detection. A) PS particle s are transported from tip to base. B) PS particle s are transported from base to tip.

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116 Figure 42. Size distribution of poly(styrene) nanoparticle s in the buffer solution. It was measured using dynamic light scattering. The red curve is Gaussian fit data. The solution was a 10 mM phospha te buffer at 7.0 also contained 10 Hoechst 33258. PS particle concentration was 50 nM. Figure 43. Current voltage curves of a single pyramidal nanopore in mica measured in different supporting electrolyte solution. Blue curve: 10 mM phosphate buffer at pH 7.0 with 10 mM KCl. Red curve: 10 mM phosphate buffer at pH 7.0 with 10 m M KCl and 15 0 20 40 60 80 100 0 20 40 60 80 100 NumberDiameter (nm) -1.0 -0.5 0.0 0.5 1.0 -10 -5 0 5 10 Current (nA)Voltage (V)

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117 Figure 44. Current time transients f or conventional resistive pulse detection of nanoparticles using a single pyramidal nanopore in mica. A) Steady state current without nanoparticles. B) A solution (10 mM phosphate buffer at pH 7.0 with 10 mM KCl) contains 50 nM PS partic les with diameter at 29 nm was place at tip side of the membrane Pyramidal nanopore dEq tip ~ 85 nm, dEq base ~ 640 nm. Transmembrane potential = 100 0 mV. Figure 45. Current time transients for coating reagent assisted resistive pulse detection of nanoparticles using single pyramidal nanopore in mica. A) Steady state current without nanoparticles. B) A solution (10 mM phosphate buffer at pH 7.0 with 10 mM contains 50 nM PS particles with diameter at 29 nm was place at tip side of the membrane. Pyramidal nanopore dEq tip ~ 85 nm, dEq base ~ 640 nm. Transmembrane potential = 1000 mV.

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118 Figure 46. Current time transients for coating reagent assisted resistive pu lse detection of nanoparticles using single pyramidal nanopore in mica. A) Steady state current without nanoparticles. B) A solution (10 mM phosphate buffer at pH 7.0 with 10 mM nm was place at base side of the membrane. Pyramidal nanopore dEq tip ~ 85 nm, dEq base ~ 640 nm. Transmembrane potential = 1000mV. Figure 47. Expanded views of current pulse for nanoparticles translocation from the opposite directions. A) From tip to base B) F rom base to tip. Tip diameter = 17 nm. Pyramidal nanopore dEq tip ~ 85 nm, dEq base ~ 640 nm. Transmembrane potential = 1000 mV.

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119 Figure 48. Histogram of current pulse amplitude for nanoparticle s translocation from the opposite direction s. ( Red) N anoparticle s move d from tip to base (Blue) Nanoparticles move d from base to tip. Figure 49. Histogram of current pulse duration for nanoparticles translocation from the opposite directions. (Red) Nanoparticles move from tip to base. (Blue) Nanoparticles move from base to tip. 200 400 600 800 1000 1200 0 20 40 60 80 Number of countsCurrent pulse amplitude (pA) 0 200 400 600 800 1000 1200 0 50 100 150 200 250 Number of countsDuration (mS)

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120 CHAPTER 5 STUDIES OF ELECTROOSMO TIC FLOW RECTIFICATI ON IN PYRAMIDAL NANOPORES IN MICA ME MBRANE S Introduction Electroosmotic flow (EOF) is an electro kinetic phenomenon that occurs when an ionic current is passed through a channel or porous medium that contains excess surface charge.124 132, 142 EOF is used to pump fluids throu gh microfluidic devices126 and capillary electrophoresis columns127. While the pore and channels typically have a symmetrical shape, there is increasing interest in transport through asymmetrical pores24, 120 and channels143, for example conically shaped pores in polymer membranes76. More importantly, t ransport in conically shaped pores is of interest because such pores act as ion current rectifiers; i.e., the magnitude of the current flowing through the pore depends on the polarity of the potential difference applied across the membrane.57 It occurred to us that this ioncurrent rectification phenomenon might produce a corresponding rectification of the EOF rate across the membrane. If this is true, such membranes could be used as EOF rectifiers, yielding high flow rates for one polarity and low flow rates when the polarity is reversed. We report here the first demonstration of this rectified electroosmotic flow effect. Experimental Materials Muscovite mica (KAl2(AlSi3)O10( F, OH)2) membranes, ~10.4 m thick and 3 cm in diameter, were obtained from Spruce Pine Co (Spruce Pine, NC). The linear accelerator at GSI (Darmstadt, Germany) was used to ion track the mica; Pb2+ ions of ~8.6 MeV kinetic energy were used. Samples with 105 track cm2, were prepared. Hydrofluoric acid (~50%) was obtained from Acros Organics USA (Morris Plains, NJ). Phenol was obtained from Sigma Aldrich (St. Louis, MO). All other chemicals were reag ent grade and used as received. Purified water,

