<%BANNER%>

Developing Resistive-Pulse Sensors Using Artificial Conical Nanopores in Track-Etched Polymer Membranes

Permanent Link: http://ufdc.ufl.edu/UFE0041611/00001

Material Information

Title: Developing Resistive-Pulse Sensors Using Artificial Conical Nanopores in Track-Etched Polymer Membranes
Physical Description: 1 online resource (243 p.)
Language: english
Creator: Horne, Lloyd
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: angle, biocompatible, biosensor, cone, conical, current, etch, ion, membrane, nanopore, peg, polyethylene, polyimide, pulse, rectification, resistive, sensing, terephthalate, track
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: DEVELOPING RESISTIVE-PULSE SENSORS USING ARTIFICIAL CONICAL NANOPORES IN TRACK-ETCHED POLYMER MEMBRANES The objective of this research is to develop sensors based on the resistive-pulse method using conical nanopores and investigate properties of such pores that impact their sensing capabilities. In the first section of this work, sensing of a model protein is demonstrated using a single, conical nanopore embedded in a track-etched polymer membrane. The pore surface was modified with a thin, conformal gold film and subsequently functionalized with thiol-modified poly(ethylene glycol)(PEG) to prevent non-specific protein adsorption. Single protein molecules were detected and counted as downward current-pulses as they were electrophoretically driven through the pore. The frequency of these current-pulses was found to vary exponentially with the applied transmembrane potential. Removal of the PEG and gold layers revealed current-pulses that went both upward and downward. Such a phenomenon had not been previously observed with resistive-pulse sensors constructed from track-etched conical nanopores. The impact of this effect on components of the current-pulse signature is discussed. Previous resistive-pulse sensors derived from track-etched polymer membranes have been configured such that the net surface charge on the analyte, the surface charge of the pore wall, and electrode at the tip opening all have the same polarity (i.e., negatively-charged). The second part of this work presents an example of a resistive-pulse system where the net surface charge on the analyte and electrode at the tip are both opposite in polarity of the pore surface (i.e., both are positively-charged). That is, the resistive-pulse sensing of a model cationic analyte, poly-L-lysine-conjugated gold nanoparticles using a single, conical nanopore in track-etched poly(ethylene terephthalate)(PET) is presented. Current-pulses were observed down to the femtomolar concentration level and exclusively upward. Such pulse direction reflects the ion current-enhancing effect of the counter-ions accompanying each nanoparticle into the nanopore sensing zone. A definition for the limit of detection in resistive-pulse sensing is presented and discussed. The third part of this work focuses on developing resistive-pulse sensors in an alternative polymer material, polyimide. Progress towards a two step-etch method for tailoring the tip diameter of conical nanopores in polyimide is introduced. Controlling the tip opening diameter during fabrication is absolutely critical to constructing functional resistive-pulse sensors. The tip diameter was observed to scale linearly with the final value of the ion current during the two-step etch. Furthermore, the extent of ion current rectification was found to be inversely proportional to tip size at the tip sizes evaluated. An approach towards loading electrolyte into conical pores in polyimide is introduced involving the use of a wetting agent, vacuum degassing, and perfusion. Carboxylated, fluorescent nanoparticles were then detected using a single, conical nanopore in track-etched polyimide. Current-pulses were exclusively upward and detected using much lower applied transmembrane potentials than those typically used for resistive-pulse sensors housed in PET membranes. This was attributed to the large cone angle and correspondingly lower pore resistance characteristic of conical pores fabricated in polyimide. The fourth part of this work introduces an alternative strategy to electroless gold deposition for functionalizing the surfaces of single, conical nanopores with PEG based on EDC/sulfo-NHS coupling chemistry. Minimizing non-specific pore surface adsorption is absolutely critical in resistive-pulse sensing. X-ray photoelectron spectroscopy and ionic pore conductance were utilized to study the non-specific adsorption of three model proteins, BSA, fibrinogen, and lysozyme, to the surfaces of single, conical nanopores before and after functionalization with amine-modified PEG. The presence of the PEG was found to reduce the non-specific adsorption of each protein to varying extents. Thus, this represents progress towards producing more biocompatible nanopores for developing resistive-pulse applications for biological analytes. The final part of this work presents cone angle studies on pores fabricated in PET membranes. The electric field strength distribution inside two single, conical-shaped nanopores having identical tip diameters but different base diameters (i.e., one large and one small) was evaluated via finite element simulations. These simulations show the electric field strength, which is directly proportional to current-pulse frequency, increases more with increasing cone angle than with increasing transmembrane potential. Thus, this provides the impetus for fabricating larger cone angle nanopores. However, before doing resistive-pulse sensing, methods for fabricating, controlling and reproducing large cone angle nanopores are needed. A high voltage etching approach in high track density PET membranes was not successful due to increased resistive-heating. Thus, a non-aqueous approach to fabricating single, conical nanopores having a larger cone angle than pores typically produced via the aqueous two-step etch method is presented. Using this approach, the effect of increasing cone angle on ionic pore conductance and ion current rectification was evaluated. Increased ionic pore conductance and decreased ion current rectification were observed with the larger cone angle pores relative to those having a smaller cone angle.
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 Lloyd Horne.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Martin, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041611:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041611/00001

Material Information

Title: Developing Resistive-Pulse Sensors Using Artificial Conical Nanopores in Track-Etched Polymer Membranes
Physical Description: 1 online resource (243 p.)
Language: english
Creator: Horne, Lloyd
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: angle, biocompatible, biosensor, cone, conical, current, etch, ion, membrane, nanopore, peg, polyethylene, polyimide, pulse, rectification, resistive, sensing, terephthalate, track
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: DEVELOPING RESISTIVE-PULSE SENSORS USING ARTIFICIAL CONICAL NANOPORES IN TRACK-ETCHED POLYMER MEMBRANES The objective of this research is to develop sensors based on the resistive-pulse method using conical nanopores and investigate properties of such pores that impact their sensing capabilities. In the first section of this work, sensing of a model protein is demonstrated using a single, conical nanopore embedded in a track-etched polymer membrane. The pore surface was modified with a thin, conformal gold film and subsequently functionalized with thiol-modified poly(ethylene glycol)(PEG) to prevent non-specific protein adsorption. Single protein molecules were detected and counted as downward current-pulses as they were electrophoretically driven through the pore. The frequency of these current-pulses was found to vary exponentially with the applied transmembrane potential. Removal of the PEG and gold layers revealed current-pulses that went both upward and downward. Such a phenomenon had not been previously observed with resistive-pulse sensors constructed from track-etched conical nanopores. The impact of this effect on components of the current-pulse signature is discussed. Previous resistive-pulse sensors derived from track-etched polymer membranes have been configured such that the net surface charge on the analyte, the surface charge of the pore wall, and electrode at the tip opening all have the same polarity (i.e., negatively-charged). The second part of this work presents an example of a resistive-pulse system where the net surface charge on the analyte and electrode at the tip are both opposite in polarity of the pore surface (i.e., both are positively-charged). That is, the resistive-pulse sensing of a model cationic analyte, poly-L-lysine-conjugated gold nanoparticles using a single, conical nanopore in track-etched poly(ethylene terephthalate)(PET) is presented. Current-pulses were observed down to the femtomolar concentration level and exclusively upward. Such pulse direction reflects the ion current-enhancing effect of the counter-ions accompanying each nanoparticle into the nanopore sensing zone. A definition for the limit of detection in resistive-pulse sensing is presented and discussed. The third part of this work focuses on developing resistive-pulse sensors in an alternative polymer material, polyimide. Progress towards a two step-etch method for tailoring the tip diameter of conical nanopores in polyimide is introduced. Controlling the tip opening diameter during fabrication is absolutely critical to constructing functional resistive-pulse sensors. The tip diameter was observed to scale linearly with the final value of the ion current during the two-step etch. Furthermore, the extent of ion current rectification was found to be inversely proportional to tip size at the tip sizes evaluated. An approach towards loading electrolyte into conical pores in polyimide is introduced involving the use of a wetting agent, vacuum degassing, and perfusion. Carboxylated, fluorescent nanoparticles were then detected using a single, conical nanopore in track-etched polyimide. Current-pulses were exclusively upward and detected using much lower applied transmembrane potentials than those typically used for resistive-pulse sensors housed in PET membranes. This was attributed to the large cone angle and correspondingly lower pore resistance characteristic of conical pores fabricated in polyimide. The fourth part of this work introduces an alternative strategy to electroless gold deposition for functionalizing the surfaces of single, conical nanopores with PEG based on EDC/sulfo-NHS coupling chemistry. Minimizing non-specific pore surface adsorption is absolutely critical in resistive-pulse sensing. X-ray photoelectron spectroscopy and ionic pore conductance were utilized to study the non-specific adsorption of three model proteins, BSA, fibrinogen, and lysozyme, to the surfaces of single, conical nanopores before and after functionalization with amine-modified PEG. The presence of the PEG was found to reduce the non-specific adsorption of each protein to varying extents. Thus, this represents progress towards producing more biocompatible nanopores for developing resistive-pulse applications for biological analytes. The final part of this work presents cone angle studies on pores fabricated in PET membranes. The electric field strength distribution inside two single, conical-shaped nanopores having identical tip diameters but different base diameters (i.e., one large and one small) was evaluated via finite element simulations. These simulations show the electric field strength, which is directly proportional to current-pulse frequency, increases more with increasing cone angle than with increasing transmembrane potential. Thus, this provides the impetus for fabricating larger cone angle nanopores. However, before doing resistive-pulse sensing, methods for fabricating, controlling and reproducing large cone angle nanopores are needed. A high voltage etching approach in high track density PET membranes was not successful due to increased resistive-heating. Thus, a non-aqueous approach to fabricating single, conical nanopores having a larger cone angle than pores typically produced via the aqueous two-step etch method is presented. Using this approach, the effect of increasing cone angle on ionic pore conductance and ion current rectification was evaluated. Increased ionic pore conductance and decreased ion current rectification were observed with the larger cone angle pores relative to those having a smaller cone angle.
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 Lloyd Horne.
Thesis: Thesis (Ph.D.)--University of Florida, 2010.
Local: Adviser: Martin, Charles R.
Electronic Access: RESTRICTED TO UF STUDENTS, STAFF, FACULTY, AND ON-CAMPUS USE UNTIL 2012-08-31

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041611:00001


This item has the following downloads:


Full Text

PAGE 1

1 DEVELOPING RESISTIVE PULSE SENSORS USING ARTIFICIAL CONICAL NANOPORES IN TRACK ETCHED POLYMER MEMBRANES By LLOYD PEYTON HORNE, JR. A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FU LFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2010

PAGE 2

2 2010 Lloyd Peyton Horne, Jr.

PAGE 3

3 To my wife, Jodie, and my son, Zackary

PAGE 4

4 ACKNOWLEDGMENTS In late 2004, I lef t a job in the pharmaceutical industry that I had held for years to return to school full time to pursue a Ph.D. At first, I was criticized by many and understood by few. Nevertheless, it was my strong interest in teaching and academic research that propel led me to make such a sacrifice. In simplest terms, the educational process by which one earns a doctorate boils down to just that, sacrifice. There are times of self doubt, high times associated with experimental success, and many low times of frustration and exhaustion. There are also frequent periods of time one must go without family and friends to conduct research. To go through this encouragement from others in your life. I am very fortunate to have had a great deal of support from my family, friends, and colleagues during my tenure at the University of Florida. First, I must thank my wife, Jodie, for her endless love and support from the beginning. Although she could not directly relate to what I was going through, she understood how important this degree is and has witnessed the emotional toll this process has taken on me and our family. Without her support, understanding, and patience, my success would not have been possible. Despite just being 2 years of age, I also thank my son, Zackary, for the many times thank my parents, Lloyd and Pattie Horne, along wit h my in laws, Gregory and Brenda Strebel, for their continued love, encouragement, and support. my academic career. I must thank the late Dr. Chia y u Li (East Carolina) for introducing me to Austin), Dr. Dennis Johnson (Iowa State), and Dr. W erner Kuhr (formerly of UC Riverside).

PAGE 5

5 Moreover, I must thank my research advisor and mentor, Dr. Charles R. Martin, for taking on a non traditional graduate student such as myself. I am truly grateful for his guidance and support during my tenure at the University of Florida. Whether it was going through the process of editing a manuscript, anticipating peer reviewer comments, scrutinizing research data, putting together great and effective PowerPoint presentations, or simply hunting down key references for grant proposals, I thank Dr. Martin for mentoring me in the many aspects of being a well rounded, professional scientist. Furthermore, Dr. Martin is a masterful storyteller at communicating science. This, along with the many research related anecdotes from his life, has been very helpful during this educational journey. I am grateful for Dr. Martin providing an environment that encourages independent and creative thinking while allowing the freedom to explore. Working in the Martin Research Group has be en challenging and truly a pleasure. I am also very grateful to Dr. David Richardson (former Chemistry Dept. Chair) and Dr. Dan Talham (current Chemistry Dept. Chair) for acknowledging my teaching efforts with 3 letters of commendation, a graduate teaching at the University of Florida. e Baker, Zuzanna Siwy, Youngseon Choi, and Hitomi Mukaibo for their insightful advice. I thank Dr. Elizabeth Heins, Dr. Lindsay Sexton, Dr. John Wharton, Dr. Pu Jin, Dr. Jai Hai Wang, Dr. Mario Caicedo, Gregory Bishop, Kaan Kececi, and Dooho Park for shari ng their experience, ideas, and offering support on my research. I also thank Dr. Heather Hille brenner and Dr. Fan Xu for creating an entertaining lab environment in the beginning. I thank the undergraduate work with, including Amanda Cotton, Lindsey

PAGE 6

6 Anderson, and Xabier Osteikoetxea for their assistance with experiments. I would also like to acknowledge Eric Lambers for his assistance with X ray photoelectron spectroscopy. I also thank Kerry Siebin and Karen Kelley for their help with scanning electron microscopy.

PAGE 7

7 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ........... 4 LIST OF TABLES ................................ ................................ ................................ .................... 10 LIST OF FIGURES ................................ ................................ ................................ .................. 11 ABSTRACT ................................ ................................ ................................ ............................. 18 CHAPTER 1 INTRODUCTION AND BACKGROUND ................................ ................................ ........ 21 Introduction ................................ ................................ ................................ ........................ 21 The Resistive Pulse Method ................................ ................................ ............................... 22 Biological Nanopores ................................ ................................ ................................ ......... 25 Artificial Nanopores ................................ ................................ ................................ ........... 28 Fabrication of Conical Nanopores in Polymer Membranes ................................ ................. 30 Ion Tra ck Etch Methodology ................................ ................................ ....................... 30 Formation of Latent Damage Tracks ................................ ................................ ........... 30 Chemical Development of Damage Tracks ................................ ................................ .. 31 Nanopore Materials ................................ ................................ ................................ ..... 35 Nanopore Characterization and Properties ................................ ................................ .......... 38 Scanning Electron Microsc opy ................................ ................................ .................... 39 Ionic Conductance Measurements ................................ ................................ ............... 41 Electric Field Strength and Distribution ................................ ................................ ....... 42 Rectification of Ion Current ................................ ................................ ......................... 44 Controlling Nanopore Surface Chemistry and Properties ................................ .................... 49 Electroless Gold D eposition ................................ ................................ ........................ 49 Chemisorption of Thiols on Gold Coated Nanopores ................................ ................... 51 Selective Functionalization using Carboiimide/N Hydroxysuccinim ide Chemistry ...... 52 Additional Strategies for Surface Functionalization and Controlling Pore Size ............ 55 Additional Sensing Strategies Based on Track Etched Conical Nanopores ......................... 56 Dissertation Overview ................................ ................................ ................................ ........ 57 2 RESISTIVE PULSE SENSING OF A MODEL PROTEIN USING A CONICAL SHAPED NANOPORE ................................ ................................ ................................ ...... 67 Introduction ................................ ................................ ................................ ........................ 67 Experimental ................................ ................................ ................................ ...................... 69 Materials ................................ ................................ ................................ ..................... 69 Fabrication of the Conical Nanopore ................................ ................................ ........... 70 Electrochemical Measurements ................................ ................................ ................... 73 Results and Discussion ................................ ................................ ................................ ....... 74 Nanopore Characterization ................................ ................................ .......................... 74

PAGE 8

8 Resistive Pulse Sensing of Streptavidin ................................ ................................ ....... 75 Effect of Applied Transmembrane Potential on Current Pulse Frequency .................... 77 Effect of Pore Surface Chemistry on Current Pulse Duration ................................ ....... 78 Effect of Pore Surface Chemistry on Current Pulse Direction ................................ ...... 79 Conclusions ................................ ................................ ................................ ........................ 83 3 RESISTIVE PULSE SENSING OF A MODEL CATIONIC ANALYTE WITH A CONICAL NANOPORE SENSOR ................................ ................................ .................... 91 Introduction ................................ ................................ ................................ ........................ 91 Experimental ................................ ................................ ................................ ...................... 93 Materials ................................ ................................ ................................ ..................... 93 Preparation of the Conical Nanopore Sensing Element ................................ ................ 93 Resist ive Pulse Sensing ................................ ................................ ............................... 94 Results and Discussion ................................ ................................ ................................ ....... 96 Nanopore Characterization ................................ ................................ .......................... 96 Why Conical Shaped Nanopores ................................ ................................ ................. 97 Proposed Definition for the Detection Limit in Resistive Pulse Sensing ...................... 97 What Order of Magnitude Detection Limit Can Be Anticipated? ................................ 99 Analysis of the Current Pulse Data ................................ ................................ .............. 99 Conclusions ................................ ................................ ................................ ...................... 103 4 RESISTIVE PULSE SENSING OF NANOPARTICLES USING A CONICAL SHAPED NANOPORE IN TRACK ETCHED POLYIMIDE ................................ .......... 111 Introduction ................................ ................................ ................................ ...................... 111 Experimental ................................ ................................ ................................ .................... 113 Materials ................................ ................................ ................................ ................... 113 Fabrication of the Conical Nanopore ................................ ................................ ......... 113 Current Pulse Measurements ................................ ................................ ..................... 114 Results and Discussion ................................ ................................ ................................ ..... 115 Determination of the Bulk Etch Rate ................................ ................................ ......... 115 Two Step Etching Method for Ion Tracked Polyimide ................................ .............. 116 Nanopore Characterization ................................ ................................ ........................ 117 Controlling the Tip Diameter in Ion Tracked Polyimide via Isotropic Etching ........... 118 Resistive Pulse Sensing of Carboxylated, Fluorescent Nanoparticles ......................... 120 Conclusions ................................ ................................ ................................ ...................... 128 5 DIRECT COUPLING OF AMINE MODIFIED POLY(ETHYLENE GLYCOL) TO PORE SURFACES OF CONICAL NANOPORES FOR PREVENTING NON SPEC IFIC PROTEIN ADSORPTION ................................ ................................ .............. 147 Introduction ................................ ................................ ................................ ...................... 147 Experimental ................................ ................................ ................................ .................... 150 Ma terials ................................ ................................ ................................ ................... 150 Fabrication of Conical Nanopores ................................ ................................ ............. 150 Ionic Pore Conductance Measurements ................................ ................................ ..... 152

PAGE 9

9 X Ray Photoelectron Spectroscopy ................................ ................................ ........... 153 EDC/Sulfo NHS Coupling of Amine Modified Poly(ethylene glycol) to PET ........... 153 Results and Discussion ................................ ................................ ................................ ..... 154 Nanopore Characterization ................................ ................................ ........................ 154 Tip Diameter Stability of Unmodified Conical Pores in PET ................................ ..... 155 XPS of Unmodified PET Membranes ................................ ................................ ........ 155 Impact of Non Specific Protein Adsorption on Tip Diameter in Unmodified PET ..... 156 Analysis of Non Specific Protein Adsorption to PET Membranes via XPS ................ 158 XPS of PEG Amine Modified Single Pore Containing PET ................................ ...... 158 Impact of Non Specific Protein Adsorption on Tip Diameter in PEG amine modified PET ................................ ................................ ................................ ........ 159 Conclusions ................................ ................................ ................................ ...................... 164 6 FABRICATION OF LARGER CONE ANGLE NANOPORES IN PET FOR STUDIES ON THE AFFECT OF CONE ANGLE ON ELECTRIC FIELD STRENGTH, IONIC PORE CONDUCTANCE, AND ION CURRENT RECTIFICATION .............................. 189 Introduction ................................ ................................ ................................ ...................... 189 Experimental ................................ ................................ ................................ .................... 191 Materials ................................ ................................ ................................ ................... 191 F abrication of the Conical Nanopores and Gold Nanocones ................................ ....... 192 Field Emission Scanning Electron Microscopy ................................ .......................... 194 Finite Element Simula tions ................................ ................................ ........................ 195 Results and Discussion ................................ ................................ ................................ ..... 196 Finite Element Simulations: The Impact of Cone Angle on the Electric Field Strength ................................ ................................ ................................ ................. 196 Nanopore Characterization ................................ ................................ ........................ 197 Etching Multiple Ion Tracked PET at High Potentials ................................ ............... 198 Etching Multiple Ion Tracked PET With Non Aqueous Etchant ................................ 200 Impact of Increased Cone Angle on Ionic Pore Conductance in PET ......................... 202 Finding Single Nanopores in Single Ion Irradiated PET Membranes for Imaging ...... 203 Impact of Increased Cone Angle on Ion Current Rectification in PET ....................... 204 Conclusions ................................ ................................ ................................ ...................... 210 7 CONCLUSION ................................ ................................ ................................ ................ 223 LIST OF REFERENCES ................................ ................................ ................................ ........ 229 BIOGRAPHICAL SKETCH ................................ ................................ ................................ ... 243

PAGE 10

10 LIST OF TABLES Table page 4 1 Tabulated data for current current pulse frequency (f p ) of 100 nM carboxylated, fluorescent nanoparticles as a function of applied transmembrane potential (E). ................................ ......................... 146 5 1 Summary of the tip opening diameter stability for 3 single, conical shaped nanopores in track etched PET over a period of 4 days. ................................ ................................ 186 5 2 Summary of XPS spectra for 6 unmodified PET membranes. ................................ ...... 186 5 3 Summary of XPS spectra of chemically etched PET about the nanopore exposed to BSA, fibrinogen, and lysozyme ................................ ................................ ................... 187 5 4 Summary of the impact of n on specific adsorption of BSA, fibrinogen, and lysozyme on single, conical shaped nanopores in track etched PET ................................ ............ 187 5 5 Summary of the effect of amine PEG 550 modification on the tip opening diameter and non specific adsorption of BSA, fibrinogen, and lysozyme. ................................ .. 188 6 1 Tabulated s ummary of the effect of increasing base diameter in pores 1 and 2 relative to that of pores 3 and 4 w ith comparable tip diameters and the effect of electrolyte concentration on the ion current rectification ratio. ................................ ...................... 222

PAGE 11

11 LIST OF FIGURES Figure page 1 1 Diagram detailing the resistive pulse method ................................ ................................ 60 1 2 The biological protein nanopore, Hemolysin, embedd ed in a lipid bilayer support. ..... 60 1 3 Schemat ic of the ion track etch method ................................ ................................ ......... 6 1 1 4 Schematic of the electr ochemical cell used for track etching and ionic conductance measurements ................................ ................................ ................................ ................ 61 1 5 Diagram of a conical nanopore in a polymer membrane showing the base and tip opening diameters ................................ ................................ ................................ .......... 62 1 6 Plot of ion current versus time recorded during the anisotropic etch step for the fabrication of a conical shaped nanopore in PET. ................................ .......................... 62 1 7 A schematic of the track etch method of fabricating a conical nanopore showing the bulk etch rate, B track etch rate, T and cone half angle, ................................ ......... 63 1 8 Plot of ion current versus time r ecorded during the isotropic etch step for tailoring the tip opening diameter in PET ................................ ................................ ..................... 63 1 9 Chemical structures of commonly used polymers for ion track etching .......................... 64 1 10 Scanning electron micrographs of nanopores tr ack etched in various materials .............. 65 1 11 Schematic of the electric field strength distribution across a c onical nanopore ............... 65 1 12 Schematic of the electr oless gold deposition procedure ................................ .................. 66 1 13 Diagram of E DC/Sulfo NHS coupling chemi stry ................................ ........................... 66 2 1 Schematic of the PEG modified c onical nanopore sensing element ................................ 86 2 2 FE SEM image of a template synthesized gold nanocone replica prepared in a conical shaped nanopore in track etch ed poly(ethylene terephthalate) ........................... 86 2 3 Current voltage curves obtained in 1M KCl used to calculate the diameter of the tip opening after each step of the resistive p ulse sensor fabrication process ........................ 87 2 4 Typical current time transient for the PEG modified, single conical nanopore sensor at an applied tra nsmembran e potential of 1000 mV without streptavidin ....................... 87 2 5 Typical current time transient for the PEG modified, single conical nanopore sensor at an applied transme mbrane potential of 1000 mV with 5 00 nM streptavidin. ............... 88