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121 obtained by passing house distilled water through a Barnstead, E pure water purification system, was used to prepare all solutions. Fa brication of Pyramidally Shaped N anopores in Muscovite Mica The method of fabrication and characterization of pyramidally shaped nanopore s were discussed in detail in Chapter 3. Briefly, a ~1 cm diameter hole was cut i nto a ~4x4 cm piece of parafilm, and the multitrack mica membrane was sandwiched between two such film s. The sandwiched membrane was placed between the two halves of the etching cell described previously. The total area of mica membrane exposed to solution is about 0.785 cm2. A 3 mL of 10 M NaOH solution was pipetted into one of the cells. A Pt wire electrode was i nserted into each half cell, and ~3 mL of 10 M HF w as then added to the other half cell. The Keithley 6487 picoammeter/voltage source was used to apply a trans membrane voltage of 10 V (with the anode in the HF solution) and measure the resulting ionic cur rent flowing through the membrane during etching. After 10 minutes the etch was terminated by placing 1 M NaOH on both sides of the membrane and applying 10 V for 5 minutes, to ensure complete neutralization of the HF in the pores. The membrane was then ri nsed with water, and water was placed in both half cells for 30 minutes to wash the NaOH from the pores. We call this procedure a 10 minute etch cycle. The same mica membrane is anisotro pically etched for one to four 10minute cycles. The current voltage curves were measured after each cycle, and the EOF experiment s w ere conducted at each pore size. Fabrication of Straight N anopores in Muscovite Mica To compare the electroosmotic flow in asymmetric nanopor e s straight nanopores with equal size openings on both sides of the membrane was prepared. A symmetric chemical etching method was used to prepare straight nanopores in the ion tracked muscovite mica membranes.

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122 Instead of using 10 M NaOH as stopping solution on one side of the membrane and 10 M HF as etching solution on the other, a ~3 mL of 5 M HF was placed on both sides of the membrane to etch mica from different directions at the same time. After 2.5 minutes the chemical etch was terminated as the sam e way we did for etching asymmetric nanopores. The nanopores etched at this condition are supposed to have almost equal size openings on both sides of the membrane.29 The same membrane was etched for another 2.5 minutes, 5 minutes and 10 minutes to achieve different pore sizes The total etch time after each step were 2.5 minutes, 5 minutes, 10 minutes and 20 minutes respectively The current voltage curves were measured in 10 mM phosphate buffer at pH 7.0 after each etches. The electroosmotic flow in these straight nanopores at different sizes was studied as well after each etch step Nanopores C haracterization For the multipore mica membranes, field emission scanning electron microscopy (FE SEM, JEOL 6335F) was used to measure the dimensions of the nanopore openings on both sides of the membranes. A s traight nanopores membrane etched for 20 minutes and a n asymmetric nanopores membrane etched for four 10minute cycles were used for SEM imaging. Even though it is not necessary to calculate the nanopore membrane conductance by using current voltage curves, it is still useful to obtain the ion current rectification infor mation from those curves. Briefly, the mica membrane containing nanopores was mounted between the two half cells, and an electrolyte solution of 10 mM phosphate buffer at pH 7.0 was placed on both sides of the membrane. A Ag/AgCl electrode was placed into each half cell solution, and a current voltage curve was obtained by scanning the potential from 10.0 V to +10.0 V at a scan rate of 10 Vmin1.