PAGE 12

12 2 6 Expanded view of a typical current pulse reflecting the tip to base translocation of 500 nM streptavidin through a PEG modified conical nanopore with a tip diameter of 12 nm. ................................ ................................ ................................ ....................... 88 2 7 Streptavidin current time transient as function of applied transmembrane potential ....... 89 2 8 Streptavidin curre nt pulse frequency versus transmembrane potential ............................ 89 2 9 Ion c urrent time transients of 500 nM streptavidin at an applied transmembrane potential of 1000 mV in unmodified and PEG modified PET nanopores ........................ 90 2 10 Histograms of streptavidin current pulse amplitude and duration ................................ .. 90 3 1 General conductivity cell setup use d for resistive pulse experiments ............................ 105 3 2 Schematic of the process by which the local ionic strength within the tip region is increased as the cationic gold nanoparticle introduces its charge balancing counterions. ................................ ................................ ................................ ................. 105 3 3 Ion c urrent time transients reflecting the translocation of cationic gold nanoparticles through the ti p opening of a conical nanopore ................................ ............................. 106 3 4 Current voltage curve determ ination of tip opening diameter ................................ ....... 106 3 5 Steady state ion current using a single, conical shaped nanopore in track etch ed poly(ethylene terephthalate) without nanoparticles ................................ ....................... 107 3 6 Ion c urrent time transients of 5 nm diameter cationic gold nanoparticles using a single, conical shaped nanopore in track etc h ed poly(ethylene terephthalate) ............. 107 3 7 Plot of current pulse frequency versus particle concentrat ion taken at 5 minute intervals ................................ ................................ ................................ ....................... 108 3 8 Ion c urrent time transients of 5 nm diameter cationic gold nanoparticles using a single, conical shaped nanopore in track etch ed poly(ethylene terephthalate) ............. 109 3 9 Scatter pl ) ................................ ................................ ................................ ............................ 110 3 10 Histograms of current pulse magnitude and duration data for cationic nanoparticles .... 110 4 1 Electron micrographs of multiple nanopores etched into multiple ion tracked polyimide me mbranes at different etch times ................................ ............................... 131 4 2 Ion cu rrent time recordings for monitoring breakthrough in polyimide and PET membr anes during the first step etch ................................ ................................ ........... 132 4 3 Ion current time recordings for tailoring the tip opening diameter during the second etch step in polyimide and PET membranes ................................ ................................ 132

PAGE 13

13 4 4 Current voltage curves obtained in 1 M KCl used to calculate the diameter of the tip opening after each round of s econd step etchin g of polyimide ................................ .... 133 4 5 Plot of tip opening diameter versus final nanopore ion current during the second isotropic, etch step for single track etched polyimide. ................................ .................. 133 4 6 Plot of rectification ratio versus tip opening diameter in 1 M KCl (pH 6) after the second step etch of a conical pore in polyimide. ................................ .......................... 134 4 7 Curre nt voltage curves obtained in 1 M KCl (pH 6) after the second step et ch and after treatment with a wetting agent and vacuum ................................ ......................... 134 4 8 Ion c urrent time transients in the absence of nanoparticles and with 100 nM carboxyl ated, fluorescent nanoparticles ................................ ................................ ....... 135 4 9 Ion c urrent time transients of 100 nM carboxylated, fluorescent nanoparticles ............. 136 4 10 Ion c urrent time transients of 100 nM carboxylated, fluorescent nanoparticles with a reversed transmembrane potential of 300 mV for 60 min. and transmembrane potential of +300 mV. ................................ ................................ ................................ .. 137 4 11 Ion c urrent time transients in the absence of nanoparticles and with 100 nM carboxylated, fluorescent nanoparticles obtained with an a pplied transmembrane potential of 50 mV. ................................ ................................ ................................ ...... 138 4 12 Ion c urrent time transients in the a bsence of nanoparticles and with 100 nM carboxylated, fluorescent nanopar ticles obtained with an a pplied transmembrane potential of 100 mV. ................................ ................................ ................................ .... 139 4 13 Ion c urrent time transients in the a bsence of nanoparticles and with 100 nM carboxylated, fluorescent nanoparticles obtained with an a p plied transmembrane potential of 300 mV. ................................ ................................ ................................ .... 140 4 14 Ion c urrent time transients in the a bsence of nanoparticles and with 100 nM carboxylated, fluorescent nanoparticles obtained with an a pplied transmembrane potential of 400 mV. ................................ ................................ ................................ .... 141 4 15 Ion c urrent time transients in the a bsence of nanoparticles and with 100 nM carboxyla ted, fluorescent nanoparticles obtained with an a pplied transmem brane potential of 500 mV. ................................ ................................ ................................ .... 142 4 16 Histo grams of current pulse duration for 100 nM carboxylated, fluorescent nanoparticles at applied transmembrane potentials of 200 mV, 100 mV, and 50 mV. ... 143 4 17 Histograms of current pulse ampli tude for 100 nM carboxylated, fluorescent nanoparticles at applied t ransmembrane potentials of 200 mV, 100 mV and 50 mV. ... 144

PAGE 14

14 4 18 Plot of current pulse amplitude versus applied transmembrane potential for 100 nM carboxylated, fluorescent nanoparticles. ................................ ................................ ....... 145 4 19 Scatter plot of current nM carboxylated, fluorescent nanoparticles at applied transmembrane potentials of 50 mV 100 mV, an d 200 mV ................................ ................................ ..................... 145 5 1 Current voltage curves obtained for 4 consecutive days in pH 6, 1 M KCl (pore 1) ...... 166 5 2 Current voltage curves obtained for 4 consecutive days in pH 6, 1 M KCl (pore 2). ..... 166 5 3 Current voltage curves obtained for 4 consecutive days in pH 6, 1 M KCl (pore 3) ...... 167 5 4 XPS spectra of unmodified PET membr ane 1. ................................ ............................. 167 5 5 XPS spectra of unmodified PET membrane 2. ................................ ............................. 168 5 6 XPS spectra of unmodified PET membrane 3. ................................ ............................. 168 5 7 XPS spectra of unmodified PET membrane 4. ................................ ............................. 169 5 8 XPS spectra of unmodified PET membrane 5. ................................ ............................. 169 5 9 XPS spectra of unmodified PET membrane 6. ................................ ............................. 170 5 10 Current voltage curves obtained in 1 M KCl fo r pore A before exposure and after exposure to BSA. ................................ ................................ ................................ ......... 170 5 11 Current voltage curves obtained in 1 M KCl for pore B before exposure and after ex posure to BSA. ................................ ................................ ................................ ......... 171 5 12 Current voltage curves obt ained in 1 M KCl for pore C before exposure and after exposure to BSA. ................................ ................................ ................................ ......... 171 5 13 Current voltage curves obtained in 1 M KCl for pore A before exposure and after exposure to fibrinogen. ................................ ................................ ................................ 172 5 14 Current voltage curves obtained in 1 M KCl for pore B before exposure and after exposure to fibrinogen. ................................ ................................ ................................ 172 5 15 Current vol tage curves obtained in 1 M KCl for pore C before exposure and after exposure to fibrinogen. ................................ ................................ ................................ 173 5 16 Current voltage curves obtained in 1 M KCl for pore A before exposure and after exposure to lysozyme. ................................ ................................ ................................ 173 5 17 Current voltage curves obtained in 1 M KCl for pore B before exposure and after exposure to lysozyme. ................................ ................................ ................................ 174

PAGE 15

15 5 18 Current voltage curves obtained in 1 M KCl for pore C before exposure and after exposure to lysozyme. ................................ ................................ ................................ 174 5 19 XPS spectra of chemically etched PET about a single nanopore exposed to B SA (pore A). ................................ ................................ ................................ ...................... 175 5 20 XPS spectra of chemically etched PET about a single nanopore exposed to BSA (pore B). ................................ ................................ ................................ ...................... 175 5 21 XPS spect ra of chemically etched PET about a single nanopore exposed to BSA (pore C). ................................ ................................ ................................ ...................... 176 5 22 XPS spectra of chemically etched PET about a single nanopore exposed to fibrinogen (pore A). ................................ ................................ ................................ ..... 176 5 23 XPS spectra of chemically etched PET about a single nanopore exposed to fibrinogen (pore B). ................................ ................................ ................................ ..... 177 5 24 XPS spectra of chemically e tched PET about a single nanopore exposed to fibrinogen (pore C). ................................ ................................ ................................ ..... 177 5 25 XPS spectra of chemically etched PET about a single nanopore exposed to lysozyme (pore A). ................................ ................................ ................................ ...................... 178 5 26 XPS spectra of chemically etched PET about a single nanopore exposed to lysozyme (pore B). ................................ ................................ ................................ ...................... 178 5 27 XPS spectra of chemically etched PET about a single nanopore exposed to lysozyme (pore C). ................................ ................................ ................................ ...................... 179 5 28 XPS spectra of chemically etched PET about a single nanopore modified with PEG 550 amine (pore A). ................................ ................................ ................................ .... 179 5 29 XPS spectra of chemically etched PET about a single nanopore modified with PEG 550 amine (pore B). ................................ ................................ ................................ ..... 180 5 30 Current voltage curves of pore 1 obtained i n 1 M KCl for an unmodified nanopore, amine PEG 550 modified nanop ore, and the same PEG 550 modified nanopore before BSA exposure and after BSA exposure ................................ ............................. 180 5 31 Current voltage curves of po r e 2 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore, an d the same PEG 550 mo dified nanopore before BSA exposure and a fter BSA exposure ................................ ............................. 181 5 32 Current voltage curves of pore 3 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore, and the same PEG 550 modified nanopore before BSA exposure and after BSA exposure ................................ ............................. 181

PAGE 16

16 5 33 C urrent voltage curves of por e 1 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore, an d the same PEG 550 modified nanopore before fibrinogen exposure and after fi brinogen exposure ................................ ............ 182 5 34 Current voltage curves of pore 2 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore, and the same PEG 550 modified nanopore before fibrinogen exposure and after fibrinogen exposure ................................ ............ 182 5 35 Current voltage curves of por e 3 obtained in 1 M KCl for an un modified nanopore ami ne PEG 550 modified nanopore, and the same PEG 550 modified nanopore before fibrinogen exposure and after fi brinogen exposure ................................ ............ 183 5 36 Current voltage curves of por e 1 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore and the same PEG 550 modified nanopore before lysozyme expos ure and after lysozyme exposure ................................ ............... 183 5 37 Current voltage curves of por e 2 obtained in 1 M KCl for an un modified nanopore amine PEG 550 modified nanopore, and the same PEG 550 modified nanopo re before lysozyme exposure and after lysozyme exposure ................................ .............. 184 5 38 Current voltage curves of pore 3 obtained in 1 M KCl for an unmodified nanopore amine PEG 550 modified nanopore, and the sam e PEG 550 modified nanopore before lysozyme exposure and after lysozyme exposure ................................ .............. 184 5 39 Current voltage curves obtained in 1 M KCl for a single, conical shaped nanopore fabricated in track etched PET having tip diameters of 42 nm after etching and 4 nm after electro less gold deposition ................................ ................................ .................. 185 5 40 Current voltage curves obtained in 1 M KCl for a single, conical shaped nanopore f abricated in track etched PET after electroless gold plating and applying a high transmembrane potential ................................ ................................ .............................. 185 6 1 Plot of electric field strength in the tip opening of the conical nanopore obt ained from finite element simulations versus applied transmembrane potential for nanopores having a large base diameter of 5000 nm and a smaller base diameter of 520 nm ................................ ................................ ................................ ........................ 212 6 2 Plot of the ba se diameter obtained for multiple ion tracked PET membranes etched at 5, 10, 15, and 20 V for 2 hours. ................................ ................................ .................... 212 6 3 SEM image of a gold replica of a pore fabricated in a multiple ion irradiated PE T membrane at an applied transmembrane potential of 20 V applied during etching for 2 hours. ................................ ................................ ................................ ........................ 21 3 6 4 SEM image of randomly distributed gold nanocone replicas of conical pores fabricated in mu ltiple ion irradiated PET membrane using a non aqueous etch method. ................................ ................................ ................................ ....................... 213

PAGE 17

17 6 5 Plot of gold nanocone base diameter versus etch time. ................................ ................. 214 6 6 Plot of gold n anocone height versus etch time ................................ .............................. 214 6 7 SEM images of randomly distributed arrays of gold nanocones obtained from pores etched at ambient temperature for 30 s, 100 s 175 s, 250 s, and 500 s. ......................... 215 6 8 SEM images of the base opening diameters of single, conical shaped nanopores fabri cated in single ion tracked PET ................................ ................................ ............ 216 6 9 Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore A) having a base diameter of 1541 nm and ti p diameter of 10 nm and a second conical pore having a base diameter of 520 nm and a tip diameter of 10 nm. ................................ ... 217 6 10 Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore B) having a base diameter of 1475 nm and tip diameter of 17 nm and a second conical pore having a base diameter of 520 nm and a tip diameter of 18 nm ................................ .... 217 6 11 Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore C) having a base diameter of 1370 nm and tip diameter of 19 nm and a sec ond conical pore having a base diameter of 520 nm and a tip diameter of 18 nm ................................ .... 218 6 12 Schematic detailing an approach for finding single nanopores in sing le ion irradiated membranes using a replica of the filter aperture mask used during single swift heavy ion irradiation. ................................ ................................ ................................ ... 218 6 13 Current voltage curves obtained in 0.01 M KCl for a larger cone angle por e 1 and a smalle r cone an gle pore ................................ ................................ ............................... 219 6 14 Current voltage curves obtained in 0.1 M KCl for a larger cone angle pore 1 and a smalle r cone angle pore ................................ ................................ .............................. 219 6 15 Current voltage curves obtained in 1 M KCl for a larger cone angle pore 1 and a smalle r cone angle pore ................................ ................................ ............................... 220 6 16 Current voltage curves obtained in 0.01 M KCl for a larger cone angle pore 2 and a smaller co ne angle pore ................................ ................................ ............................... 220 6 17 Current voltage curves obtained in 0.1 M KCl for a larger cone angle pore 2 and a smaller cone angle pore ................................ ................................ ............................... 221 6 18 Current voltage curves obtained in 1 M KCl for a larger cone angle pore 2 and a smaller cone angle pore ................................ ................................ .............................. 221

PAGE 18

18 Abstract of Dissertation Presented to the Graduate School of the University o f Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DEVELOPING RESISTIVE PULSE SENSORS USING ARTIFICIAL CONICAL NANOPORES IN TRACK ETCHED POLYMER MEMBRANES By Lloyd Peyton Horne, Jr. August 2010 Chai r: Charles R. Martin Major: Chemistry The objective of this research is to develop sensors based on the resistive pulse method using conical nanopores and investigate properties of such pores that impact their sensing capabilities. In the first section of this work, sensing of a model protein is demonstrated using a single, conical nanopore embedded in a track etched polymer membrane. The pore surface was modified with a thin, conformal gold film and subsequently functionalized with thiol modified poly(eth ylene glycol) (PEG) to prevent non specific protein adsorption. Single protein molecules were detected and counted as downward current pulses as they were electrophoretically driven through the pore. The frequency of these current pulses was found to vary exponentially with the applied transmembrane potential. Removal of the PEG and gold layers revealed current pulses that went both upward and downward. Such a phenomenon had not been previously observed with resistive pulse sensors constructed from track e tched conical nanopores. The impact of this effect on components of the current pulse signature is discussed. Previous resistive pulse sensors derived from track etched polymer membranes have been configured such that the net surface charge on the analyte, the surface charge of the pore wall, and electrode at the tip opening all have the same polarity (i.e., negatively charged). The second part of this work presents an example of a resistive pulse system where the net surface charge on

PAGE 19

19 the analyte and elect rode at the tip are both opposite in polarity of the pore surface (i.e., both are positively charged). That is, the resistive pulse sensing of a model cationic analyte, poly L lysine conjugated gold nanoparticles using a single, conical nanopore in track e tched poly(ethylene terephthalate) (PET) is presented. Current pulses were observed down to the femtomolar concentration level and exclusively upward. Such pulse direction reflects the ion current enhancing effect of the counter ions accompanying each nano particle into the nanopore sensing zone. A definition for the limit of detection in resistive pulse sensing is presented and discussed. The third part of this work focuses on developing resistive pulse sensors in an alternative polymer material, polyimide. Progress towards a two step etch method for tailoring the tip diameter of conical nanopores in polyimide is introduced. Controlling the tip opening diameter during fabrication is absolutely critical to constructing functional resistive pulse sensors. The tip diameter was observed to scale linearly with the final value of the ion current during the two step etch. Furthermore, the extent of ion current rectification was found to be inversely proportional to tip size at the tip sizes evaluated. An approach to wards loading electrolyte into conical pores in polyimide is introduced involving the use of a wetting agent, vacuum degassing, and perfusion. Carboxylated, fluorescent nanoparticles were then detected using a single, conical nanopore in track etched polyi mide. Current pulses were exclusively upward and detected using much lower applied transmembrane potentials than those typically used for resistive pulse sensors housed in PET membranes. This was attributed to the large cone angle and correspondingly lower pore resistance characteristic of conical pores fabricated in polyimide. The fourth part of this work introduces an alternative strategy to electroless gold deposition for functionalizing the surfaces of single, conical nanopores with PEG based on EDC/ s ul fo NHS

PAGE 20

20 coupling chemistry. Minimizing non specific pore surface adsorption is absolutely critical in resistive pulse sensing. X ray photoelectron spectroscopy and ionic pore conductance were utilized to study the non specific adsorption of three model prot eins, BSA, fibrinogen, and lysozyme, to the surfaces of single, conical nanopores before and after functionalization with amine modified PEG. The presence of the PEG was found to reduce the non specific adsorption of each protein to varying extents. Thus, this represents progress towards producing more biocompatible nanopores for developing resistive pulse applications for biological analytes. The final part of this work presents cone angle studies on pores fabricated in PET membranes. The electric field st rength distribution inside two single, conical shaped nanopores having identical tip diameters but different base diameters (i.e., one large and one small) was evaluated via finite element simulations. These simulations show the electric field strength, wh ich is directly proportional to current pulse frequency, increases more with increasing cone angle than with increasing transmembrane potential. Thus, this provides the impetus for fabricating larger cone angle nanopores. However, before doing resistive pu lse sensing, methods for fabricating, controlling and reproducing large cone angle nanopores are needed. A high increased resistive heating. Thus, a non aqueous approach to fabricating single, conical nanopores having a larger cone angle than pores typically produced via the aqueous two step etch method is presented. Using this approach, the effect of increasing cone angle on ionic pore conductance and ion current rectifi cation was evaluated. Increased ionic pore conductance and decreased ion current rectification were observed with the larger cone angle pores relative to those having a smaller cone angle.

PAGE 21

21 CHAPTER 1 INTRODUCTION AND BAC KGROUND Introduction Nanoscale mate rials have attracted tremendous interest in recent years. Such materials have been generally defined as structures comprised of one dimension of 100 nm or less in size. As the length scale of materials approaches that of single molecules, their inherent ph ysical properties change and differ from that of their much larger, or bulk, counterparts. Consequently, such unique characteristics have found potential applications in a variety of areas including biotechnology, medicine, electronics, and energy. 1 16 In particular, nanopore research constitutes one area of growing interest in nanoscience. This is due, in part, to the prevalence of highly selective and sensitive nanopores in the human body. Such biological nanopores, or ion channels, play a critical role in many key biological processes. 17 18 Therefore, biological ion channels provide an excellent paradigm for designing highly selective and sensitive chemical and biological sensors. However, there are several drawbacks from using biological nanopores which preclude their use in any practical sensing device ( vide infra ). As a result, there has been significant efforts towards developing artificial nanopores as a feasible alternative. 19 64 Such synthetic analogues are attractive due to their mechanical robust ness and chemical stability, controllable pore size, and easily tunable surface chemistry. Artificial nanopores also provide a vehicle in which the fundamental transport properties of biological ion channels can be investigated without dealing with their c haracteristic fragility. In recent years, several technologies have been utilized for fabricating nanopores in artificial materials. Some of these approaches include focused ion beam (FIB) methods and electron beam lithography, 23 38 micromolding, 39 44 ca rbon nanotube embedded polymers, 45 50

PAGE 22

22 femtosecond laser ablation, 51 53 base etching of silicon wafers, 54 and single ion track etching. 55 65 Nanopores have been constructed in a diverse array of materials including organic inorganic hybrid materials, silico n based inorganics, carbon nanotubes, and thin polymer membranes. There has been increasing interest in using nanopores as the sensing element for developing analytical devices based on the resistive pulse method. This approach has been used to detect meta l ions, 66,67 small molecules, 68 77 nucleic acids, 78 91 proteins, 92 95 viruses, 20,21,23 and nanoparticles. 48,96 In the Martin group, we have been investigating the fabrication and application of conically shaped nanopores in thin polymer membranes. These p ores are prepared via the track etch method. 55 65 Track etched membranes have been utilized to investigate fundamental transport properties, 74,97 110 to perform bioseparations, 111,112 and as templates for fabricating open tubular and solid nanomaterials (i .e., nanotubes and nanowires). 113,114 Single, conical shaped nanopores have also been used to construct resistive pulse sensors. 74,75,89 91,95 The research presented herein discusses recent investigations on nanopore fabrication, resistive pulse sensing, varying pore surface chemistry, and varying cone angle in asymmetric nanopores using thin polymer membranes. Chapter 1 presents the background information which supports the research. First, the resistive pulse method is discussed along with examples of tw o different nanopore constructs: (1) those derived from biological transmembrane proteins and (2) pores fabricated in artificial materials. Then, the track etch method is discussed along with pore characterization and surface functionalization. The Resisti ve Pulse Method The resistive pulse method has been around for decades. The Coulter Counter, a commercially available instrument that counts and sizes biological cells and colloidal particles, operates on this principle. In fact, approximately > 98% of a ll cell counters are of the Coulter

PAGE 23

23 type. 115 This device was developed in the 1940s by W. H. Coulter and patented in 1953. 116 The Coulter Counter contains a small diameter aperture (15 m 2 mm) that is placed in between two electrolyte solutions and subsequently filled with electrolyte. 115 An electrode is placed into each solution and used to pass an ionic current through this aperture. The solution of particles to be counted is added to one of the electrolyte solutions, and these particles are driven through the aperture by applying pressure or a potential difference (i.e., either a constant current or constant voltage). When a particle enters the aperture, it displaces a volumetric f raction of electrolyte solution that is equivalent to the volume occupied by the particle. As a result, the ionic resistance of the aperture increases while the particle is present in the aperture. This is registered as a transient voltage or current puls e by the device. The magnitude of such pulses is proportional to the size (volume) of the particle. The frequency of these pulses is proportional to the particle concentration. Coulter Counters can size particles that are 400 nm 2 mm in diameter using a pertures 15 m 2 mm. 115 The operating particle sizing range is primarily determined and limited by the aperture diameter. Thus, to detect single molecules, nucleic acids, or proteins, this suggests that much smaller aperture diameters are required. In fact, in the 1970s, DeBlois, et al., postulated that if they could fabricate smaller apertures, then smaller analytes (i.e., virus particles) could be measured. 19 22 They used apertures, or pores, fabricated via the track etch method ( vide infra ). Using a single, cyl indrical pore diameter <500 nm, Deblois and coworkers were able to detect and accurately size Mason Pfizer virus, Rauscher murine leukemia, and simian sarcoma virus particles of 140 + 3 nm, 122 + 2 nm, 110 + 3 nm in diameter, respectively. 22 Such values we re found to be in good agreement with those obtained by other methods and reported in the literature. Their device could routinely

PAGE 24

24 measure virus particle concentrations on the order of 10 9 10 11 particles/mL in just a few minutes. Although their approac h was very creative and seemed promising, it was plagued with several problems: (1) they were only able to fabricate multipore membranes (pore density >10 6 pores cm 2 (2) the cylindrical pore shape and large aspect ratio caused more than one particle to s imultaneously reside in the pore during translocation, and (3) pore blockage due to non specific adsorption of particles along the pore interior. Problems (1) and (2) have since been resolved using a single ion etching technique 57,58 and conical pore geome try, 59,95,96 respectively. Problem (3) has been addressed indirectly in multiporous membranes 117 and directly with single, conical shaped nanopores as part of the research presented herein. Nevertheless reduction of pore diameter is a critical pre requisi te for detecting smaller size analytes. The nanopore based experiment is analogous to those early experiments based on the Coulter principle. That is, a membrane containing a single, conical shaped nanopore is immobilized between two halves of an electro lyte filled, U tube cell, and filled with electrolyte (Figure 1 1). 74,75,89,90,91,95 An electrode, typically Ag/AgCl, is immersed into each half cell. A transmembrane potential difference is applied across the nanopore, thereby generating an ionic current which is measured as a function of time. When a charged analyte is driven via electrophoresis into the pore, it displaces a volumetric fraction of electrolyte that is related to analyte size (i.e., molecular volume) and increases the pore resistance. As a result, a transient decrease, or blockage, of the ionic current occurs. This phenomenon is generally observed as a downward current pulse. 74,75,89,90,91,95 That is, the ionic current during a current pulse is less than the baseline current. As mentioned pr eviously, analyte concentration is directly proportional to the frequency of such current pulses. 89,95 Analyte identity, or selectivity, is encoded in both the

PAGE 25

25 magnitude and duration of the current pulses. 89,95 The duration of these current pulses is also related to effective surface charge on the analyte. 89,95 Recent research efforts in resistive pulse sensing have been focused on the detection and characterization of smaller size analytes such as ions, 66,67 small molecules, 68 77 nucleic acids, 78 91 prote ins, 92 95 and small particles. 48,96 As discussed in a later section, there are advantages for using conical shaped nanopores derived from ion track etched materials for developing such sensors. Such artificial nanopore systems have been used to detect smal l molecules, 74,75 proteins, 95 nanoparticles, 96 and DNA. 90,91 Biological nanopores 66 70,78,79,82 88,93,94,118,119,121,126 have also been utilized as the sensing element in resistive pulse devices. Biological Nanopores Biological nanopores, or ion channels, represent a broad class of highly selective and sensitive transmembrane proteins that play critical roles in many key biological processes. 17,18 As a result, biological ion channels provide excellent archetypes for designing chemical and biological sensor s. Ion channels are generally comprised of proteins and/or subunits thereof that assemble to form a selective passage barrier between one region and another (e.g., separating the extracellular environment from the intracellular space of cells). 17,18 In sim plest terms, these channels regulate the selective transport of ions and neutral molecules into and out of the cells. 17,18 As a result, such channels mediate cellular communication. Biological ion channels are generally categorized according to how they f unction. For example, ligand most widely studied ligand gated channels is the nicotinic acetylcholine receptor. These receptors selectively bind the neurotransmitter, acetylcholine. This recognition process causes a

PAGE 26

26 conformational change i n the receptor and subsequent opening of the ion channel. 17,18 Voltage gated ion channels represent another type of ion channel which function based on a cell membrane potential. 127 Examples of such channels regulate both nerve signal transduction (i.e., a ction potentials of axons) and muscle contraction (i.e., cardiac and skeletal muscle). Additionally, ion channels were the subject of the 2003 Nobel Prize in Chemistry which was jointly awarded to Peter Agre and Roderick MacKinnon for their discoveries o n aquaporins and potassium ion channels, respectively. Aquaporins are transmembrane channels that regulate the passage of water molecules across the cell membrane. 128 131 Such channels are present in most all organs including the gastrointestinal tract, ki dneys, and nervous system. Thus, aquaporins play a key role in maintaining homeostasis and offer potential applications for addressing major medical problems. Potassium channels play a central role in many key biological processes as well. MacKinnon, et al first explained the selectivity of such channels for potassium ions. 132 138 That is, these channels only permit potassium ions to pass through. Other ions, such as sodium, are rejected due to their smaller size with a selectivity ratio (K+/Na+) of 10,000 /1. 136 Moreover, due to their widespread importance, biological transmembrane channels are one of the most widely studied platforms for developing resistive pulse sensors. Research conducted on such channels has provided the impetus for developing molecu lar scale, resistive pulse sensors. Sensors based on hemolysin, 66 70,78,79,82,83 88,93,94,118,119,121,126 maltoporin, 139 and the outer membrane protein F (OmpF) 140 have all been described, but hemolysin is the most commonly utilized and remains the benchmark by which all other resistive pulse sensors ar e compared.

PAGE 27

27 Alpha hemolysin ( HL) is a transmembrane protein exotoxin comprised of 293 acids and produced by Staphylococcus aureus HL comes in contact with a lipid bilayer membrane, it self assembles into a nanoscale pore that penetrates the memb rane (Figure 1 2). This channel provides a very narrow passageway between the cell interior and extracellular HL has been implicated in cell lysis. This nanopore also provides a highly sensitive and selective detection passa geway that has been used to detect a wide variety of target analytes via the resistive pulse method. Either native or engineered forms of HL have been utilized to detect enantiomers of drug molecules, 68 DNA, 78,79,82 88 nitroaromatic compounds, 69 metal io ns, 66 small organic molecules, 70 anions, 120 proteins, 93,94 and polymers. 124 The typical resistive pulse experiment begins with HL embedded in a planar lipid bilayer support. An appropriate electrolyte solution is placed above and below a HL embedded lipid bilayer membrane. Electrodes are inserted into each solution and an applied potential HL by the movement of cations and anions present in the electrolyte. This ionic current quickly re aches and maintains a steady state as long as analytes are not present. When an analyte is introduced into the electrolyte, it is HL where it transiently increases the pore resistance as it enters and passes through the lum en. There are several advantages for using HL to construct resistive pulse sensors. First, HL is commercially available and its structure is known. Furthermore, the pore size is reproducible and can be achieved from one bilayer to the next. The key ad vantage is that highly selective molecular recognition capabilities can be imparted into the lumen of the HL protein via chemical and genetic engineering. For instance, nucleic acids have been selectively detected HL pore that was chemically mo dified with a covalently attached single

PAGE 28

28 oligonucleotide strand. 78,141 Similarly, arginine HL lumen has provided a means to selectively detect phosphate ester anions. 120 Metal ions have also been detected by introducing four histidine resides in the lumen. 142 HL, several major drawbacks persist. As mentioned above, the fragility of the lipid bilayer membrane HL pore precludes the use o f this protein nanopore in any practical sensing technology. 119,143,144 That is, the lipid bilayer membrane only lasts for a few hours before rupture, which is too short of time for any repeat use analytical device. Furthermore, planar lipid bilayers are i ncapable of withstanding the broad range of applied potentials, temperatures, and pHs that artificial pores can endure. The use of HL in resistive pulse sensing is applicable only to very HL based devices HL remains the standard by which other resistive pulse sensors are compared Consequently, recent research efforts have focused on the development of resistive pulse sensors based on nanopores derived from artificial materials. Artificial Nanopores By replacing the biological nanopore and lipid bilayer with an artificial nanopor e, much greater chemical stability and mechanical robustness can be achieved. Nucleic acids, 26,31 36,89,90 proteins, 37,38,95 and nanoparticles 46,48,49,96 have been detected with such artificial pores. For example, Crooks, et al. embedded a single carbon na notube within an epoxy resin. 45 50 This resin was microtomed and utilized as the sensing element in a resistive pulse detector for nanoparticles of different surface charge densities. Their carbon nanotube device was also used to study DNA transport using fluorescence microscopy. The most commonly utilized fabrication methods involve the application of either ion or electron beams to create nanopores in silicon oxide and silicon nitride membranes. Golovchenko

PAGE 29

29 used a focused ion beam (FIB) method to fabrica te nanopores in Si 3 N 3 membranes, 23,24 and Letant, et al. used FIB for making pores in silicon membranes. 145 Bashir, et al., and other groups have utilized electron beam approaches to fabricating pores in SiO 2 28,30,35 Similarly, Timp, et al. employed a ele ctron beam method for creating pores in Si 3 N 3 32 Such pores have been used to primarily study DNA but some protein studies have also been conducted. Sohn, et al. reported another, rather unique method of artificial pore fabrication based on the micromold ing of poly(dimethylsiloxane) (PDMS). 40 44 Such pores have been utilized to detect phage DNA and colloidal nanoparticles, as well as directly study antigen antibody interactions on antibody conjugated colloidal particles. Nanopores in PDMS have also been used in multianalyte immunoassays to detect human granulocyte and macrophage colony stimulating factor (GM CSF) and granulocyte colony stimulating factor (G CSF) antigens. Mayer, et al. developed a laser ablation fabrication technique in which a femtoseco nd pulsed laser is utilized to drill a conical pore in glass. 51 53 Such pores have been used to detect viruses, virus antibody complexes, and particles. Additional methods of fabricating single nanopores include etching silicon wafers under alkaline condi tions, 54 and track etching of surfaces that have been tracked via swift heavy ion irradiation. 55 65 The Martin group has used such a track etch approach to fabricating single, conical shaped nanopores in a variety of different polymer membranes 74,75,89,90, 95,96 as well as muscovite mica. 106,113,146 These conical pores have been utilized for the resistive pulse detection of single stranded phage and double stranded plasmid DNA, 89 small double stranded DNAs, 90 small molecules, 74,75 proteins, 95 and nanoparticl es. 96

PAGE 30

30 Fabrication of Conical Nanopores in Polymer Membranes Ion Track Etch Methodology The ion track etch method has been practiced commercially for decades to make a wide variety of pore diameters that are available at many different pore densities. That is, membranes containing pore diameters ranging from 10 nm to as large as 10 m, with pore densities on the order of 10 5 10 9 pores cm 2 are commercially available. 147 These porous materials have been commonly utilized for filtration applications (e.g., laboratory, cell culture, and process filters), 148 151 as templates for fabricat ing tubes and wires, 113,114,152,153 and for investigating fundamental transport phenomena. 74,98 110,146,154 158 In the commercial fabrication process, the ion tracked membrane is submerged into an appropriate etching solution, or etchant, which etches pr eferentially along the damage track from both sides of the membrane. As a result, cylindrical pores are created. The pore density is determined by the ion track density, which is governed by the exposure time of the membrane to a collimated beam of high en ergy particles emanating from a cyclotron or nuclear reactor. Pore diameter is determined by exposure time to the etchant as well as etchant temperature. This method has been further modified such that membranes containing a single ion damage track can be constructed that provides a substrate for fabricating single nanopores. Such single pores are required for resistive pulse sensing studies. Formation of Latent Damage Tracks As briefly mentioned above, the track etching of nanopores begins with the irr adiation of a membrane with heavy ions (Figure 1 3). That is, the process begins when swift, high energy particles (1 10 MeV/nucleon), emitted from either a linear accelerator, cyclotron, or nuclear reactor, strafe a polymer membrane. This bombardment with heavy ions cuts completely through the polymer matrix, thereby creating a latent damage track spanning the entire thickness of the

PAGE 31

31 membrane (typically, 5 10 m). The number of such latent damage tracks formed in this process represents the approximate number of nanopores (typically cylindrical) generated by subsequent chemical etching. In other words, a single ion track yields a single nanopore embedded in the polymer surface. Multiple ion tracks produce many monodisperse nanopores. Single track membranes are produced via a technology developed at Gesellschaft fuer Schwerionenforschung (GSI) in Darmstadt, Germany. 57,58 In this method, the heavy ion beam is def ocused to lower the ion flux. Both a metallic filter containing a 200 m diameter aperture and automatic shutter, or impenetrable gate, is placed in the beam path and between the ion beam source and the membrane target. An ion detector is placed on the other side of the membrane. Polymer membranes are typically irradiated in a stack of 5 7 membranes at one time. This stack of membranes is loaded onto a plastic cartridge and placed into an autosampler that enables remote, automated control and efficiency over the irradiation process. When an ion completely traverses the filt er aperture and membrane stack, it is detected on the opposite side by the detector and the shutter closes, thereby turning off the ion beam. This prevents the membranes from being further exposed to additional ions. As a result, the polymer membrane conta ins only one latent damage track. The success of this ion irradiation technology is largely dependent on the energy of the irradiating ions, type of material being irradiated, and post irradiation storage conditions. 97 Chemical Development of Damage Tracks After irradiation, the latent damage track is etched chemically to obtain conical shaped nanopores. The process, developed by Apel, et al., begins with the immobilization of the ion tracked polymer membrane between two halves of an electrochemical, U tub e, cell (Figure 1 4). 59 A suitable etching solution that etches the damage track is placed on one side of the

PAGE 32

32 59 The etchant preferentially etches the damage tr ack from the etchant side of the membrane to the stopping solution side. When the etchant has broken through to the stopping solution, neutralization of the etchant occurs. The etching process ends by placing the nanopore membrane briefly in stopping solut ion and subsequently rinsing with water. This anisotropic etch/neutralization method results in a single, conical shaped nanopore with a large diameter solution side (Figure 1 5). During the etching process, a platinum electrode is placed into each half cell. The positive electrode (anode) is placed in the solution on the base opening (etchant) side. The negative electrode (cathode) is placed in the solution on the ti p opening (stopping solution) side. A transmembrane potential difference (typically +1 V) is applied and the ion current across the membrane is measured with a picoammeter. This process serves several key purposes. First, it provides a means of determining exactly when the etchant has broken through the membrane to the stop solution. That is, prior to breakthrough, the current through the growing pore is zero. The moment of breakthrough is signaled by a sudden increase in the ion current (Figure 1 6). Sec 59 For example, hydroxide and formic acid are commonly used as the etching and stopping solutions, respectively, for etching pores in polymers such a poly(e thylene terephthalate) (PET). When the anode is placed into this etching solution, the hydroxide anions are electrophoretically driven away, or impeded, from the tip opening. Since formic acid neutralizes this lowered hydroxide concentration at the tip, th e net effect is a decreased etch rate at the tip opening. This helps generate conical shaped nanopores having ultrasmall tip diameters

PAGE 33

33 approaching 1 5 nm. 59 Furthermore, the magnitude of the ion current is related to the pore diameter. Thus, by monitoring the ion current, an approximate pore diameter can be determined ( vide infra ). Another benefit of applying a potential during etching is that the cone angle of the conical nanopore in some polymers (e.g. polycarbonate) can be controlled at will by varying the transmembrane potential applied during etching. 64,97 That is, cone angle increases with applied potential. However, in other materials, such as high ion track density PET, this approach is problematic. As a result, etching methods have been developed t o control the cone angle in PET. For example, by introducing ethanol into the aqueous hydroxide etchant, the cone angle can be increased by increasing the ethanol/water ratio. 65 As will be discussed later, an entirely non aqueous method has been developed using potassium hydroxide dissolved in 100% methanol. 114 Additives, such as surfactants, have also been utilized to slow down what is known as the bulk etching rate, The geometry of the nanopore is determined by the track etch ratio, B whe re represents the track etch rate and is the bulk etch rate (Figure 1 7). 56,60 The track etch rate is defined as the rate in which the etchant etches down the long axis of, or parallel to, the latent damage track. The track etch rate is determined by several factors including etching conditions (e.g., etchant composition, concentration, temperature), post irradiation treatment (e.g., UV treatment), sensitivity of the polymer to ion tracking, and polymer type. 60 The bulk etch rate is the rate in whic h the etchant etches radially, or perpendicular, to the damage track. The bulk etch rate is governed by etchant concentration, composition, and temperature. Pore shape is often described using the cone half angle, which is the inverse of

PAGE 34

34 the track etch ratio, or T When T is large, the nanopore is conical shaped (large Conversely, when T is small, a cylindrical pore is obtained (small To make functional resistive pulse sensors using conical nanopores, it is absolutely critical that both the base and tip opening diameters are known and can be reproducibly fabricated and controlled. In other words, validation of any nanopore method of analysis, in terms of instrumentation variability, requires comparable nanopore dimensions from membrane control and reproducibility of the base opening diameter. This is accomplished by performing the anisotropic etch for a certain amount of time (i.e., for PET). For example, to control the base diameter in PET, the polymer is first subjected to UV irradiation ( = 320 nm) for ~ 12 hours to sensitize the latent damage track. Then, the anisotropic etch is performed by pla cing 9 M NaOH on one side of the membrane and 1 M formic acid with 1 M KCl on the other side. 59,62 A +1 V potential difference is applied across the membrane while the OH catalyzes the hydrolysis of the ester linkages of PET, thereby leaving the polymer s urface populated with carboxylate and hydroxyl groups. 110 This anisotropic etch is generally performed for a preset amount of time. In the case of PET, this process is allowed to run for 2 hours and produces a base opening diameter of 520 + 45 nm. 63 Howeve r, the tip opening diameter varies much more from membrane to membrane due to the interfacial mixing of reacting etchant and stopping solutions at the tip. Therefore, to solve this problem and fine tune the tip diameter, a second, or isotropic, etch step w as developed by Wharton, et al. 63 This isotropic etch begins with an analogous experimental setup as that used in the previous, anisotropic etch but with two differences. 63 First, a more dilute etching solution, 1 M NaOH, is placed on both sides of the me mbrane to facilitate a decreased and controllable bulk

PAGE 35

35 etch rate. It is believed that etching occurs uniformly along the entire length of the conical pore. In other words, both the base and tip opening diameters increase at the same rate. The second differ ence is that instead of etching for a predetermined amount of time, etching is stopped at a predetermined value of the ion current (Figure 1 8). Thus, the tip opening diameter can be directly correlated to the ion current value as Wharton, et al. showed. 63 If etch time was used instead of ion current, the large variability in tip opening diameter obtained from the anisotropic etch step would be preserved. As a result, this combination of anisotropic and isotropic etching steps provides precise and accurate control over all of the important dimensions of single nanopores. 63 Nanopore Materials Many materials have been utilized to fabricate nanopores via the track etch method. Polymer membranes are most commonly used because of their excellent response to the ion tracking process. 97 They also possess excellent chemical stability and mechanical robustness under a wide range of conditions and are relatively inexpensive. Polymers such as poly(ethylene terephthalate) (PET), poly(imide) (PI, Kapton), poly(carbonate ) (PC), poly(propylene) (PP), and poly(vinylidenefluoride) (PVDF) have been used to fabricate track etched nanopores. The chemical structure of the 3 most frequently used polymer membranes, PET, PC, and Kapton, are shown in Figure 1 9. In addition to poly mers, conical nanopores have been fabricated in inorganic materials such as glass, 97 muscovite mica, 106,146,156 and silicon nitride 162 via the track etch method. With such a diverse array of materials available to make nanopores, the etching conditions (i .e., etchant composition, concentration, temperature, stop solution type) vary for each material. As mentioned previously, the latent damage track in PET is etched using 9 M NaOH and a stopping solution comprised of 1 M formic acid and 1M potassium chlorid e. 59,62

PAGE 36

36 Track etching is performed at ambient temperature using an applied transmembrane potential of +1 V (i.e., anode placed into the etchant). The alkaline etchant catalyzes hydrolysis of the ester bonds in PET. 110 This reaction leaves a nanopore surfac e populated with both carboxylate and hydroxyl groups. Acid catalyzed ester hydrolysis also occurs but at a much slower rate due to a more involved reaction mechanism. 163 Similarly, conical nanopores are fabricated in ion etched poly(carbonate) using eit her 9 M NaOH or KOH as the etching solution and 1 M formic acid as the stopping solution. 64,97 The process is monitored by applying a transmembrane potential of +1 V (i.e., anode placed into the etchant). As mentioned previously, higher potentials can be a pplied to increase the base diameter and cone angle in low ion track density (50 ion tracks/cm 2 ) poly(carbonate). 64 By comparison, ion damage tracks in Kapton membranes are typic ally etched using sodium hypoch lorite (NaOCl) containing an active chlorine content of 13%, 1 2 M potassium iodide (KI) as the stopping solution, and an applied transmembrane potential of +1 V (i.e., anode placed into the etchant) at a temperature of 50 o C. 60,62,74,75,99 Upon etchant breakthrough at the pore tip opening, an oxidati on reduction reaction takes place in which iodide ions catalyze the reduction of hypochlorite ions to produce chloride ions. The reaction provides iodine which is yellow in color. Thus, this color change signals when membrane breakthrough occurs. This etch ing process proceeds via the hydrolysis of the imide bonds of Kapton and, like PET, generates a carboxylate covered pore surface. 99,163 Ion tracked glass 97 and muscovite mica membranes 106,146,156 can also be chemically etched to fabricate conical nanopor es. The latent damage track is typically etched with hydrofluoric acid (HF) with a stopping solution of calcium chloride. Such thin glass membranes are very inexpensive, have a small track etch ratio, and exhibit excellent reproducibility.

PAGE 37

37 Muscovite mica is a material that has received a great deal of attention recently due to its crystallinity, and large degree of hydrophilicity. 106,146,156 Like glass, ion tracked mica is etched using a hydrofluoric acid etchant but either a calcium chloride or sodium hy droxide stopping solution ( vide infra ). However, fabrication of conical nanopores in mica is quite challenging due to a relatively high track etch ratio (large B ). Thus, the hydrofluoric acid penetrates the entire membrane so quickly that an appreciable amount of bulk material cannot be etched away fast enough to generate any asymmetry in pore shape. Two approaches have been developed to fabricate conical p ores in mica. The first method entails etching a cylindrical pore using hydrofluoric acid. 106 Then, this pore is completely filled with a material, such a silver, which can be used to slow down the track etch velocity in a subsequent etch step. In this sec ond step, a mixture of nitric acid and hydrofluoric acid is used. The nitric acid etches away the silver nanowire along the track while the hydrofluoric acid etches away bulk material surrounding the wire. This results is a conical shaped nanopore. The s econd method for making conical pores in mica involves a multi cycle etching procedure. 146,156 During each cycle, the ion tracked mica membrane is etched in concentrated hydrofluoric acid (10 M) on one side of the membrane for a set period of time and then stopped. Sodium hydroxide is used as the stopping solution. In each subsequent etch cycle, the membrane is etched in the same manner. As a result, the degree of asymmetry slowly increases with increasing cycle number, thereby producing a conical shaped na nopore. As mentioned previously, the track etch ratio, B determines the asymmetry of the nanopore. Both the bulk and track etch rates vary amongst different materials used for pore fabrication. For instance, the bulk etch rates of Kapton and PET a re 0.42 + 0.04 m/hour and ~2.17 nm/min, respectively. 59,62,99 The track etch rate of Kapton is 3.12 + 0.65 m/hour for a

PAGE 38

38 12.5 m thick Kapton membrane. 99 For PET, the track etch rate is ~10 m/hour for a membrane having a thickness of 12 m. 59 Therefore PET membranes etch must faster (i.e., have shorter breakthrough times) than Kapton membranes of comparable thickness. However, since Kapton has a much larger value ( T ), the cone angles and base diameters of nanopores fabricated in Kapton are muc h larger than those made in PET. 59,62,99 Despite having the ability to fabricate single, conical shaped nanopores in a wide variety of materials, no material has yet emerged as the one material best suited for constructing resistive pulse sensors. Nanopor e Characterization and Properties To develop resistive pulse sensors using single, conical shaped nanopores, it is absolutely crucial that the pore dimensions and geometry are accurately known. Without knowing such key parameters as the base and tip openi ng diameters, it would be quite challenging to successfully construct a functional sensor. As will be described below, an accurate base diameter is required to calculate an accurate tip diameter. This tip diameter must be comparable to the hydrodynamic dia meter of the target analyte in order to observe a detectable analyte signal. Therefore, scanning electron microscopy is typically used to accurately determine the base diameter. As will be discussed below, there are several approaches for doing this. For measuring the tip diameter, an electrochemical method based on the ionic conductance of the electrolyte filled nanopore is utilized. Moreover, the conical shape presents several advantageous characteristics that make a conical pore more suitable than a cy lindrical geometry for resistive pulse sensing. The conical shape also provides a means for achieving ion current rectification.

PAGE 39

39 Scanning Electron Microscopy Field emission scanning electron microscopy (FE SEM) is generally used to accurately measure the base opening diameter of conical nanopores (Figure 1 10). The base diameter obtained after the anisotropic etch along with the bulk etch rate are determined from FE SEM measurements on track etched, multi pore membranes (i.e., pore density ~10 6 pores cm 2 ) Multi pore membranes are utilized for two reasons. First, it is simply easier to find a pore when the pore density is large. Hence, this makes the imaging more practical. Secondly, intramembrane base diameter and bulk etch rate reproducibility can be mor e accurately determined by taking such measurements on a large number of pores. The base opening diameter is governed by the bulk etch rate. Therefore, by multiplying the value for the bulk etch rate times the total etching time, the base opening diamet er obtained during the anisotropic etch step can be calculated. For example, as mentioned previously, the reported bulk etch rate for PET is ~2.17 nm/min. 59,63 This value was obtained from FE SEM images. Since the anisotropic etch of ion tracked PET is per formed for 2 hours, based on this etch rate, the base diameter should be ~520 nm. Experimentally, a base opening diameter 520 + 45 nm has been determined. 63 In addition to using track etched, multi pore membranes for measuring pore diameter, two approache s have been developed for imaging much lower pore density membranes. For example, pores in low pore density (50 pores cm 2 ) polycarbonate have been imaged by first sputter coating the membrane surface with a metal. 64,97 As a result, visible light can only penetrate the membrane by going through the pores. A fluorescent dye, fluorescein isothiocyanate (FITC), is then placed beneath the membrane and allowed to penetrate the pores for subsequent fluorescence microscopy. 64,97 This enables the isolation of a sin gle pore for FE SEM.

PAGE 40

40 The second approach was developed in conjunction with the research presented herein. As will be discussed later, this entails first sputter coating the track etched membrane with a metal. Then, the membrane is placed on top of a meta llic mask that has the same diameter as the membrane. This metallic mask is comparable to the filter aperture utilized for ion tracking. That is, a 200 m diameter hole resides in the center of the metallic mask. An ink pen is used to trace this hole onto the center of the sputter coated membrane. This effectively reduces the search area for subsequent FE SEM to 200 m and provides a more efficient way to find and image a pore housed in a single pore membrane. Despite measuring the base diameter via FE SEM and calculating the tip diameter using an electrochemical method ( vide infra ), it is important to accurately determine the geometry of the nanopore. The shape of the nanopore can be characterized by making gold replicas of the pores and imaging them with FE SEM. 64,65,114 These gold replicas are produced using an electroless gold deposition method. This process entails completing filling in the pores with gold and depositing a thin film of gold on both faces of the membrane. To image the gold replicas, two approaches can be used. First, the gold surface layer on both faces on the membrane can be removed, followed by dissolution of the membrane and subsequent filtration of the gold replicas. 64,65 In the second approach, only the gold surface layer on the tip side is removed. Dissolution of the membrane reveals an array of gold replicas standing up on the surface. Gold nanocone arrays have been produced this way and constitute part of the research presented herein. 65,114 Either strategy provides the means to ac curately characterize the geometry of nanopores.