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123 Phenol Transport M easurements A mica membrane containing nanopores (pore density ~105 per cm2) was mounted betw een the two half cells and a 0.7 85 cm2 portion of the mica membrane was exposed to the electrolyte solution. The rate of EOF was investigated by measuring the flux of a probe molecule (phenol) across the membrane and into the permeate half cell. A 3 mL of permeate solution with 10 mM phosphate buffer pH 7.0 was added to the cell on one side of the membrane. A 3 mL feed solution that was 10 mM phosphate buffer pH 7.0 containing 10 mM phenol was added to the other. A Pt wire electrode was placed into each hal f cell. The Pt wire in the feed solution was the working electrode (anode) and the Pt wire in the permeate solution was the reference and counter electrode (cathode). Since the mica surface is negatively charged,147 the EOF direc tion is same as the direction of cations migration (anode to cathode). A Solartron SI 1287 electrochemical interface (Farnborough, UK) was used to apply a constant cu through the multipore mica membrane After 20 minutes, the ion current was terminated and both the permeate solution and feed solution were pipette d out of the two half cells. An Agilent 8453 UV visible spectrometry system (Waldbronn, Germany) was used to measure the UV Vis absorbance of phenol in the permeate solution. The conc entration of phenol in permeate solution was calculated from the calibration curve measured at wavelength 270 nm. We called this procedure a 20minute transport cycl e. After the UV visible spectrum measurement, the permeate solution was placed back into th e original half cell for next 20minute cycle transport. To reduce the effect of oxidized phenol on transport and the UV Vis spectrum a fresh feed solution with 10 mM of phenol was placed into the cell on the original feed side. This 20minute tr ansport c ycle was repeated for six times

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124 and the total time we ap plied current was 120 minutes. The UVvisible spectrum of permeate solution was measured after each 20 minute transport cycle. Then the permeate and feed solution were switched to the other side of th e membrane and the flux from the opposite direction was me asured for another 120 minutes. The diffus ion of phenol across the asymmetric nanopore membrane without applying voltage was also measured at the same 20 minute time intervals for the total 120minu tes transport. But for the straight nanopores membrane with very small openings the diffusion f lux was too small to transfer enough phenol for accurate measurement. A much longer time interval to measure the phenol concentration was necessary, so a 2 hour transport cycle was performed in this case and the to tal transport time was 6 hours. EOF measurement was performed for asymmetric multipore mica membrane after each 1 0minute etch cycles and straight multipore mica membrane etched for 2.5, 5, 10, 20 minut es. Results and Discussion Characterizations of Nanopore S ize in Multipore Mica Membrane To calculate the size of a nanopore opening in single pore membrane, we usually measure the nanopore conductance when it is filled with electrolyte solution. However, two reasons make it inappropriate way to characterize the average nanopore size for multipore membrane. The first reason is the uncertainty of nanopore density in the mica membrane. Even though we can calculate the average n anopore density from the scanning electron microscopy images, it still brings the error for accurate calculation of nanopore size. Another reason is the uncertainty of voltage drop across the membrane. The electrochemical measurement and calculation requir e knowledge of the exact membrane conductance, which is calculated from the linear part of current voltage curves. In single nanopore membrane, the resistance of a nanopore is tremendous so that it is reasonable to assume that the voltage drop across the m embrane is equal to the

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125 voltage applied between the electrodes. However, the multipore membrane resistance is smaller and is comparable to the solution resistance. As a result, a portion of the voltage drop will be on the solution. Therefore, it is not appropriate to assume the voltage drop across the membrane is equal to the total voltage between two electrodes. Only after making a correction f or the voltage on membrane, is it possible to calculate the membrane resistance accurately. Instead, the nanopores in multipore mica can be studied by using scanning electron microscopy (SEM) To e nsure that the pore density of the membrane is the same for electroosmosis experiments we only image d the membrane sample after completion all the EOF measurement s It is a lso difficult to image the nanopores when they are too small. In these studies, we imaged the asymmetric nanopore base and tip openings after they were etched for four 10minute cycles. We also imaged the straight nanopore openings on both sides of the mem brane after they were etched for 20 minutes. Figure 51AB shows the SEM images of base and tip openings of asymmetric nanopores in a mica membrane etched for four 10minute cycles. It is obvious that the base was much larger than the tip. Figure 5 1CD illu strates that straight nanopore openings on both sides of the membrane ha d almost equal dimensions, which was the same as we predicted. It was also shown that the nanopore ha d a straight shape with equal size rhomboidal openings if the membrane was etched symmetrically from both sides.29 The lengths of major axis (al) of the rhomboidal op enings were measured from the SEM images. The equivalent diameter (dEq) of nanopore openings can be calculate d from the long diagonals using Eq uation 51, = 0 .606 ( 51) It was assumed that the etch rate was constant for base and tip respectively. The average size of