PAGE 41

41 Ionic Conductance Measurements It is important to know the diameters of both the base and tip openings, the length, and geometry of track etched nanopores. Because the base diameter is typically large (~500 nm or higher), the base size can be determined by FE SEM, as mentioned previously. However, resistive pulse sensing of small analytes often requires tip opening diameters that are too small to be determined via FE SEM. Therefore, an electrochemical method is utilized. 59,63,75,89 This approach entails mounting the nanopore containing membrane in an U tube cell. The setup is analogous to that used in track etching. An electrolyte of known ionic conductivity is placed into each half cell and allowed to fill t he conical nanopore. A Ag/AgCl electrode is immersed into each half cell solution. The transmembrane potential is scanned linearly in stepwise fashion while measuring the resulting ion current flowing through the nanopore. As a result, a current voltage cu rve is obtained in which the slope represents the ionic conductance, G (in Siemens, S), of the electrolyte filled nanopore. When the tip diameter is very small, rectification of the ion current can occur (i.e., when the thickness of the electrical double l ayer is comparable to the pore radius). In this case, the linear portion of the current voltage curve (i.e., from 0.2 V to +0.2V) is used to determine the value of G. The ionic conductance of a single, conical shape nanopore is described by: (Eq. 1.1) where d base is the base opening diameter determined from FE SEM, d tip is the tip diameter, L represents the length of the nanopore (equivalent to membrane thickness), and is the specific conductivity of the electrolyte solution (S m 1 ). Therefore, the tip diameter can be calculated from experimental determinations of G and d base The above relationship can be thoroughly

PAGE 42

42 applied to conical nanopores only after the anisotro pic etching step. 63 Tip diameters following the anisotropic etch generally range from 1 7 nm. 63 During the isotropic etch step, the pore is etched at both the base and tip openings at the same rate. 63 Therefore, this must be taken into account when calcul ating the tip opening diameter. Wharton, et al. developed a mathematical model that accounts for this change and provides the basis for calculating the tip diameter from the ionic conductance (G) obtained after the isotropic, or second, etch step. 63 This m odel shows that the change in the base diameter is negligible when the tip diameter is very small (e.g., < 50 nm). Thus, the equation above can be used for calculating small tip diameters. It is important to note that there are three assumptions made when determining the tip diameter of a single, conical shaped nanopore via Eq. 1.1. First, it is assumed that the conductivity of the electrolyte within the nanopore is comparable to the bulk electrolyte conductivity. Such an assumption is only valid at high s alt concentrations. Thus, current voltage curves are typically obtained using 1 M KCl. As the ionic strength of the bulk electrolyte decreases, the electrical double layer that forms along the interior of the pore begins to play a role in the pore conducti vity. The second assumption is that the pore geometry is that of either a perfect cylinder or cone. For this reason, pore shape is verified by fabricating gold replicas, as described previously. Lastly, it is assumed that the bulk etch rate obtained from multipore membranes is comparable and transferrable to that for a single nanopore membrane. Electric Field Strength and Distribution It is important that the nanopore sensing element be conically shaped for several reasons. 95,96 First, the conical shape focuses the electric field at the tip and extends the electric

PAGE 43

43 nanopore that is cylindrically shaped and has a pore diameter of 20 nm. When a transmembrane pot ential difference is applied, the potential is dropped across both in the bulk solution in contact with the pore and the solution inside the pore. Consequently, with such small pore diameters as this, the potential drop occurs mainly inside the pore and le ss in the bulk solution in contact with the pore. If the pore length is 12 m and a 700 mV potential difference is applied, then the electric field strength is approximately on the order of 10 4 V/m inside the entire pore. However, when the pore is conically shaped, the electric field strength is highly focused at the tip opening and reaches into the solution around the tip. 95,96 This is because the pore resistance (R pore = 1/G) is inversely proportional to the product of the base and tip diameters (Eq. 1.1). Thus, the potential drop in the solution at the base opening is insignifi cant when compared to the potential drop at the tip opening. Secondly, simulations show that the electric field strength within the tip opening is enormous. 95,96 For instance, Lee, et al. using a finite element approach to simulate the electric field stre ngth magnitude and distribution within a single, conical nanopore (L = 6 m) with tip and base opening diameters of 60 nm and 2.5 m, respectively. 96 The electrolyte was 1 M KCl with an applied potential difference of 1 V. The results of this simulation ar e shown in Figure 1 11. In Figure 1 11, we see that the electric field strengths in the solutions above and below the conical nanopore are rather small. However, in the expanded view of the tip region, we observe that the electric field strength within the tip opening and in the bulk solution contacting the tip is on the order of 10 6 V/m. In other words, the potential drop is highly focused at the tip opening of the conical nanopore. Furthermore, when the base diameter is held constant at ~520 nm and the ti p diameter is varied between 10 30 nm, such finite element simulations show that the electric field strength inside the tip increases as the tip opening diameter decreases. 95

PAGE 44

44 As a result, this field focusing effect creates an extremely sensitive sensing z one within the tip opening region. 75,89,95,96 Only analytes that migrate into this region will have an impact on the ionic current flowing through the pore. This detection mechanism has provided the means to detect small molecules, DNAs, proteins, and nano into and through the sensing zone. The length of this sensing zone represents the distance from the tip opening where most of the electric field is focused. Heins, et al. defined the sensing zone, or effectiv e length, as the length over which 80% of the voltage is dropped. 75 For example, if the base diameter is 5 m and the tip diameter is 20 nm, finite element simulations show that the conical nanopore has an effective length of 50 nm. The effective length can be tailored to match the size of a target analyte by varying the cone angle of the nanopore. 97 Rectificat ion of Ion Current Another important property of single, conical shaped nanopores is that they can rectify the ion current. This phenomenon is observed when the current voltage curve obtained for a pore is non linear. 18,19 In other words, the absolute val ue of the ion current measured at a one polarity (e.g., 1 V) is not equal to the absolute value of the ion current at the same voltage, but opposite polarity (e.g., +1 V). This asymmetry is attributed to the preferential transport of either cations or ani electronics, where it describes devices that conduct electrons in only one direction. 97 For example, under certain conditions, a conical shaped nanopore in PET or Kapto n preferentially transports cations from the tip opening towards to base opening. 100 103 This is reflected in a non linear current voltage curve. Although the details are still being debated, several models have been reported to explain ion current rectif ication in artificial nanopores. When the latent damage tracks in PET and

PAGE 45

45 Kapton membranes are etched, carboxylate groups are produced along the entire nanopore surface. 62 Above the isoelectric point (pI~3 for both PET and Kapton) for the polymer, these ca rboxylates are deprotonated and create a negative surface charge. 62 As a result, an electrical double layer (EDL) forms along the surface of the nanopore to compensate for this negative charge. The thickness of the EDL is inversely proportional to the ioni c strength of the electrolyte. 164 In other words, as electrolyte concentration decreases, the thickness of the EDL increases correspondingly. Consequently, the negatively charged nanopore becomes cation permselective when the radius of the tip opening is c omparable to the thickness of the EDL. 102 That is, the conical pore will preferentially transport cations and reject anions. Therefore, as long as the pore wall is negatively charged (electrolyte pH > polymer pI), ion current rectification occurs as observ ed by a non linear, current voltage curve. 62,99 102 In the model developed by Siwy, et al., ratchet electrostatic trap for cations is created near the tip opening at positive (i.e., anode at base opening) trans membrane potentials. 102 This electrostatic trap hinders the migration of cations, of the conical nanopore. When a negative (i.e., cathode at base opening), transmembrane potential is applied, this electrostatic trap is removed and a larger ion current is observed. This is charged, conical nanopores. If the surface charge is positive, then t he model is reversed. Three criteria must be met for ion current rectification to occur based on the ratchet model. 102 Such conditions include (1) a conical pore shape, (2) a charged pore wall, and (3) a tip opening radius comparable to the thickness of t he EDL. Studies with track etched cylindrical

PAGE 46

46 nanopores having the same limiting diameter of conical pores show that ion current rectification does not occur. 165 A second model explaining ion current rectification in artificial nanopores was developed by Cervera, et al. 166 Like the ratchet model discussed above, this model requires a conical pore geometry and charged pore wall. This model is based on accumulation and depletion modes of ion transport that are controlled by electrode polarity. For example, negatively charged nanopore. When the positive electrode (anode) is on the tip side of the pore, cations are transported from the tip side to the base side of the membrane. Anions are transported from the base side towards the tip side. However, anions cannot effectively pass through the tip opening due to electrostatic repulsion caused by the negatively charged pore wall. Consequently, anions build up in the nanopore, thereby increasing the local concentration of anions. In ord er to retain electroneutrality, the local concentration of charge balancing cations must also increase the tip region. As the electrolyte concentration increases, the membrane resistance decreases. As a result, under such conditions (i.e., anode a tip ope A different effect is observed when the anode is switched to the base opening of the pore. 166 When the positive electrode is placed on the base side of the pore, cations are transported from the base side towards to tip opening. Anions present within the nanopore are retracted from the pore towards the anode by the electric field. On the tip side of the membrane, anion transport into the tip opening is greatly reduced by e lectrostatic repulsion from the negative charged pore wall. Thus, the retracted anions with the pore cannot be effectively replenished and a local salt

PAGE 47

47 observed with much lower ion current. Bund, et al. reported a similar model based on ion accumulation (i.e., high conductive state) and depletion (i.e., low conductive state). 155 The above models are applicable to single, conical nanopores having tip opening diameters that are very small. However, some studies have been done on conical pores with larger tip diameters. For instance, Kovarik, et al. observed ion current rectification with a conical nanopore (in track etched PET) having a tip opening diameter of 380 nm. 16 7 They believe this may be due to geometric affects and/or an electroosmotic flow but further work remains before this phenomenon can be explained. Yusko, et al. also observed ion current rectification in conical micropores (in borosilicate glass) having tip diameters 500 times larger than the Debye length. 168 Ion current rectification was achieved by introducing dimethyl sulfoxide (DMSO) into the aqueous electrolyte on the tip side of the pore, thereby increasing the viscosity and reducing the ionic cond uctance at the tip. Yusko and coworkers proposed an electroosmotic flow contribution to rectification. 168 That is, when the anode is at the tip, or low conductance, side of the pore, electroosmotic flow drives the low conductance electrolyte into the tip o pening. In contrast, when the anode is at the base, or high conductance, side of the pore, electroosmotic flow drives the high conductance electrolyte into the pore. Jin, et al. demonstrated the rectification of electroosmotic flow in a multi pore mica m embrane containing conical shaped nanopores. 156 This phenomenon occurs as a consequence exists a local depletion of electrolyte with the tip region of the pore This increases the resistivity of the solution. As a result, the velocity of electroosmotic flow ( eof ) increases because eof is proportional to solution resistivity.

PAGE 48

48 In addition to modeling ion current rectification and its impact on electroo s mosis, several studies have been reported on modifying the pore surface to either augment or control ion cu rrent rectification. Other studies have focused on using ion current rectification to developing biosensing applications. Harrell, et al. controlled ion current rectification by attaching thiol terminated DNAs to a gold coated, conical nanopore in polycarb onate. 104 By controlling the DNA chain length, the degree of rectification can be controlled. That is, the extent of ion current rectification increases with increasing DNA chain length by increasing the negative surface charge within the tip opening and d ecreasing the tip diameter. Umehara, et al. reversed the direction of ion current rectification by modifying a conical nanopipette (in quartz) with a positively charged poly L lysine coating. 169 Fu, et al. modified a conical nanopipette (in glass) with a fourth generation poly(amido amine) dendrimer (G4 PAMAM). 170 This dendrimer creates a cationic surface which can bind polyanionic DNA via electrostatic adsorption. As a result, DNA adsorption and hybridization can be detected by monitoring changes in ion c urrent rectification. Vlassiouk, et al. used ion current rectification to develop a sensor for poly D glutamic acid ( DPGA) from Bacillus anthracis by modifying a conical pore in track etched PET with the monoclonal antibody for DPGA. 171 Similarly, sens ors for the proteins, avidin and streptavidin, were developed by modifying conical pores with biotin. Furthermore, Vlassiouk and coworkers demonstrated that ion current rectification can be used to determine the isoelectric point of proteins. This was achi eved by modifying the tip opening with a small amount of protein and monitoring the change in ion current rectification as the pH is varied. Sexton also used ion current rectification to closely approximate the isoelectric points for bovine serum albumin, amyloglucosidase, and phosphorylase B by modifying track etched conical nanopores in PET with such proteins. 172

PAGE 49

49 Controlling Nanopore Surface Chemistry and Properties As previously mentioned, the fabrication of conical nanopores in artificial materials pro vides chemical stability and mechanical robustness under a wide range of conditions. Although this is very important, the analytical utility of such pores is largely determined by controlling the pore surface chemistry and properties. For instance, the non specific adsorption of biological molecules often has an adverse impact on the sensing capabilities of conical nanopores. Thus, the pore surface is chemically modified to present a biocompat ible surface that is more amen able to biosensing. Furthermore, th e pore surface can be modified with molecular recognition agents (e.g., antibodies, aptamers, DNA, proteins) to introduce selectivity into the pore. Several methods exist for controlling the pore surface chemistry ( vide infra ) The first approach utilize s electroless gold deposition to coat the pore wall with a t hin layer of gold which is amen able to subsequent thiol based functionalization. 163 Secondly, the carboxylate groups created during the track etching of PET are activated and made amine reactive u sing 1 ethyl 3 [3 dimethylaminopropyl]carboimide/N hydroxysulfosuccinimide (EDC/sulfo NHS). 173 178 As a result, an amine terminated species can be covalently linked to the pore surface via an amide bond. In the case of glass nanopores, sol gel and silane c hemistry can be utilized to control the pore surface chemistry. 179 181 Electroless Gold Deposition Electroless deposition of metals, such as gold, inside nanoporous structures provides a useful way for controlling pore diameter and introducing various che mistries into the pore. This approach is also useful for fabricating hollow and solid nanostructures via the template synthesis method. In general, template synthesis entails the deposition of a material into the pores of a template. 65,113,114,152,153,182 201 Electroless gold deposition represents a template synthesis method

PAGE 50

50 for depositing gold into nanopores by using a chemical reducing agent to deposit gold from a gold solution onto the pore and membrane surfaces. 163 This method begins with the exposure of the track etched membrane to a sensitizer, Sn 2+ (Figure 1 12). 163 This is achieved by briefly rinsing the nanopore membrane in methanol and then immersing the membrane into a solution of 0.026 M SnCl 2 and 0.07M trifluoroacetic acid in 50/50 water/metha nol for 45 minutes. As a result, the Sn 2+ binds via electrostatic complexation to the negatively charged functional groups on the pore and membrane surfaces that are created during track etching. 163 For instance, track etching of PET produces carboxylate g roups along the surface. 62 After coating the surface with Sn 2+ the membrane surface is thoroughly rinsed with methanol to remove any excess SnCl 2 and subsequently immersed into an aqueous ammoniac solution of 0.029 M AgNO 3 for 7.5 minutes. As a result, a surface redox reaction occurs in which Ag + is reduced to elemental Ag concurrently with the oxidation of surface bound Sn 2+ to Sn 4+ This generates a membrane surface layer of silver nanoparticles. 163 The silver coated membrane is rinsed in methanol to r emove excess silver nitrate. Then, the membrane is placed into a low temperature (4 o C), gold plating solution comprised of 0.127 M Na 2 SO 3 0.625 M formaldehyde, and 7.9 x 10 3 M Na 3 Au(SO 3 ) 2 and adjusted to pH 10 with dropwise addition of 1 M H 2 SO 4 Since the standard reduction potential of Au is more positive than that of Ag, Au galvanically displaces the silver nanoparticles on the membrane surface, thereby initiating the formation of a surface layer of gold nanoparticles. 163 These gold nanoparticles prov ide excellent catalytic sites to catalyze the subsequent oxidation of formaldehyde with concurrent reduction of Au(I) to Au(0) via: 163 2 Au(I) + HCHO + 3OH HCOO + 2H 2 O + 2 Au(0)

PAGE 51

51 These catalytic sites on the pore wall are important be cause to form a surface layer, the reduction of the metal ion needs to occur at the pore surface. This process occurs spontaneously, requires no electrodes, and relies entirely on redox chemistry. Thus, it is appropriately referred to as an deposition process. In the case of conical nanopores, electroless gold deposition results in a gold, conical nanotube and a gold layer on both faces of the membrane. This gold layer is typically too thin to block the nanopore tip opening. To isolate the g old, conical nanotube, the gold surface layer on the membrane faces can be removed via tape removal (i.e., with Scotch tape) or ethanolic swabbing. 64,65,95,114 The diameter of the resulting gold nanotube can be varied at will by varying the deposition tim e. In other words, thicker gold layers, or small pore diameters, can be obtained using longer plating times. Gold nanotubes having inner diameters on the order of molecular dimensions (1 nm) can be obtained utilizing this method. Furthermore, by extending the plating time for very long periods of time, solid, gold nanocones can be fabricated. Gold nanocones are described as part of the research presented herein. The advantage of coating a conical nanopore with gold resides in the fact that the gold layer pr ovides an effective means for chemical and biological functionalization using very well known and versatile gold thiol chemistry. Chemisorption of Thiols on Gold Coated Nanopores To construct functional sensors using conical nanopores, it is important to control the surface chemistry of the pore. By modifying the pore surface, the transport properties of the nanopore can be controlled at will. For instance, chemisorption has been used to covalently attach various thiol molecules to the surfaces of gold co ated nanotubes. The process of chemisorption occurs when thiols come in contact with a gold surface. The lone pair electrons on the sulfur atom form a covalent bond with the electrons from the electron rich gold surface. This process is commonly utilized t o generate self assembled monolayers anchored via thiols to gold

PAGE 52

52 surfaces. 202 205 It is also used to functionalize gold nanoparticles with thiol modified molecules, such as DNA. 206 209 Experimentally, chemisorption simply involves immersing the membrane c ontaining a gold nanotube into a solution of the desired thiol for a period of time. The thiol coated nanotube is then rinsed to remove excess thiol solution. The tip opening diameter of the nanotube can be measured using the electrochemical method, as des cribed previously. Using gold thiol chemistry, molecules have been attached to nanopores to introduce (1) pH control over ion selectivity, 165,200,210 212 (2) chemical selectivity, 200,211,213 216 (3) size based selectivity, 95,117,217 ,218 and (4) selectivity based on the hybridization of nucleic acids. 109 Thiol chemisorption has also been used to attach molecular recognition agents onto the pore wall that target specific proteins. To detect biological molecules with single nanopores, the pore wall must be mo dified to prevent non specific adsorption. 95,117,202 205,219 221 In multi pore membranes, this has been accomplished by coating the gold plated pore wall with thiol modified poly(ethylene glycol) (PEG). 117 PEG surfaces are typically used to prevent surface adsorption of biomolecules because they are uncharged and hydrophilic. Such PEG coatings have been used on quantum dots 222 and SPR (Surface Plasmon Resonance) surfaces 223 to prevent non specific adsorption. The effectiveness of PEG coatings in single, con ical shaped nanopores is part of the research presented herein. Selective Functionalization using Carboiimide/N Hydroxysuccinimide Chemistry An alternative approach to controlling the surface chemistry of conical nanopores is by using a coupling method b ased on 1 ethyl 3 [3 dimethylaminopropyl]carbodiimide (EDC)/ N hydroxysulfosuccinimide (sulfo NHS) chemistry. 173 175 This approach is commonly used to couple primary amines, including small molecules, amine modified DNAs, and proteins, to carboxylates. 90,17 2 178,224 229 As previously mentioned, track etching of both PET and Kapton

PAGE 53

53 produces free carboxylates along the pore wall. 62 As a result, the pore surface can be modified with primary amines via EDC/sulfo NHS coupling chemistry. The procedure generally entails two steps, (1) activation/stabilization and (2) amine conjugation (Figure 1 13). 173 First, the free carboxylate groups along the pore wall are activated with EDC. That is, the negatively charged oxygen of the carboxylate attacks the electropositive carbon located between the two adjacent nitrogens on EDC. It is electropositive due to inductive withdrawal of electron density by these two nitrogen atoms. As a result, an o acylisourea ester is formed which is unstable because of the carbon with three e lectronegative atoms bonded to it. In other words, this is a high energy intermediate. A number of directions in electron movement would take this intermediate down in energy, thereby producing a more stable species. Thus, the half life of the o acylisoure a ester is very short in aqueous solutions. Unless the desired amine reacts with this ester very quickly, the ester is usually converted back to the carboxylate via hydrolysis. 173 Therefore, EDC alone lacks a high degree of efficiency for coupling carboxyl ates and primary amines. Consequently, the solution to this problem is to convert the unstable o acylisourea ester to a more stable, amine reactive NHS ester by adding sulfo NHS. The formation of a semi stable intermediate is driven by the formation of th e urea byproduct which is very stable relative to EDC and the o acylisourea ester. 173 A primary amine is then added to the reaction. Nucleophilic attack from the amine occurs at the electropositive carbon on the carbonyl of the ester. This carbon is electr opositive because of all of the electron withdrawing groups nearby (e.g., on the succinimide). Thus, if the pH is high enough such that an appreciable amount of amines are unprotonated (i.e., nucleophilic), then the most stable product, the amide, will dom inate at equilibrium. As a result, the desired primary amine is conjugated to the pore wall via a stable amide bond. 173

PAGE 54

54 Several studies have been conducted on coupling primary amines to conical nanopores using the EDC/sulfo NHS approach. For instance, Ali et al. changed the surface polarity of conical nanopores in track etched Kapton from negative to positive by coupling ethylenediamine to the pore wall using EDC/NHS. 197 As a result, the ion permselectivity of the nanopore was switched from cation select ive to anion selective. This provides a means for controlling the direction of ion current rectification. Vlassiouk, et al. altered the surface charge of conical nanopores in track etched PET by local modification of the region just inside the tip opening with ethylenediamine. 228 As a result, the direction of ion current rectification was controlled and diode like behavior observed. In another study by Ali, et al., conical nanopores in track etched PET were individually modified with ethylenediamine and p ropylamine using EDC/pentafluorophenol (PFP) coupling chemistry. 229 PFP was used instead of NHS due to a reportedly higher coupling efficiency. The ethylenediamine changed the polarity of the PET surface from negative to positive. The propylamine provided a more hydrophobic pore surface due to the terminal propyl ( C 3 H 7 ) group. As result, the direction of ion current rectification was controlled. Furthermore, bovine serum albumin (BSA) was adsorbed to the different pore surfaces and detected via current vol tage curves. This provides a means of studying BSA adsorption as a function of pH and pore surface chemistry. As previously mentioned, both Sexton and Vlassiouk, et al. used EDS/sulfo NHS to couple proteins to conical nanopores in track etched PET to deter mine the isoelectric points of proteins. 171,172 Kececi, et al utilized EDC/sulfo NHS to attach ethanolamine to conical nanopores in PET to reduce the negative charge of the pore wall in order to detect small DNAs via the resistive pulse method. 90 Thus, ED C/sulfo NHS provides a very versatile route to modifying nanopore surfaces populated with free carboxylates.