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126 nanopore openings on both sides of the membrane were calculated from the etch rate on each side and the total etch time. The radial etch rate of straight nanopore using 5 M HF is about 1.3 nm min1, which is comparable to the literature.29 The radial etch rate of base an d tip for pyramidal nanopores were about 3.75 nm min1 and 0.5 nm min1, respectively. These values are close to the data we reported in chapter 3. The calculated equivalent diameters of openings for straight nanopores etched for different time s are listed in Table 5 1. T he nanopore equivalent diameters of base and tip openings for asymmetric nanopores after each etch cycle were calculated and shown in Table 52. Ion Current Rectification in Asymmetrically Shaped Multipore Mica M embrane Figure 52 shows the current voltag e curves of straight and asymmetric multipore mica membrane s etched for different time s For straight nanopore s the conductance of the membrane increased when the nanopore size became larger and there was no significant ion c urrent rectifi cation observed for any pore size. However, strong ion current was observed when the nanopore s were asymmetrically etched for only one and two 10minute etch cycles. At longer etch time, the ion current rectification was not significant because of the larg er pore size and the multipore membrane resistance became comparable to the solution resistance. We define the ion current rectification ratio as the ratio of absolute ion cur rent value recorded at 10 V to the current value at +10 V. The ion r ectification ratio is about 1 for different pore size s for straight nanopore membrane s Phenol Transport Measurements Electroosmotic flow in straight multipore mica membrane The electroosmotic flow in the straight multipore mica membrane with different pore sizes were studied. When the nanopores were smaller, the membrane resistance was larger. From the Ohms law, a

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127 larger voltage was needed to maintain the constant current for the nanopore with larger resistance. For example, an average voltage of 9.0 V was applied to maintain the constant current during the transport experiment when the nanopore Lower voltages were applied when the nanopores were larger and membrane resis tance is lower. The applied voltage was about only 3.5 V when the average nanopore equiv alent diameter was about 52 nm. The Helmholtz Smoluchowski equation defines the relationship between the electroosmotic velocity (veof) and the linear electric field gr adient (E(x)) across the membrane = ( )/ ( 52) electroosmotic velocity veof is proportional to the electric field strength E(x) A larger electroosmotic ve locity will give a larger electroosmotic flux. However, the voltage drop across the multipore membrane varied with multiple nanopore membrane resist ance, it was hard to know the accurate electric field strength in the pore. Equation 53 that deduced from the Helmholtz Smoluchowski equation might make the explanation easier. = / ( 53) where Japp is the constant applied current density. It is obvious that value of Japp was the same no matter which direction the current flowed through the pore Figure 53 shows the transport rate of phenol across the straight multipore membrane with pore equivalent diameter at 7 nm. It was observed that the diffusion rate of phenol across the membrane is very small and the transport rate with applied current was much larger. It is obviously that the electroosmotic flow in the se nanopores greatly enhanced the transp ort rate of

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128 the neutral molecule phenol. We also observed exactly equal transport rate when phenol was transported from the opposite directions Equation 53 predict s that the electroosmotic flow velocity would be smaller if we increase the nanopore size. across the membrane and the current density Japp decreased as the total nanopore crosssection area increased. As will be discussed be low, the experiments proved this prediction. Calculation of electroosmotic flow velocity in nanopore membrane Nernst Plank equation can be used to describe the mass transfer in a one dimensional case as137 ( ) = ( ) ( ) + ( ) ( 54) where Ji(x) is the flux of species i, Di is the diffusion coefficient, i(x) / is the concentration gradient, (x)/i and Ci are the charge and concentration of species i respectively. v (x) is the velocity of a volume element. The three te r ms in the equation c orrespond to the diffusion, migration and convection of species i. In this experiment, Phenol (pKa = 9.99) will be transported mainly by diffusion and EOF because the molecule is neutral at pH 7.0. The magnitude of electroosmotic ve locity (veof) was determined via an analysis of the phenol flux with and without the applied transmembrane current as described previously.128, 152 Briefly, an enhance ment factor is given by = / ( 5) i.e., the ratio of the flux in the presence of an applied current t o the flux without any current. F ollowing Srinivasan and Higuchi152, the peclet number (Pe) can be calculated from E via = / (1 exp ( ) ) ( 6) Pe can then be used to calculate veof using

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129 = / ( 7) where l is the thickness of the membrane and D is the diffusion coefficient (~8.9106 cm2s1). Electroosmotic flow in asymmetric multipore mica membrane The current voltage curves in Figure 52B clearly demonstrates that there was ion current recti fication occurred in the asymmetric nanopore mica membrane. The current was larger when the anode was on the nanopore tip side of the membrane (on state) and current was lower at the opposite electrode polarity. Because the nanopores surface is negatively charged and the size of nanopores size is comparable to the thickness of electrical double layer the membrane bec ame cation permselective.30 According to literature 24, 74 Ramrez and White et al. have demonstrated a possible explanation for this ion current rectification phenomenon in asymmetric nanopore. As shown in Figure 54, when the an ode is on the tip side of the nanopore, cations are transported from the tip side (anode) to the base si de (cathode). Anions are electrophoretically driven from the external solution at the base side of the membrane towards the tip by the electric field. H owever, they cannot pass through the nanopore because of electrostatic repulsion from the negatively charged pore wall close to the tip region. As a result, anions accumulate in the nanopor e. To maintain electrostatic neutral ity more cations are needed in this tip region to balance the extra anions. Therefore, the local salt concentration increases and the resistance of the pore solution decreases Thus, a higher ion current was obtained when a transmembrane potential was applied in this way We refer to this situation as the ion accumulation mode. Since the mica nanopore surface is negatively charged141, electroosmosis will proceed in the same direction as that of cations electromigration. Therefore, in this ion accumulation mode, the electroosmotic flow is from the tip opening to base opening in the nanopore.

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130 On the contrary, when the anode is placed on the base side of the nanopore (Figure 5 4 right) cations are transported from the base opening to the tip opening of the nanopore. Anions inside the nanopore are vacated from the nanopore and into the bulk solution on the base side by the electric field. However, the anions on the tip side are less likely to enter the nanopore because of electrostatic repulsion from the anionic pore wall close to tip. As a result, the local salt concentratio n in the nanopore decreases, and t he resistivity of pore solution increases. When a transmembrane potential was applied in this way a lower ion current was obtained. We refer to this situation as the ion depletion mode and the EOF direction in this mode is from base to tip in the nanopore. In other word, t he nanopore solution resistivity is larger in th e ion depletion mode and it is smaller in the ion accu mulation mode. When the EOF was driven from base to tip, the pore solution resistivity was high because it was in the ion depletion mode. We expected to observe a larger electroosmotic flow velocity in this c ase according to Equation 5 3. When the EOF wa s driven from tip to base the pore solution resistivity was lower because it was in the ion accumulation mode and we were expected to observe a lower electroosmoti c flow velocity. The experimental results pro ved these prediction s As shown in Figure 55, a much higher phenol transport rate was observed when the flux was from the nanopore base to tip. The calculated the electroosmotic flow velocity veof was about 3.8 mms1 and 0.37 mms1 when phenol was transported from the different directions (Table 5 3) It is important to point out that because the field strength in an asymmetric pore varies with position down the length of the pore, veof will also vary down the pore length. The veof values reported here are the net velocities in that they were obtained from the net rate of transport of phenol from the feed solution to the permeate solution.

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131 It was also observed that the electroosmotic flow velocity became smaller if we increased nano the current density Japp decreased as the total nanopore crosssection ar ea increased. Another reason that th e EOF velocity decreased so rapidly for the transport fr om base to tip is because the ion depletion effect decreased so much wh en the nanopore size increased. Relationship betwee n the ion current rectification and electroosmotic flow rectification It is clearly demonstrated that the rectified electroosmotic flo w is the direct consequence of ion current rectification. The veof values obtained are shown in Table 53. The extent of EOF rectification was quantified by defining a parameter called the EOF rectification ratio, reof, which is veof for the off state po larity divided by veof for the on state polarity. We see that reof increases with decr easing size of the pore (Table 5 3); i.e. the extent of EOF rectification is greater in smaller pores. While we will have more to say about this point in the future, th is is certainly reasonable since the chance of achieving the requisite h igh cation transference number is greater in a smaller pore. The extent of ioncurrent rectification is quantified by the ion current rectification ratio, ric, defined here as the abso lute value of the current at 10 V divided by the current at +10 V. The ric values (Table 5 3) show that like reof, ion current rectification is greater in smaller pores We also see that reof scales with ric, which is reasonable since both phenomena deriv e from the change in pore solution resistivity with sign of the applied transmembrane potential. The proportionality between reof and ric indicates that a simple measurement of ric (Figure 5 2) can be used to predict whether a membrane will be a good EOF rectifier. Conclusions We have show n for the first time that asymmetric nanopore mica membran e rectifies EOF. As theory predicts, and as we have shown expe rimentally, EOF rectification was observed in