PAGE 55

55 Additional Strategies for Surface Functionalization and Controlling Pore Size In addition to electroless gold deposition and EDC/sulfo NHS couplin g, other methods have been used to chemically modify the pore surface of nanopores and control pore size. Sol gel chemistry has been used previously to fine tune the pore diameter and surface chemistry. 179 181,230 Briefly, in the sol gel method, tetraethyl orthosilicate is dissolved in an acidic solution and undergoes hydrolysis for a fixed time interval (e.g., 30 min). 179 181 The nanopore is then briefly immersed in this solution and sonicated. Then, the silica sol is cured by removing and rinsing the memb rane and placed it in an oven at typically > 100 o C. As a result, a relatively uniform, thin layer of silica is applied to the nanopore surface. This entire method can be performed repeatedly, thereby adding subsequent silica layers and providing a means of controlling the pore diameter. Hillebrenner, et al. 179 and Buyukserin, et al. 180,181 used sol gel chemistry to fabricate silica nano test tubes in alumina membranes for eventual use in target specific delivery and imaging applications. To control the surf ace chemistry of the nanopore, silanes have been used. For instance, PEG silanes have been used to enhance surface hydrophilicity and reduce non specific adsorption. Aldehyde silanes have been used as the first step in coupling molecular recognition agents to the pore wall. First, an aldehyde terminated silane is coupled to the pore surface. Molecular recognition agents are then coupled via primary amines to the aldehyde group of the silane using Schiff base chemistry. The stability of the Schiff base can b e improved by reducing the imine bond with NaCNBH 3 Hillebrenner, et al. introduced free amine groups to the open tubular ends of silica nano test tubes via an amine terminated silane. 179 These amine groups were then reacted with aldehyde modified latex nanoparticles using Schiff base chemistry. As a result, imine bonds

PAGE 56

56 were formed which linked the nanoparticles to the open ends of the nano test tubes, thereby capping them in a manner akin to corking laboratory test tubes. In a similar manner, Buyukse rin, et al. introduced free amine groups to the interior wall of silica nano test tubes housed in a porous alumina template using an amine terminated silane. 181 The amine coated tube wall was then reacted with Alexa 488 carboxylic acid succinimidyl ester w hich cross linked the fluorescent Alexa 488 dye to tube interior while producing N hydroxysuccinimide as a reaction by product. The nano test tubes were then liberated from the alumina template, thereby exposing the tube exterior. These outer walls of the nano test tubes were functionalized with aldehyde groups using an aldehyde silane. This provided a route to cross linking antibodies (in this case, rabbit polyclonal IGF IR ), via the free amine sites on the antibodies, to the aldehyde coated tube wall usi ng Schiff base chemistry. Such antibody coated nano test tubes were used to selectively target MDA MB 231 breast carcinoma cells and image them via fluorescence microscopy. Additional Sensing Strategies Based on Track Etched Conical Nanopores In addition to resistive pulse sensing, other sensing methods using track etched conical nanopores have been reported. For instance, Siwy, et al. developed a protein biosensor that 157 That is, a single, conical shaped nanopore was first fabricated via the track etch method in a polymeric membrane. The pore walls were subsequently coated with a thin, conformal gold layer via electroless deposition. As a proof of concept experiment, the gold surface was functionalized with thiol modif ied biotin which is a highly selective molecular recognition agent for the protein, streptavidin. The detection paradigm entails passing an ion current through the biotin coated nanopore. However, unlike the resistive pulse method, transient current pulses are not observed. Instead, the protein analyte (in this case,

PAGE 57

57 streptavidin) selectively binds to the biotin immobilized at the tip opening of the conical nanopore. Since the protein and tip opening have comparable diameters, binding of the protein effecti vely blocks the nanopore tip. This molecular recognition event is detected as a permanent introduction of the target protein analyte, the ion current was swit paradigm was subsequently applied to two other molecular recognition agent/target analyte systems including protein G/immunoglobulin (IgG), and anti ricin/ricin. Furthermore, this approach can likely be extended to a wide range of molecular recognition agent/target analyte systems. In another sensing approach, ion current rectification was used to monitor the adsorption of analyte drug (i.e., Hoechst 33258) molecules to the pore walls in a single track etched polyimide membrane. 158 Polyimide is relatively hydrophobic, but has free carboxylate groups along the pore wall. The anionic surface, due to these carboxylates, caused the conical pore to rectify the ion current passing through it. Hoechst 33258 is also hydrophobic, but cationi c. When the conical pore was exposed to this drug, it adsorbed to the pore surfaces. As a result, the negative surface charge of the pore was reduced along with the extent of ion current rec t ification At higher drug concentrations, the surface charge pola rity was reversed to positive along with a corresponding reversal in the direction of rectification. Such changes in ion current rectification were related to the drug concentration. Thus, this represents a sensing paradigm incorporating hydrophobic effect based selectivity using track etched conical nanopores. 158 Dissertation Overview The objective of the research efforts presented herein was to investigate resistive pulse sensing, nanopore properties associated with resistive pulse sensing and ion transp ort, as well as nanopore fabrication. Chapter 1 presents an overview of pertinent background information that

PAGE 58

58 supports these research efforts. This includes the resistive pulse method, prior sensing work with biological and artificial nanopores, ion irradi ation of polymer membranes, the track etch method, pore materials, nanopore characterization, ion current rectification, and methods for controlling pore size and surface chemistry. In Chapter 2, an approach for detecting proteins using the resistive puls e method is reported using a model protein analyte, streptavidin. A single, conical shaped nanopore in track etched PET was modified via electroless gold deposition and subsequent chemisorption of thiol modified PEG. This provided the sensing element to de tect individual protein molecules. Current pulse direction was evaluated as a function of pore surface chemistry. The impact of applied transmembrane potential on the current pulse frequency was also studied. In Chapter 3, efforts to detect cationic analy tes via the resistive pulse method are introduced. That is, the resistive pulse method was applied to the detection of a model cationic analyte, cationic protein coated gold nanoparticles. These particles were detected using an unmodified, conical nanopore in track etched PET. This represents a departure from current examples of resistive pulse sensing in PET, which involved surface modified nanopores. The impact of particle concentration on current pulse frequency was investigated. A definition for the det ection limit in resistive pulse sensing was proposed and discussed. Current pulse direction, duration, and amplitude were also examined. In Chapter 4, the resistive pulse method is applied to the detection of polymeric nanoparticles in an alternative pol ymer type, polyimide. These particles were detected using an unmodified, conical nanopore in track etched polyimide. A lower ionic strength electrolyte, relative to electrolytes used previously with polyimide resistive pulse sensors, was used. A pore loadi ng process based on the use of a wetting agent, with vacuum degassing and perfusion, was

PAGE 59

59 introduced. Efforts towards developing a two step etch method for single, conical shaped pores in polyimide were presented. The impact of tip diameter of ion current r ectification was examined. The relationship between current pulse amplitude, duration, and frequency with applied transmembrane potential was evaluated. In Chapter 5, an alternative approach to electroless gold deposition for functionalizing the pore surf aces of single, conical shaped nanopores with poly(ethylene glycol) using EDC/sulfo NHS coupling chemistry was introduced. The effectiveness of this approach towards reducing non specific protein adsorption was investigated using three prototype proteins t hat present different surface reactivity types. Current voltage curves and X ray photoelectron spectroscopy (XPS) were used to evaluate protein adsorption. In Chapter 6, a non aqueous approach to fabricating and increasing the cone angle of conical nanopo res in track etched PET is presented. This approach provides a route to fabricating single, conical shaped nanopores with larger cone angles than the commonly used aqueous two step etch method. The impact of larger cone angle on the electric field strength was modeled via finite element simulations and discussed. The effect of increased cone angle on ionic pore conductance and ion current rectification was examined by holding the tip diameter constant and varying the base diameter. A n approach for efficient ly finding single pores in single track etched membranes for imaging was presented. Furthermore, efforts to construct large cone angle pores in high track density PET membranes were examined and discussed. Fabricating randomly distributed gold nanocone arr ays of controllable cone height and base diameter was also presented.

PAGE 60

60 Figure 1 1. Diagram detailing the resistive pulse method ( pore not drawn to scale; drawn data used to show concept ). Figure 1 2. The biological protein nanopore, Hemolysin, embedded in a lipid bilayer support. [adapted from Bayley, H.; Jayasinghe, L. Molecular Membrane Biology 2004 21 209 220.]

PAGE 61

61 Figure 1 3. Schematic of the ion track etch method. A) Irradiation of a thin membrane with high energy, heavy met al ions results in B) the formation of latent damage tracks along the path of each ion. C) Chemical etching proceeds along each damage track creating pores. Figure 1 4. Schematic of the electrochemical cell used for track etching and ionic conductance m easurements [adapted from Wharton, J. E.; Jin, P.; Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Mino, W. K.; Martin, C. R. Small 2007 3 1424 1430.]

PAGE 62

62 Figure 1 5. Diagram of a conical nanopore in a polymer membrane showing the base and tip opening diame ters (drawing not to scale). [adapted from Wharton, J. E.; Jin, P.; Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Mino, W. K.; Martin, C. R. Small 2007 3 1424 1430.] Figure 1 6. Plot of ion current versus time recorded during the anisotropic etch ste p for the fabrication of a conical shaped nanopore in PET. The moment of breakthrough is signaled by the sudden increase in ion current around 75 minutes.

PAGE 63

63 Figure 1 7. A schematic of the track etch method of fabricating a conical nanopore showing the bu lk etch rate, B track etch rate, T and cone half angle, Figure 1 8. Plot of ion current versus time recorded during the isotropic etch step for tailoring the tip opening diameter in PET. The ion current increases with increasing etch time as the tip opening diameter increases.

PAGE 64

64 Figure 1 9. Chemical structures of commonly used polymers for ion track etching. A) poly(carbonate) (PC), B) poly(ethylene terephthalate) (PET) and C) poly(imide) (PI Kapton ).

PAGE 65

65 Figure 1 10. Scanning electron microgra phs of nanopores track etched in various materials. A) Glass, B) PC, C) PET, and D) Kapton Figure 1 11. Magnitude and distribution of the electric field across a conical nanopore. The nanopore used for simulations had a base opening diameter of 2.5 m, a tip opening diameter of 60 nm, and a pore length of 6 m. 1 V was applied across the nanopore using 1 M KCl. White hash marks are added to the section of the nanopore where the majority of the electric field is focused. [adapted from Lee, S.; Zhang, Y.; White, H. S.; Harrell, C. C.; Martin, C. R. Analytical Chemistry 2004 76 6108 6115.]

PAGE 66

66 Figure 1 12. Schematic of the electroless gold deposition procedure. [adapted from Menon, V. P.; Martin, C. R. Analytical Chemistry 1995 67 1920 1928.] Figure 1 13. Diagram of EDC/Sulfo NHS coupling chemistry. Formation of a stable amide bond occurs between a carboxylate molecule and a molecule with a terminal primary amine group via EDC/Sulfo NHS chemistry. [adapted from Pierce Biotechnology, http://www.piercenet.com ]

PAGE 67

67 CHAPTER 2 RESISTIVE PULSE SENSING OF A MODEL PROTEIN USING A CONICAL SHAPED NANOPORE Introduction There is increasing interest in developing resistive pulse devices for the rapid detection and quantificati on of small molecules and biological analytes using biological 66 73,78 88,93,94,118,119,121,126,231 233 and artificial nanopores. 24 31,36 54,75,89 91,95,157,158,234 236 While sometimes referred to as stochastic sensing, 66 70,78,79,93,94,118 120 the resisti ve pulse method 235 begins with a membrane containing a single nanopore having a limiting diameter comparable to the size of the target analyte. This membrane is immobilized between two electrolyte solutions. A transmembrane potential difference is applied and the resulting ion current flowing through the electrolyte filled nanopore is measured. When the target analyte is added to one of the electrolyte solutions, analyte molecules are driven into the nanopore where they displace a volumetric fraction of ele ctrolyte and transiently block the ion current. The ion current returns to the steady state when the molecule exits the nanopore, thereby resulting in downward current pulses. The frequency of the current pulses is proportional to analyte concentration, an d analyte identity, or selectivity, is determined by both the magnitude and duration of the current pulses. 50,143,234,235,237 Most resistive pulse sensing data reported in the literature have been obtained using hemolysin ( HL), a biological nanopore that self assembles into supported lipid bilayer membranes. A wide variety of analytes including small molecules, 68 70 metal ions, 66 DNA, 78,79,82 88 and proteins 93,94 have been detected using HL. As a result, the HL nanopore approach represents the benchmark by which devices using alternative pore

PAGE 68

68 materials are evaluated. However, this approach is very limited due to the large fragility of the lipid bilayer membrane containing the HL pore. 119,143,144 Consequen tly, this renders lipid bilayer based systems as impractical. As a result, there is significant research interest in developing artificial analogues (i.e., an abiotic nanopore housed in a chemically stable and mechanically robust artificial membrane) of su ch biological pores. For instance, ion or electron beam fabrication methods have been utilized to construct nanopores in silicon nitride and silicon films. 23 25 Such films have been primarily used for the detection of DNA via the resistive pulse method. A n increasingly popular alternative to biological pores are nanopores fabricated by the track etch method. 55 65 This approach has been utilized for decades for the commercial production of nanopores in artificial polymer membranes used for filtration applic ations. 148 151 Such membranes have a large nanopore density (i.e., often >10 6 pores cm 2 ). However, the resistive pulse method requires a membrane containing one nanopore. Fortunately, a company in Germany, GSI, developed a single ion irradiation technique that allows for the fabrication of single nanopores in thin polymer membranes (e.g., polyimide, polycarbonate, poly(ethylene terephthalate), 5 12 m thick). 57,58 Both cylindrical and conical shaped nanopores can be fabricated in such membranes. Such coni cal nanopores have been used to detect proteins, 95 DNA, 89,90 nanoparticles, 96 and small molecules. 74,75 In this chapter, a resistive pulse sensor for a model protein, streptavidin, using a conical shaped nanopore in track etched poly(ethylene terephthalat e) (PET) as the sensing element is described. A conical shaped nanopore is comprised of two openings, a large diameter opening at one side of the membrane and a small diameter opening

PAGE 69

69 located at the other side of the membrane. 74,75,89,90,95,96 The large di ameter opening is diameter opening is known as the The protein, streptavidin, was driven via electrophoresis through the conical nanopore in the direction of tip to base. Prot ein translocation events were detected as transient blocks, or current pulses, in the ion current flowing through the nanopore. The frequency of the current pulses increased with the magnitude of the applied transmembrane potential. The nanopore sensing el ement was coated with a thin layer of gold via electroless gold deposition. 163 Subsequent chemisorption of a thiol modified poly(ethylene glycol) on the gold surface was used to reduce non specific protein adsorption. 95,117,202 205,219 221 Analogous to cla ssic Coulter Counting, in the PEG modified nanopore, current pulses were predominantly downward. In contrast, in an unmodified conical nanopore of comparable tip size, the current pulses were upward and downward. The results suggest a protein adsorption/d esorption event occurring on the unmodified pore wall as protein s translocate the sensing zone. Experimental Materials Poly(ethylene terephthalate) (PET) films (3 cm diameter, 12 m thick) were obtained from GSI (Darmstadt, Germany). These films were each irradiated with a single, swift heavy ion to create a single damage track through the entire film. The model protein, streptavidin (SA), was obtained from Sigma Aldrich and used as received. SA has a molecular weight (MW) of ~60 kDa. A thiol modified poly (ethylene glycol) (PEG thiol, MW 5 kDa) was obtained from Laysan Bio (Huntsville, AL). All other chemicals were of reagent grade or better and used as received. All solutions were prepared using

PAGE 70

70 purified water (i.e., obtained by passing in house deionized water through a Barnstead, E Fabrication of the Conical Nanopore A conical shaped nanopore was etched into the single ion tracked poly(ethylene terephthalate) membrane by anisotropic, and subsequent isotropic, che mical etching of the swift heavy ion irradiated poly(ethylene terephthalate) film. Using the two step etch method developed by Wharton, et al., 63 the irradiated film was mounted between two halves of an U tube cell made of Kel F. An etching solution, 9 M s odium hydroxide (NaOH), was added to one half cell and a stop solution, 1 M formic acid and 1 M potassium chloride (KCl), was added to the other half cell. The ion induced damage track was etched preferentially from the membrane face in contact with the et ching solution towards the membrane face in contact with the stopping solution. Etching was performed until the etch solution completely penetrated through the membrane to the stop solution on the other side. 63 To detect exactly when breakthrough occurr ed, a platinum wire electrode was placed into each half cell and a transmembrane potential difference of +1 V was applied during etching using a Keithley 6487 voltage source/picoammeter (Keithley Instruments, Cleveland, OH). The electrodes were configured such that the positive electrode (anode) was located in the etching solution side of the membrane. The negative electrode (cathode) was located in the stopping solution side of the membrane. By applying a transmembrane potential difference, the etching pro cess was monitored in real time. 63 The ion current was initially zero. At the moment of membrane breakthrough, the ion current suddenly increased. For poly(ethylene terephthalate) membrane used herein, breakthrough usually occurred ~1.5 hour. This anisotro pic etch procedure was performed for 2 hours. In addition to

PAGE 71

71 fabricating a conical shaped nanopore, the chemical etching process thinned the membrane slightly from 12 to ~11.76 m. After etching, the etching solution is removed and replaced with neutralizi ng stopping solution. Then, both halves of the U tube were emptied and rinsed thoroughly with water. The membrane was stored overnight in water. To tailor the tip opening diameter, the second etch step was performed using an analogous experimental set up as that used for the anisotropic etch process described above. 63 However, a more dilute etching solution, 1 M NaOH, was placed onto both sides of the membrane. A transmembrane potential difference of +1 V was applied across the membrane with the anode loca ted on the base opening side of the membrane and cathode at the tip opening side. The resulting ion current was measured as a function of etching time. The two step etch method provides the means for fabricating very reproducible base and tip diameters. 63 As Wharton, et al. demonstrated, the ion current correlates to tip opening diameter. 63 Thus, by stopping the isotropic etching process at an ion current value of 25 nA, the tip diameter was approximated to be 50 nm. The base diameter obtained after the ani sotropic etching step and used for this work was 520 + 45 nm, as obtained via field emission scanning electron microscopy (FE SEM), per Wharton, et al. 63 In addition to measuring the base diameter, FE SEM (Hitachi S 4000) was utilized to evaluate the geom etry of the conical nanopores This was achieved by completely filling in the nanopores (multiporous PET, 10 6 pores/cm 2 ) with gold using a previously described electroless gold deposition procedure. 163 As a result, gold nanocone replicas of the nanopores ar e produced. Furthermore, both membrane faces of the poly(ethylene terephthalate) membrane are coated with thin layers of gold during the

PAGE 72

72 deposition process. To image these nanocone replicas, the gold surface layers on both faces of the membrane were remove d via mechanical polishing away the gold using a cotton swab wetted with ethanol. The polymer membrane was then removed via dissolution using 1,1,1,3,3,3 hexafluoroisopropanol (HFIP). The liberated nanocones were filtered through a commercially available a nodized alumina membrane filter that had been sputter coated with Au Pd. 64,65 The same electroless gold deposition method was used to deposit gold along the pore walls of the nanopore to produce a gold coated conical nanopore. 95,163 Again, the gold platin g process produces a thin, gold surface film on both faces of the membrane which was subsequently removed by mechanically polishing away the gold using a cotton swab wetted with ethanol. As a result, a single, conical shaped gold nanopore is created. A cu rrent voltage curve obtained after electroless gold deposition was used to measure the tip opening diameter of the resulting gold nanopore ( vide infra ). 95 No significant change in the base opening diameter occurred due to gold plating. The gold surface of the nanopore was modified with PEG thiol (MW 5 kDa) in order to prevent non specific protein adsorption. 95,117,217 This was achieved by placing the gold nanopore containing membrane into a 100 M solution of PEG thiol in purified water for ~12 hours at 4 o C. The membrane was then carefully rinsed in 2 L of purified water to remove excess PEG thiol. That was accomplished by suspending the membrane atop of 2 L of purified water and slowly stirring via stirbar. The tip opening diameter of the PEG modified nanopore was then measured using an electrochemical method based on current voltage curves ( vide infra ).

PAGE 73

73 Electrochemical Measurements For measuring tip opening diameter, the single, conical shaped nanopore membrane was mounted in an U tube cell made of Kel F and both half cells were filled with an electrolyte solution of known conductivity (10.5 11.5 S/m) that was 1 M KCl in purified water (pH ~6). A Ag/AgCl electrode was placed into the electrol yte in each half cell. A transmembrane potential difference was applied across the electrolyte filled nanopore and the resulting ion current measured. The applied potential was linearly increased in stepwise fashion from 1 V to +1 V while measuring the io n current at each potential step. The slope of the resulting current voltage was utilized to calculate the tip diameter of the nanopore ( vide infra ). 59,75,89 For current pulse measurements, the PEG modified, conical nanopore membrane was mounted in a U tu be cell in similar fashion. A schematic of the PEG coated nanopore sensing element is illustrated in Figure 2 1. Both half cells were filled with ~3.5 mL 0.1 M KCl that was pH 7.4 using a 10 mM phosphate buffer solution. A commercially available Ag/AgCl e lectrode (Bioanalytical Systems/BASi, West Lafayette, IN) was placed into the electrolyte in each half cell and connected to an Axopatch 200B patch clamp amplifier (Molecular Devices Corp., Union City, CA). The Axopatch 200B was utilized to apply a desired transmembrane potential and monitor the corresponding ion current flowing through the electrolyte filled nanopore. Current recordings were obtained in voltage clamp mode using a low pass Bessel filter at 2 kHz bandwidth. The signal was digitized using a D igidata 1233A analog to digital converter (Molecular Devices Corporation). Data were recorded and analyzed using pClamp 9.0 software (Molecular Devices Corporation). Unless stated differently, the electrodes were configured as follows: the positive Ag/AgCl electrode (anode) was placed into the

PAGE 74

74 electrolyte in the half cell located on the base opening side of the conical nanopore and the negative Ag/AgCl electrode (cathode) was placed into the electrolyte at the tip opening side. Because the pH of the sensing buffer (pH ~7.4) used here is above the isoelectric point of streptavidin (pI ~7.0), 238 the protein has a net negative surface charge. Therefore, the protein solution was added at the cathode side of the membrane facing the tip opening of the conical nano pore. Results and Discussion Nanopore Characterization Wharton, et al. demonstrated that the two step etch method reproducibly produces a base opening diameter of 520 nm for a conical shaped nanopore in track etched poly(ethylene terephthalate). 63 Knowin g the base diameter allows the tip opening diameter to be calculated from an experimental determination of the ionic conductance of the electrolyte filled nanopore. 59,75,89 It the nanopore possesses a true conical geometry, then the ionic conductance of th e nanopore (G) is related to the base diameter (d base ), the tip diameter (d tip ), the specific conductivity of the supporting electrolyte ( ), and the pore length (membrane thickness after etching, L) via the following equation: 89 (Eq. 2.1) The value of G was experimentally determined by simply measuring the linear current voltage curve for the electrolyte filled nanopore. That is, G is the slope of the linear current voltage curve. With this value of G, all of the other variables in Eq. 2.1 are known. Therefore, the tip opening diameter can be calculated. To further evaluate the geometry of the nanopore, an electroless gold depositio n method was used to completely fill the conical nanopores produced in a comparably

PAGE 75

75 etched multi tracked PET membrane with gold. 163 Multi tracked PET membranes are used since a single gold replica obtained from a single pore PET membrane is difficult to fi nd. As a result, gold replicas that represent the geometry of the nanopores are produced from multiporous membranes. 64,65,114 A representative image of one such replica showed ideal conical geometry (Figure 2 2). Furthermore, such replicas can be used to a pproximate cone angle. In the case of the PEG modified nanopore used here, the two step, anisotropic and isotropic, etch method produced a conical nanopore having a base opening diameter of 520 + 45 nm and a tip opening diameter of ~48 nm, as determined by the current voltage curve in Figure 2 3. Electroless gold deposition was used to plate a thin gold film along the walls of the nanopore to provide a reactive surface for PEG thiol chemisorption. The tip opening diameter after gold deposition was determine d via a current voltage curve to be ~23 nm (Figure 2 3). Thiol modified PEG (MW 5 kDa) was then attached to the gold coated nanopore and the tip diameter re measured in similar fashion to be ~12 nm (Figure 2 3). 95,117,217 Resistive Pulse Sensing of Strepta vidin A key characteristic of the PEG modified, conical nanopore sensor is that the ionic current flowing through the pore generates an electric field that is highly focused at the tip opening. 95,96 Finite element simulations performed by Lee, et al. show ed that, when a potential difference of +1 V is dropped across the nanopore sensing element, the magnitude of the electric field strength in and around the tip opening is on the order of 10 6 V m 1 96 As a result of this field focusing phenomenon, the ion c urrent flowing through the nanopore tip region becomes very sensitive, thereby creating an analyte sensing zone. 75,89,96 Therefore, the ion current in this sensing zone becomes highly

PAGE 76

76 sensitive to the presence of a target analyte, such as the protein, stre ptavidin. This field focusing feature of conical nanopores makes conical shaped pores more amendable to resistive pulse sensing than cylindrical shaped pores. In the absence of protein, an applied transmembrane potential of +1000 mV resulted in a steady state ion current of ~700 pA (Figure 2 4). Without protein, no transient current pulse events were observed (Figure 2 4). Upon addition of 500 nM streptavidin to the electrolyte solution facing the tip opening of the nanopore, numerous transient current pu lses were observed (Figure 2 5). These current pulses are due to the electrophoretic transport of streptavidin into the sensing zone at the nanopore tip and through the base of the nanopore. Similar results were previously observed using bovine serum album in (BSA) as the target analyte for resistive pulse sensing using a comparable conical nanopore. 95 Such current pulse data have been interpret ed as transient current blocking events that represent the protein molecules entering and translocating the nanopor e sensing element. 95 Furthermore, single nanopores prepared via track etching have produced similar transient current pulse events attributed to the electrophoretic transport of double stranded DNAs 89 and porphyrin molecules 75 through tip opening diameters of 40 nm and 4 nm, respectively. However, in these cases, the tip opening diameters were different than that used here because the analytes had different sizes. Figure 2 6 displays an expanded view of a streptavidin current pulse. The ion current decreas es very sharply at the beginning and then gradually tails upward towards the baseline with increasing data acquisition time. The current represents the ion current interval between the ion current value at the lowest point in this shar p decrease and the ion current value at the steady state. The current pulse duration ( )

PAGE 77

77 represents the time interval between the very sharp decrease in the ion current and the time when the ion current returns to its steady state value. Comparable current pulse peak shapes have been observed previously with other charged analytes, such as bovine serum album (BSA), 95 BSA/anti BSA complexes, 95 poly(styrene sulfonate), 146 and single stranded phage DNA, 89 as they were electrophoretically driven in the directio n of tip to base through conical shaped nanopores. This peak shape is reasonable because it represents the effectiveness in which analytes block the ion current based on analyte location within the nanopore. That is, the analyte has the largest impact on t he ion current while it resides in the tip region. As a result, the analyte is most effective in blocking the ion current in this region. In contrast, the analyte has the smallest impact on the ion the analyte has very little impact on the ion current at the base of the conical nanopore. Another way of looking at this is to consider the ratio (A protein /A pore,x ) of the cross sectional areas of the protein (A protein ) to that of the nanopore (A pore,x ) at any point, x, 239 This ratio is greatest at the pore tip opening when the protein first enters the tip. Thus, at large A protein /A pore,x the protein is most effect ive at blocking the ion current. As the protein translocates the sensing zone towards the base, this ratio approaches zero. As a result, the extent in which the protein can affect the ion current diminishes correspondingly. Effect of Applied Transmembrane Potential on Current Pulse Frequency The effect of voltage on the current pulse frequency was determined using applied transmembrane potentials of 400 mV, 600 mV, 800 mV, and 1000 mV (Figure 2 7). The current pulse frequency (f p ) was obtained by counting the number of current