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132 pores that show the more familiar ion current rectification. We demonstrate that such rectified EOF is the direct consequence of the ionic current rectification at a constant applied current. The flux is larger when the EOF direction is from the base of pyramidal nanopore to the tip side. The ratio of the EOF flux from opposite dire ction is related to the level of the observed ion current rectification in such asymmetric nanopore When there is no ioncurrent rectification, su ch as in the case of straight pores, we observe no difference between the EOF fluxes from the opposite directions. It is important to point out that the extents of both ioncurrent and EOF rectification increase with charge density on the pore wall. As a result, mica is an especially propitious material for such rectifiers since th e anionic charge density on mica147 exceeds by a factor of ~2 of the charge density on the more commonly studied polymeric membranes .148

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133 Fig ure 51. FESEM images of asymmetric nanopore and straight nanopore openings in mica membranes used in EOF experiments. A) Base openings in asymmetric nanopore membrane B) Tip openings in asymmetric nanopore membrane. CD) S traight nanopore openings on opposite side s of the membrane.

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134 Figure 52. Current voltage curves of multipore mica membrane with straight or pyramidal nanopores e tched for different time. A) S traight nanopore membrane. B) Pyramidal nanopore membrane -10 -5 0 5 10 -1.00 -0.75 -0.50 -0.25 0.00 0.25 0.50 0.75 1.00 ACurrent (mA)Voltage (V) 2.5 minutes 5 minutes 10 minures 20 minures -10 -5 0 5 10 -1.5 -1.0 -0.5 0.0 0.5 1.0 1.5 BCurrent (mA)Voltage (V) 10 minutes 20 minutes 30 minutes 40 minutes

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135 Figure 53. Amount of phenol transported across a straight multipore mica membrane Equivalent diameter deq= 7 nm (Blue ) Phenol t ransport ed from different (Red ) P henol diffused across the membran e. 0 20 40 60 80 100 120 0 50 100 150 200 250 Amount of Phenol (nmol)Time (minute)

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136 Figure 54. Schematic of ion accumulation and ion depletion in asymmetric nanopore and direction of corresponding electroosmotic flow. Figure 55. Amount of phenol transported across an asymmetric multipore mica membrane. Equivalent diameter of tip deq T = 10 nm and equivalent diameter of base deq B = 7 4 nm. (Blue) Phenol transported from different directions with applying constant ( base to tip, tip to base). (Red ) Phenol diffus ed across the membrane. 0 20 40 60 80 100 120 0 200 400 600 800 1000 Amount of Phenol (nmol)Time (minute)

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137 Table 5 1. Estimated equivalent diameters of straight nanopore openings. Etch time (minutes) Average opening on both sides d eq (nm) 2.5 ~7 5 ~13 10 ~26 20 52 5 (SEM) Table 5 2. Estimated equivalent diameters of asymmetric nanopore base and tip openings. Etch time (minutes) Base d Eq B (nm) Tip d Eq T (nm) 10 ~74 ~10 20 ~148 ~19 30 ~ 222 ~29 40 ~296 7 (SEM) ~38 6 (SEM) Table 5 3. EOF velocities and ioncurrent and EOF rectification ratios for the membranes studied here. Tip d Eq T ( nm) Base d Eq B (nm) v eof (mm s 1 ) r eof r ic Base to Tip Tip to Base 10 74 3.8 0.37 10.3 5.3 19 148 1.7 0.35 4.9 2.7 29 222 0.55 0.32 1.7 1.3 38 296 0.32 0.23 1.4 1.2 7 7 12 12 1.0 1.0

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138 CHAPTER 6 CONCLUSIONS The goal of this research was to develop asymmetricall y shaped nanopores in thin film materials and to investigate the potential applic a tions of these devices as resistive pulse sensors. Chapter 1 introduced and reviewed all relative background information for these studies, including the track etch method, characterization of asymmetric nanopore s ion current rectification phenomenon in asymme tric nanopore and nanochannel, resistive pulse sensing with biological and artificial nanopores. In Chapter 2, polyelectrolyte sodium poly(styrene sulfonate) was used as a model analyte to study s ome fundamental parameters of the resistive pulse sensing. In this work, a single conically shaped nanopore in PET membrane was fabricated by using twostep chemical etch method. It was shown that the polyelectrolyte translocation through the nanopore tip could be detected using such nanopore resistive pulse detector. It was fo und that the current pulse frequency increased at high analyte concentration and applying a higher transmembrane potential could increase the pulse frequency. These studies provide information about improving the det ection limit of resistive pulse method by using high voltage. In Chapter 3, a multi cycle anisotropic chemical etch method was developed to fabricate asymmetrically shaped nanopores in muscovite mica membrane The effects of the etchant concentration, transmembrane potential and the number of etch cycle on the nanopore opening size and geometry were studied in detail and the etch condition was optimized. Asymmetric nanopores were prepared in both the multi track and single track mica membrane s A strong ion current rectification phenomenon was observed in the pyramidally shaped nanopore embedded in mica membrane especially in the low ionic strength solution. We proposed that the strong ion current rectification in mica is because of the high surface charge density on mica A much

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139 weaker ion current rectification was obtained when the charge s on the mica nanopore surface w ere partially removed. In Chapter 4, a new mechanism based on the local charge inversion induced ion current rectification switch was propo sed for resistivepulse detection. This mechanism was tested by using a single asymmetric nanopore in muscovite mica membrane. A coating reagent was used to temp orarily change the surface charge condition and resulted in ion current rectification change. W e observed strong current pulse signal s when the model analyte poly( styrene ) nanoparticle s were transport ed through the nanopore The current pulse shape, pulse amplitude and pulse duration were greatly different when the nanoparticle s w ere transported fro m the opposite directions in the nanopore. We believe that the nanopore local charge inversion is the key in this mechanism. However, the details about this proposed mechanism are still under investigation In Chapter 5, an interesting electro kinetic phenomenon, electroosmotic flow rectification in asymmetric nanopore was reported. This phenomenon was studied by monitor ing a probe molecule, phenol transported across asymmetric nanopores embedded in mica membran e. It was found that the transported rate of phenol across the membrane was higher from base to tip and it was lower from the opposite direction. We think this is because the electroosmotic flow velocity was different in each case. It was found that this rectified electroosmotic flow was because o f the well known ion current rectification in asymmetric nanopore s T he ion accumulation and ion depletion in the asymmetric nanopore s affected the pore solution resistivity and caused such rectified electroosmotic flow when a constant current was passed through the asymmetric nanopore membrane s However, this rectified electroosmotic flow was not observed in the straight nanopore s

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140 The work presented here has demonstrated that single asymmetric nanopores can be used for resistive pulse sensing studies. Resistive pulse sensing of different analyte s using synthetic nanopore is relative new area with lots of phenomena and theories need to be investigate d and proved. In this study, the successful fabrication of asymmetric nanopore s in crystal materials mica mem brane shows opportunities for many research based on its capability to strongly rectify the ion current flowing through the nanopore. Although, further studies will be needed to co ntinue to exploring the applications the work presented here may hopefully ser ve as a foundation for such research

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149 BIOGRAPHICAL SKETCH Pu Jin was born in Kunming with his parents are chemists. Under the influence of his parents, he entered Xiamen University in Xiamen, China in 1997. He spent 4 years on undergraduate study and obtained a Bachelor of Science degree in materials c hemistry in July 2001. After that, he went to University of Science and Technology of China in Hefei and started his research in the fields of functional nanomaterials synthesis under the guidance of Dr. Qianwang Chen. In August 2004, after he obtained his Master of Science degree in i norg anic c hemistry, Pu Jin joined Dr. Charles R. Martin research group in the Department of Chemistry at University of Florida. He continued his research in the fields of nanomaterials, especially on the fabrication of nanostructure in polymeric and inorganic thin film and their application in sensing and separation. He completed his research in December 2009, obtaining a Doctor of P hilosophy degree.