PAGE 78

78 pulses occurring in 5 min. time intervals and averaging the number of current pulses from 3 such intervals. As shown in Figure 2 8, there is a threshold potential below which streptavidin current pulses were not observed. This has bee n previously observed for other charged analytes using both biological 83 and artificial nanopores. 75,89,95 The current pulse frequency increases exponentially with applied voltage above the threshold potential. This exponential relationship is attributed t o an entropic barrier that streptavidin molecules must overcome to enter a nanopore having a tip opening diameter that is comparable to the size of streptavidin. 75,83,89,95 Therefore, since streptavidin is charged, it lowers its entropy by being driven via electrophoresis into the tip opening of the nanopore. 75,83,89,95 Effect of Pore Surface Chemistry on Current Pulse Duration Current pulse durations shorter than 100 ms are typically observed with molecular resistive pulse sensors. 79,89 For example, curren t pulses less than 20 ms were observed for DNA using the biological nanopore, HL. 79 Conical nanopore sensors in track etched polycarbonate produced current pulses less than 80 ms for large, single stranded phage DNA. 89 This is interesting because the diameter of the DNA in solution (~128 nm) is over 3 times larger than the diamete r of the nanopore tip (~40 nm). 89 However, such current pulse durations for DNA were much smaller than current pulse duration for the streptavidin (average = 1 s) studied here and other proteins studied previously. 95 Such long current pulse durations li kely stem from non specific interactions between the streptavidin and the unmodified gold layer along the pore wall. That is, anionic streptavidin transiently adsorbs to the underlying gold surface via non specific adsorption. Since a large MW PEG thiol wa s used, with respect to the small pore size and

PAGE 79

79 curvature of the pore wall, it is unlikely that a perfectly close packed monolayer of PEG forms. Thus, for each PEG thiol, the PEG chain assumes a random coil atop a single thiol, thereby leaving a portion of the underlying gold surface exposed. Any portions of make the pore wall susceptible to non specific protein adsorption. This phenomenon has been observed previously wi th bovine serum albumin using a comparable nanopore. 95 Thus, pore surface chemistry plays a role in current pulse duration. Effect of Pore Surface Chemistry on Current Pulse Direction Despite the conceptual simplicity of constructing resistive pulse senso rs from track etched conical nanopores, modifying the pore wall via electroless gold deposition and subsequent PEG thiol chemisorption introduce practical complexity in terms of reproducibly creating a functional sensing element having long term stability. Therefore, alternative approaches to doing resistive pulse sensing have been investigated. For instance, a different method for attaching PEG directly to the pore wall using EDC/sulfo NHS coupling chemistry 173,174,175 is introduced in Chapter 5. Here, t he resistive pulse detection of streptavidin using an unmodified, conical nanopore in track etched poly(ethylene terephthalate) was investigated. Such pores that are not gold plated offer several advantages. As will be discussed in more detail later, at hi gher applied transmembrane potentials, the underlying gold layer becomes detached from the pore wall and, after a period time, is completely removed. This is likely due to increased resistive heating that occurs at higher electric field strengths. 64 The us e of higher potentials offers several advantages in resistive pulse sensing. Theoretically, a higher current pulse frequency can be obtained at higher electric field strengths that result from increasing the applied transmembrane potential. 89,95,172 Conseq uently,

PAGE 80

80 improvements in the limit of detection may be achieved at such higher current pulse frequencies. A high electric field strength also has been shown to reduce the current pulse duration. 23,89,172 Such a reduction may improve selectivity. Thus, the resistive pulse sensing of streptavidin in an unmodified PET nanopore, which is more amendable to higher transmembrane potentials, was studied. Using the two step etch method described previously, 63 a single, conical shaped nanopore was fabricated having a base diameter of 520 + 45 nm and tip diameter of ~12 nm. Using an analogous resistive pulse experimental setup as that used for the PEG modified nanopore, streptavidin (500 nM) was electrophoretically driven into the tip opening of the nanopore and detect ed as transient current pulses. However, instead of observing the typical downward current pulses observed for the PEG coated pore, the current pulses went both upward and downward (Figure 2 9). That is, the ion current sharply increased above the baseline for a certain time interval and subsequently decreased sharply below the baseline for a certain time interval before returning to the steady state. This suggests that some additional factor is contributing to the current pulse aside from what occurs with classic Coulter counting. Coulter counting is built on the underlying assumption that an analyte displaces a corresponding volume of electrolyte and, as a result, always results in an increase in ionic resistance (i.e., downward current pulses). However, at the nanoscale, it is believed that surface chemistry plays a larger role in the resistive pulse detection of smaller, nanoscale analytes. Several reports have reported the observation of upward current pulses instead of downward current pulses for the resistive pulse detection of DNA using nanopores. 35 Such upward current pulses reflect a decrease in the ionic resistance of the pore upon

PAGE 81

81 DNA translocation. This was attributed to DNA entering the pore along with its charge balancing counterions. Therefo re, due to these additional charge carriers, there is an increase in the local conductivity of the electrolyte within the pore when the DNA is present. As a result, the ion current increases as observed experimentally by an upward current pulse. The downwa rd current pulse following the upward pulse can be attributed to pore blocking analogous to the Coulter case. This explains why current pulses go downward pulses observed for the unmodified pore h ere were not observed using the previously used PEG modified pore. The results suggest that a transient protein adsorption desorption process is occurring along the pore wall within the sensing zone at the nanopore tip opening. With PEG present, such a pro cess cannot readily occur because non specific adsorption has been reduced by the bulky, high MW PEG. It is reasonable to assume that a transient interaction between the unmodified pore wall and the protein would slow down protein translocation, thereby in creasing the current pulse duration. Hence, this is exactly what was observed experimentally. That is, a larger current pulse duration was observed for the unmodified nanopore compared to that for the PEG modified pore. The same streptavidin concentration electrolyte, and transmembrane potential were used here as used previously for the PEG modified pore. As shown in Figure 2 9, the current pulse frequency for streptavidin in the unmodified pore was lower (12 pulses 5 min 1 ) than the pulse frequency obse rved with the PEG modified pore (23 pulses 5 min 1 ). One plausible explanation is that the threshold potential for the translocation of streptavidin in the unmodified nanopore is greater than the threshold potential of the

PAGE 82

82 PEG modified pore. 172 This differ ence could be attributed to the negative surface charge of the unmodified pore wall relative to the net negative charge of streptavidin. That is, electrostatic repulsion between the carboxylates created along the pore wall as a byproduct of chemical etchin g and the negatively charged streptavidin could impede translocation and increase the entropic barrier to translocation. Modifying the pore wall with gold and PEG thiol reduces some of the surface charge. As a result, there is less electrostatic repulsion and a lower entropy barrier to overcome. Therefore, the current pulse frequency is somewhat higher in the PEG modified pore at the applied transmembrane potential of +1000 mV used here. Furthermore, the presence of the bulky, high MW PEG likely reduces the number of available adsorption sites along the pore wall. With less adsorption sites available, less interactions with the pore wall can occur in PEG coated pores relative to unmodified pores. Interestingly, if we compare the current pulse frequency for b ovine serum album (BSA) reported by Sexton, et al. 95 to that for streptavidin obtained at the same tip opening diameter (~12 nm), applied transmembrane potential (1000 mV), and electrolyte using a PEG modified conical nanopore, we observe 6 + 1 pulses/min. for streptavidin versus ~3 pulses/min. for BSA. However, the concentration of BSA (100 nM) was 5 times smaller than the concentration of streptavidin (500 nM). Current pulse frequency, f p can be described by the equation: 89,95 (Eq. 2.2) where z is the effective surface charge on the analyte, D t is the diffusion coefficient associated with analyte transport through the tip opening, C is analyte concen tration, E is the electric field strength, r tip

PAGE 83

83 and the other variables have their usual meanings. Several factors could be attributed to the higher pulse frequency of streptavidin versus that of BS A. Certainly, the higher concentration of streptavidin contributes to the higher current pulse frequency observed for streptavidin. The cross sectional area of the tip opening relative to size of the protein is also an important factor. In the case of stre ptavidin, the protein is about 5 nm in diameter and the pore is 12 nm. Thus, when streptavidin enters the tip opening, it occupies roughly 50% of the cross sectional area of the tip. In contrast, BSA has a long axis of ~14 nm and a short axis of ~4 nm. 95 T herefore, with a tip opening diameter of 12 nm, BSA transport through the tip becomes hindered because BSA loses a degree of rotational freedom in tips smaller than its long axis 95 Streptavidin does not experience this. Although a quantitative determinati obtained for either BSA or streptavidin, surface charge undoubtedly impacts the current pulse frequency as well (Eq. 2.2) The isoelectric points of BSA and streptavidin are ~4.8 and ~7.0, respectively. 238,239 Thus both proteins have excess negative surface charge in the pH 7.4 sensing buffer. Conclusions In this work, it was shown that single, conical shaped nanopores can be fabricated by chemical etching of ion tracked polymer membranes. Such nanopores were used to detect a model protein, streptavidin, via the resistive pulse method in both PEG modified and unmodified nanopores in poly(ethylene terephthalate). In PEG coated nanopores, the current pulses were predominantly downward. However, removing the PEG and u nderlying gold layers resulted in current pulses going both upward and downward. This difference was attributed to non specific interactions (i.e., adsorption and desorption) between the streptavidin and the pore wall. Furthermore, a combination of pore bl ocking

PAGE 84

84 and enhanced local ionic strength in the sensing zone due to the presence streptavidin also contributed to the phenomenon observed in bare PET. Such local ionic strength enhancement has been previously observed with resistive pulse sensors for DNA i n artificial nanopores. 35 The current pulse frequency was observed to vary exponentially with increasing applied transmembrane potential. This was due to the entropic penalty that the streptavidin must overcome to enter the tip of the nanopore. The curren t pulse frequency of comparable concentrations of streptavidin in the unmodified pore was less than that of the PEG modified pore. This suggests that the threshold potential for obtaining current pulses in the unmodified pore is greater than that for the P EG coated pore. 95,172 One of the distinct advantages of track etched conical pores is that such pores can be coated with gold via electroless gold deposition and subsequently modified with thiols to tailor the pore surface chemistry. As a result, the tran sport properties of the pore can be controlled. In this work, PEG thiol was attached via this approach to reduce non specific adsorption. Another advantage of conical pores is the characteristic conical geometry which causes a field focusing affect within the tip opening that effectively facilitates analyte detection and translocation. A key requirement for obtaining current pulses for streptavidin is having the capability to fabricate a tip opening diameter that is comparable to size of streptavidin. The t wo step fabrication method provides a way to fine tune the tip diameter to the size of the analyte. 63 Using an electrochemical method, the tip opening diameter can be measured after each step of the fabrication and pore modification process. 59,75,89,90,95 This successful detection of streptavidin is a promising step towards expanding the application

PAGE 85

85 of conical nanopores to the resistive pulse detection of streptavidin bioti n complexes (e.g., streptavidin biotin DNA, streptavidin biotin aptamer protein), and additional proteins (e.g., biomarkers).

PAGE 86

86 Figure 2 1 Schematic of the PEG modified co nical nanopore sensing element ( d rawing not to scale ). Figure 2 2 FE SEM image of a template synthesized gold nanocone replica prepared in a conical shaped nanopore in track etched poly(ethylene terephthalate). The replica represents the geometry of the nanopore sensing element after removal from the PET template.

PAGE 87

87 Figure 2 3. Current voltage curves obta ined in 1M KCl used to calculate the diameter of the tip opening after each step of the resistive pulse sensor fabrication process. Blue: track etched PET, tip diameter ~48 nm, Orange: after deposition of gold surface layer, tip diameter ~23 nm, Black: aft er attachment of PEG thiol to gold nanopore walls, tip diameter ~12 nm. Figure 2 4 Typical current time transient for the PEG modified, single conical nanopore sensor at an applied transmembrane potential of 1000 mV. Electrolyte only; no streptavidin

PAGE 88

88 Figure 2 5 Typical current time transient for the PEG modified, single conical nanopore sensor at an applied transmembrane potential of 1000 mV. Electrolyte contained 500 nM streptavidin. Figure 2 6. Expanded view of a typical current pulse r eflecting the tip to base translocation of 500 nM streptavidin through a PEG modified conical nanopore with a tip diameter of 12 nm.

PAGE 89

89 Figure 2 7. Streptavidin current time transient as function of applied transmembrane potential. Tip diameter = 12 nm. [st reptavidin] = 500 nM. PEG coated, conical nanopore. Applied transmembrane potentials: (A) 1000 mV, (B) 800 mV, (C) 600 mV, (D) 400 mV. Figure 2 8. Streptavidin current pulse frequency versus transmembrane potential. Tip diameter = 12 nm. [streptavidin] = 500 nM. Error bars represent standard deviations obtained by averaging the number of current pulses during three 5 minute intervals of current pulse data.

PAGE 90

90 Figure 2 9. Current time transients of 500 nM streptavidin at an applied transmembrane potentia l of 1000 mV using a n (A) unmodified conical nanopore in PET, tip diameter ~12 nm, and (B) PEG modified conical nanopore in PET, tip diameter ~12 nm Figure 2 10. Histograms of streptavidin current pulse amplitude (left) and duration (right). Tip diame ter = 12 nm (with PEG attached). [streptavidin] = 500 nM. Applied transmembrane potential = 1000 mV.

PAGE 91

91 CHAPTER 3 RESISTIVE PULSE SENSING OF A MODEL CATIONIC ANALYTE WITH A CONICAL NANOPORE SENSOR Introduction There is increasing interest in the concept o f using nanopores in artificial 24 31,36 54,75,89 91,95,157,158,234 236 or biological 66 73,78 88,93,94,118,119,121,126,231 233 membranes as resistive pulse sensors for small molecule and biopolymer analytes. The resistive pulse method, 235 which when applied to such target analytes is sometimes called stochastic sensing, 66 70,78,79,93,94,118 120 entails mounting a membrane containing a nanopore between two electrolyte solutions, applying a transmembrane potential difference, and measuring the resulting ion cu rrent flowing through the electrolyte filled nanopore. In simplest terms, when the analyte enters and translocates the nanopore, it transiently blocks the ion current, resulting in a downward current pulse. The frequency of such analyte induced current pul se events is proportional to the concentration of the analyte. Analyte identity, or selectivity, is encoded in the magnitude and duration of current pulses. 50,143,234,235,237 The majority of such molecular resistive pulse sensing work has been done using a biological nanopore, hemolysin ( HL), embedded in a supported lipid bilayer membrane as the sensor element. 66,68 70,78,79,82 88,93,94 Numerous analytes including metal ions, 66 DNA, 78,79,82 88 proteins, 93,94 and various small molecules 68 70 have been detected with the HL n anopore. These studies have shown unequivocally that resistive pulse sensing using a nanopore as the sensor element is a promising sensing paradigm. However, because the supported lipid bilayer membrane that houses the HL nanopore is very fragile, 119,14 3,144 it seems unlikely that any practical, real world, sensing devices will be possible with this bilayer technology. One approach for solving this

PAGE 92

92 problem is to replace the bilayer membrane and biological nanopore with a mechanically robust and chemicall y stable artificial membrane containing an artificial micro or nanopore. Prototype artificial micro and nanopore sensors have been prepared by the track etch method, 55 65,75,89,90,95 by inserting a carbon nanotube into an epoxy/silicon nitride support, 45 50 by electron beam lithography in silicon membranes, 28 by ion beam sculpting in silicon dioxide and nitride membranes, 26,38,43 and by a femtosecond pulsed laser method in a glass membrane. 51 53 There artificial nanopore sensors have been used, with the r esistive pulse method, to detect DNA, 27,32 37,42 polystyrene nanoparticles, 48,96 small molecules, 75 proteins, 38,39,42,95 and virus particles. 51 Furthermore, like the HL based sensor, 78,120,141,142 chemical selectivity can be introduced by biofunctionalization of the arti ficial nanopore sensor element. 104,157 In addition to selectivity, a key question that must be addressed for any proposed new sensing method is wh at detection limits can be achieved with this technology? To date, the lowest detection limits that have been reported with nanopore resistive pulse sensors for ionic, 142 small molecule, 120 protein, 94,95 or DNA analytes 141 are at the nanomolar level, altho ugh quantitative evaluations of the detection limit are often not reported. This work represents a step towards such a description. That is, this research explores the issue of resistive pulse detection limits using a single, conical shaped nanopore, prepa red by the track etch method, in a poly(ethylene terephthalate) (PET) membrane as the sensor element. Results are reported here for an ideal, nano sized analyte, 5 nm diameter poly L lysine coated cationic gold nanoparticles. Particles were detected at sub nanomolar concentrations, which are lower than that reported in any ionic, molecular, or macromolecular resistive pulse sensing experiment.

PAGE 93

93 Experimental Materials Poly(ethylene terephthalate) (PET) membranes (3 cm diameter, 12 m thick), that had been irradiated with a single, swift heavy ion to create a single damage track through the membrane, were obtained from Gesellshaft fur Schwerionenforschung (GSI), Darmstadt, Germany. Poly L lysine conjugated, cationic gold nanoparticle s (5 nm diameter, particle concentration: 5 x 10 13 particles/mL ) were obtained from Ted Pella. All other chemicals were of reagent grade. Solutions were prepared in purified water (Barnstead, E pure) and the buffer used to prepare the analyte solutions was filtered through Durapore (Millipore) filters. Preparation of the Conical Nanopore Sensing Element The single damage track in the PET membrane was converted into a conical shaped nanopore using the two step chemical etching procedure described previously 63 Briefly, the first etch step entails placing the membrane between at 9 M NaOH etch solution and a stop etch solution comprised of 1 M formic acid and 1 M KCl in a U tube cell made of Kel F (3.5 mL half cell volume). This yields a single, conical shaped nanopore with the large diameter (or base) opening facing the etch solution and the small diameter (or tip) opening facing the stop etch solution. To determine when the etchant had broken through to the stop etch solution, and a contiguous pore had been o btained, a platinum wire electrode was placed in each half cell solution and a potential difference of +1 V (using a Keithley 2487 voltage source/picoammeter, Keithley Instruments, Cleveland, OH) applied across the membrane. The electrodes were configured such that the positive electrode (anode) was placed in the half cell containing the etch solution and the negative electrode (cathode) was placed in the half cell containing the stop solution.

PAGE 94

94 Before breakthrough, the transmembrane ion current was zero and breakthrough was signaled by a sudden rise in the ion current. The first etch step yielded a conical nanopore with a base diameter of 520 + 45 nm, as determined by scanning electron microscopy. Wharton, et al. previously validated the first step etch for reproducibly producing base diameters of 520 + 45 nm in single ion irradiated PET membranes. 63 The second etch step is used to adjust and fine tune the size of the tip opening. 63 This step entails placing a more dilute (i.e., 1 M NaOH) etch solution on bo th sides of the membrane. Again, a potential difference of +1 V was applied across the membrane and the transmembrane ion current was monitored during the etching process. Excellent reproducibility in the tip diameter (relative standard deviation less than 10%) can be obtained by stopping the second etch at a prescribed value of the transmembrane ion current. For the research reported here, the second etch was stopped at an ion current value of ~4.6 nA yielding a conical nanopore with a tip diameter of ~10 nm. The tip diameter was determined using an electrochemical method 59,75,89 based on current voltage curves (i.e., ionic pore conductance) as described below. Resistive Pulse Sensing The single, conical nanopore membrane was placed between two halves of a Kel F conductivity cell comparable to that used for pore fabrication (Figure 3 1). Both half cells were filled with a pH 7, 10 mM phosphate buffer solution that also contained 0.1 M KCl. A Ag/AgCl electrode (Bioanalytical Systems/BASi, West Lafayette, IN) was placed into each half cell solution, and an Axopatch 200B (Molecular Devices Corp., Union City, CA) was used to apply the desired transmembrane potential and measure the resulting ion current flowing through the electrolyte filled nanopore. The electr ode polarity was configured such that the cathode (negative electrode) was in the solution

PAGE 95

95 facing the base opening and the anode (positive electrode) was in the solution facing the tip opening. The Axopatch amplifier was used in voltage clamp mode with a l owpass Bessel filter (2 kHz bandwidth). Data were obtained using a Digidata 1322x analog to digital converter (10 kHz sampling frequency) and pClamp 9.0 software (both from Molecular Devices Corp.) The 5 nm diameter, poly L lysine conjugated gold nanopart icles (a model cationic analyte) were diluted in the pH 7 buffered electrolyte described above. Because the pH is below the pKa of the lysine amine groups (pKa = 10.0) 240 the analyte nanoparticles have excess cationic surface charge. The analyte solution was placed in the half cell facing the tip opening, and the nanoparticles were driven by electrophoresis through the nanopore from the tip side to the base side of the conical pore containing membrane. Typically, in resistive pulse sensing, translocation of the analyte through the nanopore sensor element results in a downward (decreasing below the baseline) current pulse; i.e., the analyte transiently blocks the ion current flowing through the nanopore. 75,89,90,95 However, there have been recent reports of upward (increasing above the baseline) current pulses associated with translocation of highly charged analytes through the nanopore sensor element. 35 Upward current pulses are observed because the highly charged analyte brings its charge balancing counter ions with it as it translocates the pore (Figure 3 2). This results in a transient increase in the ionic strength, and thus the ionic conductivity, of the solution within the nanopore. Upward current pulses were observed for the cationic nanoparticles inve stigated here (Figure 3 3). The current pulses in three 5 minute recordings were analyzed at each concentration of analyte used. This

PAGE 96

96 analysis yielded the average current (f p ). Results and Discussion Nanop ore Characterization Knowing the tip diameter of the conical nanopore is critical to developing functional resistive pulse sensors. As Sexton, et al. demonstrated, the current pulse frequency depends on tip diameter within a certain range. 95 In order to d etermine the tip opening diameter, the base opening diameter must be known. Wharton, et al. showed that the two step etch method, used in this work, reproducibly produces base diameters of 520 + 45 nm. 63 Knowing this value for the base diameter, the tip di ameter can be obtained via calculation based on an electrochemical determination of the ionic conductance of the electrolyte filled nanopore. 59,75,89,90,95 Since previous studies have shown that pores etched with the two step etch method produce truly coni cal shaped pores, 63,95 the ionic conductance of the pore (G) is related to the base diameter (d base ), tip diameter (d tip ), specific conductivity of the electrolyte ( 10.5 11.5 S/m for 1 M KCl), and the length of the pore (L, membrane thickness) via the following equation: 56,75,89,90,95 (Eq. 3. 1) The value for G was determined by obtaining the linear current voltage curve for the electrolyte filled nanopore. This was done by mounting the pore containing membrane between both halve cells of a U tube cell comparable to that used for pore fabricat ion and filling each half cell with electrolyte (1 M KCl). A Ag/AgCl electrode was placed into each half cell and the applied transmembrane potential was linearly scanned from 1 V to

PAGE 97

97 +1 V while measuring the ion current flowing through the conical pore at each voltage step (Figure 3 4). The slope of the resulting current linear between 1 V and +1 V, then the slope obtained from a smaller voltage range (e.g., 200 mV to +200 mV) is used. 95 Why Conical Shaped Nanopores In a conical shaped nanopore, the voltage drop caused by the ion current flowing through the electrolyte filled pore is focused to the electrolyte solution in the tip opening. 95,96 Indeed, the electric field strength in the solution within the nanopore ti p can be greater than 10 6 V m 1 when the total voltage drop across the nanopore membrane is only 1 V. 96 A consequence of this field focusing effect is that the nanopore ion current is extremely sensitive to analyte species in the nanopore tip. That is, th 75,89,95,96 This property makes conical shaped nanopores more ideally suited for resistive pulse sensing applications than cylindrical shaped pores which lack this field fo cusing effect. This has been demonstrated with prototype conical nanopore sensors for analyte species ranging in size from small molecules, 75 to DNA, 89,90 proteins, 95 and nanoparticles. 96 Proposed Definition for the Detection Limit in Resistive Pulse Sens ing Because the cationic analyte nanoparticles are driven by electrophoresis through the nanopore, the flux (J) of analyte through the pore is given by: 89 (Eq. 3.2) where z is the effective charge on the particle, D is the diffusion coefficient associated with particle transport through the tip opening, C is the particle concentration, E is the

PAGE 98

98 electric field strength, and the o ther terms have their usual meanings. Multiplying both sides of Eq. 3.2 by the cross sectional area of the nanotube tip (A t ) converts the flux into (A g ) converts this t o the number of molecules translocating the nanopore tip per second, which is equivalent to the frequency of the analyte induced current pulses, fp by: 89 (Eq. 3.3) Equation 3.3 suggests that f p should be linearly related to the analyte concentration. This has been verified in studies of DNA analytes, as well as other studies. 89 The linear dependence of f p on concentration means that the de tection limit for the resistive pulse method must be defined in terms of how long the analyst is willing to wait to detect a current pulse due to the analyte. At low analyte concentrations, the frequency becomes prohibitively low, or put another way, the a verage time interval between pulses becomes prohibitively long. To define the detection limit concentration, C dl an agreement must be reached on how long the analyst is willing to wait to see a current pulse due to the analyte. It is proposed here that t he detection limit be defined as that concentration that yields, on average, a current pulse every 60 sec. This definition was chosen because analysts will undoubtedly want to record and analyze at least 5 to 10 analyte current pulses. Hence, with this def inition, the total analysis time per sample would be 5 to 10 minutes. If the analysts can tolerate lower sample throughput than this, then a more liberal definition of the detection limit (e.g., a pulse every 2 minutes) could be employed.

PAGE 99

99 What Order of Mag nitude Detection Limit Can Be Anticipated? With the definition proposed here, C dl is that analyte concentration that gives a current pulse frequency of 0.017 Hz; we call this the detection limit frequency, f dl and rearranging Eq. 3.3 yields: (Eq. 3.4) If we assume that E is 10 4 V cm 1 that D for our 5 nm cationic nanoparticles is 9.7 x 10 7 cm 2 s 1 (calculated from the Stokes Einstein on the particle is z = 100, the theoretical C dl for our particles using a conical nanopore having a tip diameter of 10 nm is 1 pM. However, while we use the same applied transmembrane potential of +1 V as used in the determination of the electric field strength via finite element simulations, E is undoubtedly higher in our nanopore than the 10 4 V cm 1 calculated by these simulations because our tip diameter is smaller. Finite element simulations by Sexton, et al. showed that the electric field strength increases with decreasing tip opening diameter and constant base diameter. 95 Furthermore, we do not yet have an accurate value for z for our nanoparticles. Hence, this C dl of 1 pM should be regarded only as a rough a pproximation. Analysis of the Current Pulse Data With an applied transmembrane potential of +1 V, the conical nanopore sensor yielded, in the absence of analyte, a steady state ion current of ~1050 pA (Figure 3 5). Addition of the analyte nanoparticles re sulted in upward current pulses associated with translocation of the particles through the nanopore tip (Figure 3 6), and as expected, current pulse frequency, f p increases with analyte concentration (Figure 3 7). Although the current pulse frequency was very low, current pulses were observed at 100 fM

PAGE 100

100 concentration level (Figure 3 8). However, over the concentration range studied in Figure 3 7), f p does not increase linearly with concentration, as predicted by Equation 3.3. Instead, over this concentratio n range, f p is related to C via the empirical relationship: y = 80.23664 exp( x/0.10971) + 84.03351 (Eq. 3.5) which was determined via curve fit using Origin Software, version 8.1 SR2 (OriginLa b, Massachusetts ). Despite the lack of data points between 100 pM and 10 nM concentration levels, Eq. 3.5 and Fig. 3 7 suggest that a saturation phenomenon is occurring at higher concentrations for the cationic nanoparticle analyte. This was not the case f or the DNA analyte investigated previously, where the predicted linear relationship (Eq. 3.3) was observed; however, a much smaller concentration range, 5 to 25 nM, was investigated. 89 The nature of this saturation phenomenon is the subject of on going res earch. Similar saturation of ion conductance is known to occur in glycine receptor and sarcroplasmic reticulum channels. 17 That is, with these channels, a comparable saturation phenomenon occurs when the binding unbinding steps associated with ion permeati on become rate limiting. Such behavior has been described empirically using Michaelis Menton curves 17 By analogy, it is possible that the cationic nanoparticles undergo electrostatic binding unbinding with the anionic pore wall. Thus, at some very high na noparticle concentrations, it is possible that the rate of particle entry into the pore exceeds the maximum rates of unbinding with the pore wall. Figure 3 9 shows a scatter plot of the current ), for the current pulses obtained at the 10 nM nanoparticle concentration (Fig. 3 6). Figure 3 10 show histograms representing the current pulse magnitude and duration at different particle concentrations. When the scatter plot is

PAGE 101

101 compared to analogous scatter plots obtained by us and others for DNA analytes, 89 we find that the spread in was unexpectedly higher for the nanoparticles. For example, for a single varied over a factor of 25(5 ms to 75 ms) and r a factor of 3.7 (300 pA to 1100 pA). 89 analyte varied by only a factor of 0.5 (190 pA to 260 pA) (Fig. 3 varied over a wide range from 60 ms to 4360 ms which is much worse than that observed for DNA. 9) undoubtedly results because the nanoparticle is a hard sphere, and thus does change conformation as it approaches and enters the nanopore tip. This is not the case for the DN A analyte, which had a radius of gyration larger than the radius of the nanopore tip. 89 Hence, the Au nanoparticle analyte is, in this regard, a better model system for fundamental investigations of nanopore resistive pulse sensing such as those reported h ere. A related advantage is that such nanoparticles can be obtained commercially over a large size range and with a variety of different surface charges and chemistries. 241,242 for the cationic nanoparticles relative to that for DNA likely results from electrostatic interactions between the negatively charged pore wall and the positively charged nanoparticles. The resistive pulse sensing of the DNA was conducted using a high ionic strength electrolyte (1 M KCl) 89 whereas the nanoparticles were detected using a much lower ionic strength electrolyte (0.1 M KCl and 0.010 M phosphate). As a result, in the case of the pore used to detect DNA, the negative surface charges of the pore wall were screened by the high salt concentration. 164 In

PAGE 102

102 contrast, for the nanopore used to detect the nanoparticles, the charges along the pore adily with the particles having opposite surface charge. Therefore, an electrostatic binding and release for the cationic nanoparticle analyte. To get the best of both worlds (i.e., narrow distri surface passivation technique is required for future studies. Furthermore, three aspects of the conical nanopore system used here may augment the electrophoretic flux of the cationic nanoparticles. First, single, conical sha ped nanopores in track etched PET membranes having small tip diameters (i.e., < 15 nm) are known to rectify the ionic current flowing through the electrolyte filled nanopore in low ionic strength electrolyte (e.g., 0.01 M 0.1 M KCl) when the electrolyte pH is above the isoelectric point (pI) of the membrane surface (pI ~3 for PET). 102 As a result, cations, such as the cationic particles, are preferentially transp orted and anions are rejected. Secondly, based on the model of ion current rectification prop osed by Cervera, et al. 166 when the electrodes are configured such that anode is a placed at the tip opening and the cathode is placed at the base opening, migrating anions are driven from the base opening towards the tip opening. Since the pore surface h as fixed anionic surface charge, 62 anions cannot effectively pass through the tip due to electrostatic repulsion with the pore wall. Consequently, the local concentration of anions just inside the tip increases. 166 In order to maintain electroneutrality, t he local concentration of charge balancing cations increases at the tip as well. Thus, the local ionic strength of the electrolyte in the tip region increases and a higher ionic conductance state is observed. 166

PAGE 103

103 of the conical nanopore. A comparable electrode configuration was used to detect the cationic nanoparticles. Thus, it is reasonable to expect some degree of augmentation of the electrophoretic flux of the particles due to this higher ion conductance state. Lastly, Jin, et al. showed that electro o smotic flow occurs with conical shaped nanopores in both the tip to base (i.e., anode at the tip) and base to tip (i.e., anode at base) directions. 146,156 Although the impact of these 3 phenomena on electrophoretic flux were not investigated here, based on previous studies, it is believed that they contribute to an enhanced current pulse frequency in the case where the analyte and electrode polarity at the tip side of the membrane are both opposite in polarity to the pore wall (as was the case for the cationic nanoparticles). Conclusions The model cationic, nanoparticle analyte studied here was detected at sub nanomolar concentration levels which are lower than the lowest concentrations previously reported for small molecule 120 and macromolecule 94,95,141 analytes obtained via resistive pulse sensing. A simple definition for the detection limit in resistive pulse sensing was proposed and a simple model for calculating what detection limits should be possible via this s ensing paradigm was presented. A narrow distribution in current pulse amplitude was obtained which was attributed to the use of a hard sphere nanoparticle analyte of narrow size distribution that does not have the conformational flexibility akin to many biological analytes. However, a broad distribution in current pulse duration was observed. This was likely due to electrostatic binding and release between the cationic nanoparticle analyte and the anionic pore surface during translocation. It is believed that modifying the pore wall in a way that prevents such electrostatic interactions will undoubtedly improve the

PAGE 104

104 distribution in current pulse duration, thereby making metallic nanoparticles the ideal analyte system for fundamental studies on resistive pul se sensing. Furthermore, a saturation in the electrophoretic flux was observed at a high particle concentration. This suggests that at high concentrations, the flux becomes rate limiting. This phenomenon remains the subject of on going research. It is bel ieved that ion current re c tification and electro o smotic flow may contribute to varying extents to an enhanced current pulse frequency in the case where both (1) the analyte and (2) electrode polarity at the tip side of the membrane are opposite in polarity to the pore wall (as was the case for the cationic nanoparticles). This represents a departure from previous resistive pulse studies using conical nanopores, like those with BSA 95 and streptavidin (Chapter 2) where the pore wall, analyte, and electrode p olarity at the tip were all of the same polarity (i.e., negative for studies on BSA and streptavidin).

PAGE 105

105 Figure 3 1. General conductivity cell setup used for resistive pulse experiments Figure 3 2. Schematic of the p rocess by which th e local ionic strength within the tip region is increased as the cationic gold nanoparticle introduces its charge balancing counterions.

PAGE 106

106 Figure 3 3. Upward current pulses reflecting the translocation of cationic gold nanoparticles through the tip opening of a conical nanopore. Expanded view of a typical current pulse. Tip diameter: ~10 nm; Base diameter: 520 + 45 nm. Figure 3 4. Current voltage curve determination of tip opening diameter. Slope of the linear current voltage curve is equivalent to the ionic conductance (G) of the pore which, with a known base diameter of 520 + 45 nm, was used to calculate the tip diameter.

PAGE 107

107 Figure 3 5. Steady state ion current using a single, conical shaped nanopore in track etched poly(ethylene terephthalate). No particles present. Electrolyte: pH 7, 10 mM phosphate buffer that was also 0.1 M KCl. Applied transmembrane potential: +1 V. Figure 3 6. Resistive pulse sensing of 5 nm diameter cationic gold nanoparticles using a single, conical shaped nanopore in track etched poly(ethylene terephthalate). Particle concentration: 10 nM. Electrolyte: pH 7, 10 mM phosphate buffer that was also 0.1 M KCl. Applied transmembrane potential: +1 V.

PAGE 108

108 Figure 3 7. Plot of current pulse frequency versus particle c oncentration taken at 5 minute intervals. Smaller plot (inset) is an expansion of the lower concentration data points contained in the larger plot. Curve fit performed using Origin version 8.1 SR2.

PAGE 109

109 Figure 3 8. Resistive pulse sensing of 5 nm diameter cationic gold nanoparticles using a single, c onical shaped nanopore in track etched poly(ethylene terephthalate). Particle concentration: 100 fM. Electrolyte: pH 7 10 mM phosphate buffer that was also 0.1 M KCl. Applied transmembrane potential: +1 V.

PAGE 110

110 Fi gure 3 ). Particle concentration: 10 nM. Applied transmembrane potential: +1 V. Figure 3 10. Histograms of current pulse magnitude (left) and duration (right) data for cationic nanoparticles. Particle concentration: 10 nM (red) 100 pM (green), 40 pM (blue).

PAGE 111

111 CHAPTER 4 RESISTIVE PULSE SENSING OF NAN OPARTICLES USING A C ONICAL SHAPED NANOPORE IN T RACK ETCHED POLYIMIDE Introduction The utilization of sensing para digms derived from nanopores has become increasingly popular in recent years. Such nanopore based sensing devices generally employ a biological 66 70,78,79,82 88,93,94,118 120,124 126,231 233 or artificial 23 30,35 54,75,89 91,95,157,158,234 236 nanopore emb edded in a membrane which is immobilized between two halves of a U tube cell. As a transmembrane potential difference is applied, an ionic current passes through the electrolyte filled nanopore which drives the electrophoretic translocation and correspondi ng detection of analyte molecules as they transiently block and traverse the pore sensing zone. The magnitude and duration of such transient blocks in the ion current provide identity information of the analyte molecules. 50,143,234,235,237 This approach is often referred to as resistive pulse sensing; however, the technique has been called stochastic sensing as well. The most commonly used biological nanopore, hemolysin, 66 70,78,79,82 88,93,94,118,119,121,126 is a bacterial protein which self assembles into lipid bilayer membranes. Both native and genetically engineered forms of hemolysin have been used to detect DNA, 78,79,82 88 polymers, 124 small molecules, 68,70 proteins, 93 94 and metal ions 66 via the resistive pulse method. Resistive pulse sensing ba sed on hemolysin provides an excellent demonstration of the nanopore based sensing concept; however, replacement of the fragile lipid bilayer membrane 119,143,144 with an artificial pore construct is required to develop more mechanically robust and chemic ally stable devices.

PAGE 112

112 A wide variety of approaches have been developed to fabricate such artificial nanopores for sensing applications including focused ion beam sculpting of silicon oxide and nitride, 26,38,43 embedded carbon nanotubes, 49 50 electron beam lithography and chemical etching of silicon membranes, 28,54 femtosecond pulsed laser drilling of glass, 51 53 and track etching of polymeric membranes. 74,75,89,90,95,96 Such artificial pores have been used to detect DNA, 26,31 36,41 polystyrene nanoparticle s, 48,96 small molecules, 75 proteins, 38,39,42,95 and viruses. 51 Track etched conical nanopores in polymeric membranes are of particular interest because of the control and reproducibility of pore size, tailorable pore surface chemistry, 63 and cost effective ness (i.e., in terms of fabrication and materials). Conical nanopores have been fabricated in a variety of ion tracked polymers including poly(ethylene terephthalate), 59,62,63,90,91,95 polycarbonate, 64,89,96,97 and polyimide. 60,62,74,75,158 Polyimide is particularly attractive because of its better transport properties (i.e., less noisy and more stable baseline) compared to poly(ethylene terep h thalate ). 99 To date, only two analytes, anionic porphyrins 75 and double stranded DNA, 243 have been sensed via the resistive pulse method using single, conical shaped nanopores in track etched polyimide. Both of these analytes were detected under high salt conditions (i.e., 1 M KCl). 74,243 This work presents an example of resistive pulse sensing of a prototype analyte 20 nm diameter fluorescent nanoparticles, using a conical nanopore in track etched poly i mide under lower salt conditions (i.e., 0.1 M KCl). A 2 step etching process and pore loading procedure based on perfusion are also presented.

PAGE 113

113 Experimental Materia ls Polyimide membranes (Kapton 50 HN, DuPont, 3 cm diameter, 12 m thick) which contained a single, heavy ion induced damage track, were obtained from Gesellschaft fuer Schwerionenforschung (GSI), Darmstadt, Germany. The carboxylated, fluorescent nanoparticles (carboxy modified Fluospheres, cat. #F8787, particle diam eter = 20, concentration = 2.63 x 10 15 particles/mL) were obtained from Invitrogen (Eugene, OR). Sodium hypochlorite (13% active chloride ion, Sigma) and all other chemicals (certified A.C.S. grade, Fisher Scientific) were used as received. Solutions were prepared using purified (18 M house distilled water though a Barnstead E pure water purification system). Fabrication of the Conical Nanopore A conical shaped nanopore was etched into the single ion tracked polyimide membrane via anisotropic chemical e tching of the heavy ion induced damage track. 60,62,75,99 This process entails mounting the irradiated membrane between two halves of a U tube cell. An etch solution (sodium hypochlorite) was added to one half cell and a neutralizing, or stop, solution (2 M KI) was added to the other half cell. 60,62,75,99 Chemical etching was performed at 50 o C using a temperature feedback controlled hotplate. To monitor etching progress and detect membrane breakthrough, a platinum wire electrode was placed into each half ce ll solution and a potential difference of 1 V was applied during etching using a Keithley 6487 voltage source/picoammeter (Keithley Instruments, Cleveland, OH). The electrodes were configured such that the positive electrode (anode) was placed in the etch solution and the negative electrode (cathode) placed in the stop solution.

PAGE 114

114 During this etching process, the latent damage track was preferentially etched from the membrane face in contact with the etch solution towards the membrane face in contact with t he stop solution. 60,62,75,99 Before breakthrough, the transmembrane ion current was initially zero, but increased exponentially after the etch solution breaks through the membrane into the stop solution ( vide supra ). The etching process was stopped upon r eaching an ion current of 100 pA. The etch time required to reach this current value varied greatly but typically took between 2 4 hours. The membrane was then rinsed briefly with stop solution and subsequently with purified water. This procedure produces a single, conical shape d nanopore having a larger diameter, or base, opening on the etch solution side of the membrane and a small diameter, or tip, opening on the stop solution side of the membrane. The base diameter (in this case, 1255 nm) was determined via calculation based on the bulk etch rate for polyimide as experimentally measured by field emission scanning electron microscopy (FE SEM). FE SEM (JEOL JSM 6335F) was used to measure pore diameter of multi ion tracked polyimide membranes that were chemically etched for diffe rent times (1, 2, and 3 hours). A previously described electrochemical method was used to determine the tip diameter. 59,75,89,90,95 As will be discussed below, a second, or isotropic, etch step was developed for polyimide to tailor the tip opening to the d esired diameter. After this step, the final tip opening diameter was 21 nm. Current Pulse Measurements The single, conical nanopore membrane was mounted into a U tube cell comparable to the cell used for pore fabrication. Both half cells were filled with 3.5 mL of electrolyte solution comprised of 10 mM phosphate buffer solution (pH = 7.4) and 100

PAGE 115

115 mM KCl. A Ag/AgCl electrode (Bioanalytical Systems/BASi, West Lafayette, IN) was immersed into each half cell solution. The electrodes were connected to an Axopatch 200B (Molecular Devices Corp., Union City, CA) patch clamp amp lifier. The Axopatch served as the voltage source/picoammeter to apply a constant transmembrane potential difference and measure the resulting ion current flowing through the electrolyte filled nanopore. The ion current was recorded in voltage clamp mode o n the Axopatch with a low pass Bessel filter at 2 kHz bandwidth. A Digidata 1233A analogue to digital converter (Molecular Devices Corp.) was utilized to digitize the signal at a sampling rate of 10 kHz. Data were recorded and analyzed using pClamp 9.0 sof tware (Molecular Devices Corp.). The Ag/AgCl electrodes were configured such that the Ag/AgCl anode was placed in the electrolyte solution facing the base opening and the Ag/AgCl cathode in the solution facing the tip opening. The carboxylated nanopartic les have a negative effective surface charge in the pH 7.4 sensing buffer. 244 Thus, the nanoparticles were added to the electrolyte solution facing the nanopore tip and subsequently driven via electrophoresis in the direction of tip to base through the con ical nanopore. Results and Discussion Determination of the Bulk Etch Rate To determine the bulk etch rate of polyimide, 3 ion tracked polyimide membranes (ion track density = 10 6 ions/cm 2 ) were chemically etched for etch times of 1, 2, and 3 hours. Each m embrane was then analyzed via FE SEM and the pore size measured. The 1 hour membrane had a mean pore diameter of 478 + 16 nm (Figure 4 1A). The 2 hour and 3 hour membranes had mean pore diameters of 981 + 29 nm (Figure 4 1B) and 1426 + 59 nm (Figure 4 1C), respectively. The bulk etch rate was determined to be 0.48 + 0.02

PAGE 116

116 m/hour which is slightly faster that the published value of 0.42 + 0.04 m/hour. This value was then used to calculate the base diameter of the conical nanopore used for resistive pulse sensing. Two Step Etching Method for Ion Tracked Polyimide In resist ive pulse sensing, a tip opening diameter comparable to the size of the target analyte is required. 75,90,95 To achieve this, control and reproducibility of the tip opening diameter are a critical requisite of the pore fabrication process. Wharton, et al. i ntroduced a two step etching method for ion tracked poly(ethylene terephthalate) (PET) which provides a process for controlling and reproducing tip diameters of 50 nm or less. 63 In step one, PET is anisotropically etched (with 9 M NaOH) from one membrane f ace for a fixed, predetermined amount of time (i.e., 2 hours) to achieve a certain base diameter. In step two, PET is isotropically etched from both membrane faces using a dilute etch solution (i.e., 1 M NaOH, or 11% of the step one etchant concentration) Using a similar approach, a two step etching approach for fabricating conical nanopores in ion tracked polyimide was developed. In step one, a heavy ion irradiated polyimide membrane is etched to a predetermined value of the ion current around 100 pA. U sing a fixed etch time for the first step (as used for PET) is problematic for polyimide for two reasons. First, for polyimide, when the etch solution breaks through to the stop solution, there is an exponential increase in the transmembrane ion current al ong a short time scale (Figure 4 2A). Thus, the tip opening diameter changes dramatically during a brief time interval. In contrast, the ion current detected upon breakthrough in PET increases at a much lower rate (Figure 4 2B). Secondly, the breakthrough time varies greatly between polyimide

PAGE 117

117 membranes (Figure 4 2A). With PET, the breakthrough time is more consistent. As a result, in step one, polyimide is etched to an ion current value of 100 pA. In step two, a slower bulk etch rate is desired. 63 Since the bulk etch rate is largely governed by etchant concentration, 99 the optimal etch solution concentration had to be determined. To accomplish this, 4 ion tracked polyimide membranes were etched via step one to an ion current value of 100 pA. Each membrane was subsequently etched isotropically using 1 M KCl spiked with different concentrations (i.e., 1%, 3%, 6%, and 10%) of the step one etchant at ambient temperature using an applied trans membrane potential of 1V. Figure 4 3A shows the impact of etch time on the transmembrane ion current at each etch solution concentration. At the 1% level, very little change in the ion current occurred. However, at the 10% level, the ion current increased very dramatically (65 nA in 1 hour). By comparison, the transmembrane ion current during a typical second etch step for PET using 11% of the first etch step etchant concentration is illustrated in Figure 4 3B. With PET, the ion current at this level increases 25 nA in 1 hour. Thus, two intermediate etch solution concentrations at 3% and 6% were used to etch polyimide and slower etc h rates of 5 nA and 20 nA in 1 hour were observed, respectively (Figure 4 3A). Nanopore Characterization In order to determine the tip opening diameter, the base opening diameter must be known. The base diameter for conical nanopores is determined primari ly from the bulk etch rate and FE SEM. 63,75,89,95 For the conical nanopore used in the sensing work described here, the base diameter was determined, using the bulk etch rate for polyimide, to be 1255 nm. With this value, the tip diameter can be calculated via experimental

PAGE 118

118 determination of the ionic conductance (G) of the electrolyte filled conical nanopore via the following equation: 59,63,75,89,90,95 (Eq. 4.1) where d base represents the base diameter, d tip 11.5 S/m), and L is the pore length (or membrane thickness). The value of G was determined experimentally by a linear scan of the applied transmembrane potential from 1 V to +1 V and measuring the resulting ionic current flowing through the pore at each potential step. As a result, a current voltage curve is obtained in which the slope is G. Controlling the Tip Diameter in Ion Tracked Polyimide via Isotropic Etching Single, ion tracked polyimide was etched using the first step etch described above and stopped less than 100 pA. A current voltage curve was obtained using 1 M KCl (pH 6);however, no ion current was observed (blue trace, Figure 4 4). This was likely due to stopping the first etch step short of complete breakthrough occurring. The membrane was etched isotropically using the 3% etchant solution in 1 M KCl until a transmembrane ion current value of 6 nA was achieved. The tip opening diameter was calculated via current voltage curve to be 13 nm (red trace, Figure 4 4). The conical nanopore was subsequently etched to final ion current values of 9 nA, 16 nA, and 19 nA with corresponding tip diameters of 15 nm (grey trace, Figure 4 4), 25 nm (black trace, Figure 4 4), and 28 nm (green trace, Figure 4 4), respectively. Figure 4 5 shows the relationship between final nanopore ion current and tip opening diameter. This demonstrates the great promise of applying the two step etch method to controlling the tip opening di ameter of track etched, conical nanopores in polyimide.

PAGE 119

119 Interestingly, the conical nanopore exhibited ion current rectification at all tip diameters studied using 1 M KCl (Figure 4 4). Ion current rectification occurs when there is asymmetric flow of cati ons and anions through the electrolyte filled nanopore. 100,101,102,103 It is observed experimentally as a non linear current voltage curve. In general, this phenomenon occurs when the pore radius is comparable to the thickness of the electrical double laye r. 102 To increase the extent of ion current rectification, either the pore radius must be decrease (e.g., by decreasing the tip diameter 102 ) and/or the thickness of the electrical double layer must be increased (e.g., by lowering the ionic strength of the electrolyte 164 ). In this case, the electrical double layer thickness is negligible because of the high ionic strength of the 1 M KCl electrolyte. 164 The extent of ion current rectification is measured by determining the rectification ratio (R. R.) using th e following equation: 104 (Eq. 4.2) where i E= 1 represents the value of the transmembrane ion current at an applied potential of 1 V, and i E=+1 is the value of the ion current at +1 V. In Figure 4 6, the rectification ratios in 1 M KCl were plotted as a function of tip opening diameter. The largest rectification ratio was found to be 4 for the smallest tip diameter (13 nm) and d ecreased linearly with increasing tip diameter through a tip diameter of 28 nm (Figure 4 6). Ion current rectification under high ionic strength electrolyte (i.e., 1 M KCl) conditions is due to the increased negative surface charge density along the pore s urface. Jin, et al. observed a large degree of ion current rectification in 1 M KCl using conical nanopores in mica membranes. 156 Harrell, et al. tailored the pore surface of conical nanopores in track etched polycarbonate with DNA chains of varying length s attached to the pore wall.

PAGE 120

120 The degree of ion current rectification was observed to increase with increasing DNA chain length (i.e., increasing negative surface charge). Resistive Pulse Sensing of Carboxylated, Fluorescent Nanoparticles An important prop erty of the conical nanopore sensing element described here is that the voltage drop attributed to the ion current flowing through the electrolyte filled nanopore is focused in and around the nanopore tip opening. 95,96 Finite element simulations performed by Lee, et al. indicate that the focused electric field strength located in the region just inside the tip is on the order of 10 6 V/m when a transmembrane potential of +1 V is applied. 96 As a result, a very sensitive detection zone forms at the tip opening The ion current flowing through this detection zone is very sensitive to the presence of any molecular species present in or near this region. 75,89,95,96 This is one advantage of using conical shaped nanopores for resistive pulse sensing applications ins tead of cylindrical shaped nanopores. A conical nanopore was fabricated in polyimide using the 2 step method described previously ( vide supra ). The base opening diameter was 1255 nm and the tip opening diameter was determined via a current voltage curve t o be 28 nm (blue trace, Figure 4 7). No current pulses were initially observed using this conical nanopore. It was believed that this was due to the poor wetting behavior of polyimide. Therefore, a wetting agent, methanol, was used to first wet the nanopor e for 10 minutes. The methanol was removed and quickly replaced with sensing buffer that was pH 7.4 10 mM phosphate buffer and 100 mM KCl. The U tube cell was placed briefly into a vacuum chamber and a vacuum applied for 10 minutes. The sensing buffer was alternately removed and replaced with new buffer on each side of the cell several times via perfusion while keeping the membrane wet. After this process, a second current voltage curve was

PAGE 121

121 obtained and the tip diameter determined to be 41 nm (red trace, Fi gure 4 7). The difference between this tip value and the previous tip value of 28 nm is believed to be due to a more complete filling of the pore with electrolyte after wetting. In other words, the a surface conduction mechanism along the negatively charged pore wall 62 dominated the previously measured ion current. At an applied transmembrane potential of +200 mV, in the absence of analyte, a steady state ion current of 2500 pA that was free of current pulses was observed (Figure 4 8A). Upon addition of 100 nM carboxylated, fluorescent nanoparticles (20 nm diameter) to the solution on the tip side of the membrane, transient current pulse events were observed (Figure 4 8B). Current pulses have bee n observed for proteins 95 and DNA 89,90 using track etched, conical nanopores in PET. Similarly, current pulses have been observed for small molecules 75 and DNA 243 using conical pores in polyimide. Such current pulse data have been attributed to the transie nt blockage of the transmembrane ion current as the analyte translocates the detection zone of the nanopore. 75,89,90,95,243 Figure 4 9B shows an expanded view of typical current pulses for the fluorescent nanoparticles. The ion current increases sharply at the start and is sustained for a certain pulse duration, and then decreases sharply back to the baseline. That is, the current pulses are upward (i.e., increasing above the steady state baseline) and square shaped. Upward current pulses have been observ ed previously with DNA 35 and cationic, protein coated nanoparticles (Chapter 3) and have been attributed to an increase in the local ionic strength of the electrolyte in the tip opening upon nanoparticle translocation. That is, the negatively charged, carb oxylated nanoparticle carries its charge balancing counterions

PAGE 122

122 with it into the tip opening of the nanopore. These additional charge carriers result in a transient increase in the local electrolyte concentration in the tip region upon nanoparticle entry an d translocation. As a result, ion current enhancement is observed as an upward current pulse. Another plausible explanation is local charge inversion. 245 This phenomenon occurs when a charged analyte transiently increases or decreases the magnitude of th e surface charge along the pore wall within the tip opening. In the case of the fluorescent nanoparticles used here, the conical nanopore in track etched polyimide rectifies the ion current in 1 M KCl. Therefore, the negative surface charge of the pore wal l must be very large. 146 Although a quantitative determination for the surface charge of the pore wall is not available, it is possible that local charge inversion occurs upon the transient adsorption/desorption of a less negative particle onto the pore wa ll within the tip opening. 245 Interestingly, the only two reports of resistive pulse sensing using polyimide nanopores used a high ionic strength electrolyte (pH 7 8, 1 M KCl). 75,243 In these cases, analyte (i.e., porphyrin 75 and double stranded DNA 243 ) translocation was observed as transient current pulses that were downward, as opposed to the upward pulses observed here for nanoparticles in 0.1 M KCl (pH 7.4). This suggests that the current pulse direction is very sensitive to electrolyte concentration and ionic strength along the pore wall. In Figure 4 8A, the steady state background ion current was 2500 pA using an applied transmembrane potential of +200 mV. In contrast to a conical nanopore in track etched PET, the transmembrane ion current is two times larger than that observed with

PAGE 123

123 comparable PET pores for which a higher potential of 1 V and iden tical supporting electrolyte were used. One cause for this difference is the lower pore resistance in the polyimide nanopore. The ionic resistance of the nanopore (R pore ) is related to the tip opening diameter (d tip ), and base diameter (d base ) via the foll owing equation: 59,63,64,75,89,90,95 ( Eq. 4.3 ) where L, and G are as previously described. Eq. 4.3 shows that R pore is inversely proportional to the product of the tip and base opening diameters. Thus, for example, for a PET nanopore having a d base of 520 nm, d tip of 17 nm, and L of 12 m, the ionic resis tance of the pore is higher than that for the polyimide nanopore used in this work (d base = 1255 nm, d tip = 41 nm, L = 12 m). Consequently, higher ion currents can be achieved at lower R pore and larger cone angles (Chapter 6) Another benefit of using con ical nanopores with large cone angles is that lower potentials can be used to drive electrophoresis. Figure 4 10 shows that the current pulses observed for the nanoparticles are due to electrophoretic transport. At an applied potential of +300 mV, when th e electrode polarity was reversed for 1 hour, no current pulses attributed to nanoparticle translocation were observed. This is because with the polarity reversal, the anionic nanoparticles are driven electrophoretically away from the nanopore. Interesting ly, some large spikes in the ion current occurred while the electrode polarity was reversed. While the origin of these spikes is unknown, they appear to occur much more frequently upon electrode polarity reversal and less frequently using the normal electr ode polarity (i.e., anode at the base opening and cathode at the tip opening) and in the presence of anionic nanoparticles. The

PAGE 124

124 difference in the steady state ion current between electrode polarities (i.e., reversed vs. normal) was due to ion current recti fication. As previously discussed, ion current rectification was observed by measuring current voltage curves under high ionic strength conditions (1 M KCl) (Figure 4 4). In the case of resistive pulse sensing, the ionic strength of the sensing buffer is m uch lower (pH 7.4 10 mM phosphate and 100 mM KCl). Therefore, ion current rectification undoubtedly occurs because the electrical double layer thickness increases as the ionic strength of the supporting electrolyte decreases from 1 M KCl to 0.1 M KCl. Upon returning the electrodes to the normal polarity, an induction time was observed. That is, a period of time prior to the start of current pulses occurred and was less than 4 min. The change in current pulse signature as a function of applied transmembran e potential was determined at potentials of 50 mV, 100 mV, 200 mV, 300 mV, 400 mV, and 500 mV. In Figures 4 11A to 4 15A (see Figure 4 8A for E = 200 mV), the steady state background ion current at each value of applied potential is shown. At each potentia l, the background ion current was free of current pulses attributed to nanoparticle translocation. However, in a few cases (i.e., at E = +50 mV and E = +100 mV), a few of the current spikes comparable to those observed during the electrode polarity reversa l were observed. These could be simply due to someone walking near the instrument during data acquisition since the Axopatch 200B is highly sensitive to the local environment. Steady state background ion currents of approximately 700 pA, 1325 pA, 2500 pA, 3600 pA, 4600 pA, and 5500 pA were observed at applied transmembrane potentials of 50 mV, 100 mV, 200 mV, 300 mV, 400 mV, and 500 mV, respectively (Figure 4 8A, 4 11A to 4 15A).

PAGE 125

125 Upon addition of 100 nM anionic nanoparticles to the solution side facing th e tip opening of the pore, upward current pulses were observed at each applied potential (Figures 4 8B, 4 11B to 4 15B). The current pulse signature is comprised of the current pulse amplitude and duration. The current pulse amplitude ( ion current above or below the steady state baseline current. The current pulse duration ( ) represents the time interval between the beginning of each current pulse and when the ion current returns to the baseline. Table 4 1 s hows the current pulse amplitude and duration values at applied transmembrane potentials of 50 mV, 100 mV, 200 mV, 300 mV, 400 mV, and 500 mV. At the lower potentials of 50 mV, 100 mV, and 200 mV, the current pulse duration decreased from 418 + 245 ms (a t E = 50 mV), to 210 + 71 ms (at E = 100 mV), and 164 + 54 ms (at E = 200 mV). Figure 4 16 shows histograms of current pulse duration at these potentials. This decrease was attributed to the increase in the electric field with increasing applied transmembr ane potential. This has been observed previously in resistive pulse sensing studies on DNA. 23 The current pulse duration ( ) is inversely proportional to the electric field strength via the following equation: 89,95 ( Eq. 4.4 ) where l D temperature, z is the effective surface charge on the nanoparticle, E is the e lectric field strength, and D is the diffusion coefficient associated with nanoparticle transport through the tip. Interestingly, the percent relative standard deviation in the current pulse amplitude decreased from 59% at 50 mV to 34% and 33% at 100 mV an d 200 mV, respectively.

PAGE 126

126 The current pulse amplitude increased from 73 + 2 pA (at E = 50 mV), to 113 + 5 pA (at E = 100 mV), and 173 + 4 pA (at E = 200 mV). Figure 4 17 shows histograms of the current pulse amplitude at 50 mV, 100 mV, and 200 mV. This in crease was due to the increase in the ion current at these increasing applied transmembrane potentials. In other words, the volumetric fraction of electrolyte displaced by the presence of a nanoparticle in the tip region is the same at each potential (i.e. nanoparticle and tip size are constant). However, since the ion current increases at these applied potentials, the translocation of each nanoparticle is reflected by a larger current pulse amplitude. Furthermore, the relative standard deviation in curren t pulse amplitude ranged from 2 4 % at these 3 potentials. This is undoubtedly expected because the current pulse amplitude reflects the size of the analyte. In the case of the anionic nanoparticles studied here, the particles generally do not adopt multip le conformations akin to biological analytes (e.g., some proteins 244 ). Consequently, this resulted in a highly consistent (i.e., low percent relative standard deviation) current pulse amplitude. Furthermore, as the applied transmembrane potential was incre ased further to 300 mV, 400 mV, and 500 mV, the current pulse amplitude continued to increase to 232 + 5 pA, 289 + 5 pA, and 332 + 6 pA, respectively. Figure 4 18 shows the relationship between current pulse amplitude and applied transmembrane potential. T he plot shows the relationship observed at 50 mV, 100 mV, and 200 mV was extended to 300 mV, 400 mV, and 500 mV. A scatter plot of current pulse amplitude versus current pulse duration at potentials of 50 mV (green), 100 mV (red), and 200 mV (blue) is sho wn in Figure 4 19. From this plot, the occurrence of current pulses having longer pulse durations decreases with increasing transmembrane potential. Additionally, this scatter plot shows the

PAGE 127

127 narrow distribution of current pulse amplitude which reflects th e use of a nanoparticle analyte. The current pulse frequency, which relates to analyte concentration, describes the rate of electrophoretic transport of the analyte through the conical nanopore. This is related to the electrophoretic flux (J, in mol s 1 cm 2 ) via the following equation: 89,95 ( Eq. 4.5 ) tant, D t is the diffusion coefficient associated with diffusive transport through the tip opening, C is the nanoparticle concentration, E is the electric field strength focused at the tip opening, R is the universal gas constant, and T is temperature. By m ultiplying both sides of Eq. 4.5 sectional area of the tip opening ( r tip 2 ), Eq. 4.5 becomes the following equation for current pulse frequency (f p ): 89,95 ( Eq. 4.6 ) From Eq. 4.6, the current pulse frequency is directly proportional to t he electric field strength focused at the tip opening of the conical nanopore. As the applied transmembrane potential increased, the current pulse frequency for the anionic, fluorescent nanoparticles increased from 53 events/min at 50 mV, to 72 events/min and 85 events/min at 100 mV and 200 mV, respectively (Table 4 1). However, as the potential was increased further to 300 mV, 400 mV, and 500 mV, the current pulse frequency decreased and pulse amplitude increased. This presents a perplexing issue that warr ants further investigation before any definitive conclusions can be made. One possibility is that the applied electrophoresis current pins the nanoparticles on the

PAGE 128

128 polyimide surface on the tip side of the membrane. By increasing the electrophoresis current at higher potentials, the electrophoretic force acting on these nanoparticles increases as well. Therefore, the entry of these nanoparticles into the tip opening of the conical nanopore is impeded. Lee, et al. observed the pinning of nanoparticles to the membrane surface. 96 However, their particles were larger than the tip opening diameter. Another plausible explanation is that at higher potentials, de wetting of the nanopore occurs and the seal between half cells is either lost or reduced. In fact, the ba ckground ion current in the presence of nanoparticles at the highest potential studied, 500 mV (Figure 4 15), decreased with increasing data acquisition time. Conclusions A single, conical shaped nanopore was fabricated in polyimide using a two step etch ing method that was developed to tailor the tip opening diameter. Despite variability in the track etch rate that leads to variability in the membrane breakthrough times, this two step process is an important step towards reproducibly fabricating conical n anopores of desired dimensions in ion tracked polyimide. This approach was used to fabricate a conical nanopore which was used for the resistive pulse sensing of 20 nm diameter carboxylated, fluorescent nanoparticles. Unlike the two previous reports of res istive pulse sensing using conical nanopores in polyimide with a high ionic strength sensing buffer, 75,243 a lower ionic strength sensing buffer was used for sensing nanoparticles. At first, no current pulse events were observed. However, after a wetting a gent was utilized to facilitate more efficient filling of the nanopore with sensing buffer via perfusion, upward current pulse events were observed. These upward events were attributed to either (1) a local enhancement of the ionic strength at the tip open ing due to the transient

PAGE 129

129 introduction of additional charge carriers 35 or (2) local charge inversion 245 induced by the translocating anionic nanoparticles. Compared to the small cone angle typically observed in PET, the cone angle inherent to nanopores fab ricated in track etched polyimide is larger due to a faster bulk etch rate and slower track etch rate. 59,60,62,99 As a consequence, lower applied transmembrane potentials were used to electrophoretically drive nanoparticles through the nanopore tip opening Current pulses were observed at applied potentials of 50 mV, 100 mV, 200 mV, 300 mV, 400 mV, and 500 mV. In agreement with theory, 89,95 the mean current pulse duration decreased and pulse frequency increased as the potential was increased from 50 mV to 1 00 mV and 200 mV. The current pulse amplitude increased in near linear fashion with increasing potential over the entire range of applied transmembrane potentials studied. At the higher values (i.e. 300 mV, 400 mV, and 500 mV) of applied potential, the cu rrent pulse duration worsened (i.e., increased both in magnitude and distribution) and the pulse frequency decreased. Such perplexing results could be attributed to de wetting of the pore or pinning of nanoparticles to the membrane face on the tip side of pore due to increased electrophoretic force present at increasing transmembrane potentials. However, further studies are needed to investigate this phenomenon further. Moreover, this report represents the first resistive pulse sensor using a conical sha ped nanopore in track etched polyimide for (1) the detection of an analyte other than double stranded DNA 243 and porphyrins 75 and (2) detection under lower ionic strength sensing conditions which is desirable for macromolecules (e.g., proteins, disease

PAGE 130

130 bio markers). Thus, this work is a progressive step forward in the development of track etched conical nanopores in polyimide for use in resistive pulse sensing devices.

PAGE 131

131 Figure 4 1. Electron micrographs of multi ple nanopores etched into multiple ion tracked polyimide membranes at different etch times. Etch times were (A) 1 hour, (B) 2 hours, and (C) 3 hours. Mean pore diameters were 478 + 16 nm (A), 981 + 29 nm (B), and 1426 + 59 nm. A B C

PAGE 132

132 Figure 4 2. Ion current time recordings for monitoring breakthrough in (A) polyimide and (B) PET membranes during the first step etch. For polyimide (A), 3 different membranes were etched as reflected by the different colored traces. Figure 4 3. Ion current time recordings for tailoring the tip opening diameter during the second etch step in (A) polyimide and (B) PET membranes. For polyimide (A), 4 different membranes were second step etched using different concentrations of the first step etchant, 10% (blue trace), 6% (gre en trace), 3% (light blue trace), and 1% (red trace) in 1 M KCl For PET (B), 11% of the first step etchant concentration was used in the second step etch.

PAGE 133

133 Figure 4 4. Current voltage curves obtained in 1 M KCl used to calculate the diameter of the tip opening after each round of second step etching of polyimide Blue: first step etch stopped short of breakthrough (no ion current flow). Red: second step etch to 6 nA (d tip 13 nm). Grey: second step etch to 9 nA (d tip 15 nm). Black: second step etch to 16 nA (d tip 25 nm). Green: second step etch to 19 nA (d tip 28 nm). Figure 4 5. Plot of tip opening diameter versus final nanopore ion current during the second isotropic, etch step for single track etched polyimide.

PAGE 134

134 Figure 4 6. Plot of rectifi cation ratio versus tip opening diameter in 1 M KCl (pH 6) after the second step etch of a conical pore in polyimide. Figure 4 7. Current voltage curves obtained in 1 M KCl (pH 6) after the second step etch (blue, d tip 28 nm) and after treatment with a wetting agent and vacuum (red, d tip 41 nm).

PAGE 135

135 Figure 4 8. Ion c urrent time transients (A) in the absence of nanoparticles and (B) with 100 nM carboxylated, fluorescent nanoparticles. Applied transmembrane potential = 200 mV.

PAGE 136

136 Fi gure 4 9. Ion c urrent time transients of (A) 100 nM carboxylated, fluorescent nanoparticles and (B) expanded view of (A). Applied transmembrane potential = 200 mV.

PAGE 137

137 Figure 4 10. Ion c urrent time transients of 100 nM carboxylated, fluorescent nanoparticl es with (A) a reversed transmembrane potential of 300 mV for 60 min. and (B) transmembrane potential of +300 mV.

PAGE 138

138 Figure 4 11. Ion c urrent time transients (A) in the absence of nanoparticles and (B) with 100 nM carboxylated, fluorescent nanoparticles Applied transmembrane potential = 50 mV.

PAGE 139

139 Figure 4 12. Ion c urrent time transients (A) in the absence of nanoparticles and (B) with 100 nM carboxylated, fluorescent nanoparticles. Applied transmembrane potential = 100 mV.

PAGE 140

140 Figu re 4 13. Ion c urrent t ime transients (A) in the absence of nanoparticles and (B) with 100 nM carboxylated, fluorescent nanoparticles. Applied transmembrane potential = 300 mV.

PAGE 141

141 Figure 4 14. Ion current time transients (A) in the absence of nanoparticles and (B) with 100 nM c arboxylated, fluorescent nanoparticles. Applied transmembrane potential = 400 mV.

PAGE 142

142 Figure 4 15. Ion current time transients (A) in the absence of nanoparticles and (B) with 100 nM carboxylated, fluorescent nanoparticles. Applied transmembrane potential = 500 mV.

PAGE 143

143 Figure 4 16. Histograms of current pulse duration for 100 nM carboxylated, fluorescent nanoparticles at applied transmembrane potentials of (A) 200 mV, (B) 100 mV, and (C) 50 mV.

PAGE 144

144 Figure 4 17. Histograms of current pulse amplitude for 100 nM carboxylated, fluorescent nanoparticles at applied transmembrane potentials of (A) 200 mV, (B) 100 mV, and (C) 50 mV.

PAGE 145

145 Figure 4 18. Plot of current pulse amplitude versus applied transmembrane potential for 100 nM carboxylated, fluorescent nanop articles. Figure 4 19. Scatter plot of current current for 100 nM carboxylated, fluorescent nanoparticles at applied transmembrane potentials of (A) 50 mV (green), (B) 100 m V (red), and (C) 200 mV (blue).

PAGE 146

146 Table 4 1. Tabulated data for current current pulse frequency (f p ) of 100 nM carboxylated, fluorescent nanoparticles as a function of applied transmembrane potential (E).

PAGE 147

147 CHAPTER 5 DIRECT COUPLING OF AMINE MO DIFIED POLY(ETHYLENE GLYCOL) TO PORE SURFACES OF CON ICAL NANOPORES FOR P REVENTING NON SPECIFIC PROTEIN ADS ORPTION Introduction In recent years, there has been increasing interest is utilizing nanoscale pores as resistive pulse sensor s to develop new analytical tools for a wide variety of target analytes. Such nanopores have been developed in materials comprised of both biological 66 70,78,79,82 88,93,94,118 120,124 126,231 233 and artificial 23 30,35 54,75,89 91,95,157,158,234 236 build ing blocks. Biological nanopores, such as hemolysin, have been utilized to detect DNA, 78,79,82 88 small molecules, 68,70 and metal ions. 66 Abiotic, or artificial, nanopores have been used to detect DNA, 26,31 36,41,89,90,243 proteins, 38,39,42,95 small mole cules, 75 viruses, 51 and particles. 48,96 Despite the excellent reproducibility in pore diameter provided by biological pores, biological pores suffer from several limitations. 119,143,144 For example, the most widely used biological pore, hemolysin, self assembles and inserts itself into lipid bilayer membranes which are very fragile. 119,143,144 Such membranes cannot endure the increased transmembrane potentials required to provide a sufficient level of sensitivity without rupture. Additionally, the limit ing pore diameter (i.e., 2 nm) of hemolysin restricts its analytical utility to ions, small molecules, and threading nucleic acids (i.e., single stranded DNA) via the resistive pulse method. 66,68,70,78,79,82 88 Such drawbacks can be circumvented by using a pore construct that is more che mically stable and mechanically robust over a much wider range of applied potentials and that provides a means of tailoring the pore diameter, thereby expanding application to a broader size range of target analytes. Hence, artificial materials, such as si licon nitride and

PAGE 148

148 oxide, 26,38,43 carbon nanotubes, 49 50 silicon, 28,54 glass, 51 53 and polymeric membranes 74,75,89,90,95,96 have been used to fabricate nanopores for developing resistive pulse devices. Track etched polymeric membranes are of particular in terest due to the relative ease and cost effectiveness of fabrication and controllable pore size. 63 Furthermore, the pore walls of such nanopores can be modified at will to control the surface properties of the nanopore as well as introduce selectivity. 90, 95,109,117,171,172,197,200,210 218,228,229 One method of modifying the pore wall involves the electroless deposition of a thin gold film 163 along the pore wall and subsequent chemisorption of thiols onto the gold surface. 95,109,117,200,210 218 For instance Siwy, et al. coated a single, conical shaped nanopore in poly(ethylene terephthalate) with gold, followed by gold surface modification with thiol modified biotin. 157 This provided a mechanism for the selective recognition of the protein, streptavidin. In a similar fashion, Harrell, et al. modified a gold coated conical pore in polycarbonate with thiol modified DNA for studies on ion current rectification. 104 Sexton, et al. 95 and Yu, et al. 117,217 coated single and multi nanopore membranes with gold, foll owed by thiol modified poly(ethylene glycol), respectively, in order to prevent non specific protein adsorption. A similar approach was used in Chapter 2 for constructing resistive pulse sensors for streptavidin. In recent years, there has been increasing interest in directly coupling molecules to the pore wall of track etched nanopores, thereby obviating the need for electroless gold deposition. Such direct coupling approaches take advantage of the functional groups produced as a result of track etching. 6 2 That is, the chemical etching of the latent damage track produces a pore surface populated with functional groups. Thus, simple coupling

PAGE 149

149 methods can be utilized to conjugate molecules directly to these functional groups on the pore surface. For example, free carboxylate groups are produced on the pore surface as a by product of etching on both poly(ethylene terephthalate) and polyimide nanopores. 62 A coupling reaction, such as that using EDC/sulfo NHS, 173,174,175 can be used to couple amines to these carb oxylate groups on the pore wall. For instance, Kececi, et al. modified conical pores in track etched poly(ethylene terephthalate) with ethanolamine via EDC/sulfo NHS to reduce to negative surface charge of the pore surface. 90 This strategy facilitated more effective detection of small DNAs. Using comparable coupling chemistry, Vlassiouk, et al. coupled ethylenediamine to nanopores in poly(ethylene terephthalate) to switch the polarity of the pore wall from negative to positive charge, thereby creating a nan ofluidic diode. 228 Coating surfaces with poly(ethylene glycol) to prevent undesirable surface adsorption is a widely studied phenomenon. 117,202 205,217,219 221 However, no systematic study focused on non specific protein adsorption in single, conical shap e nanopores has been conducted. To advance studies for using nanopores in biosensing applications, minimizing, or eliminating, the non specific adsorption of biomolecules is absolutely critical. The primary reason for this, in resistive pulse sensing, is b ecause the current pulse frequency is proportional to the cross sectional area of the tip opening. 89,95 It is believed that any reduction in the electrophoretic flux at the tip opening due to non specific interactions between the translocating analyte mole cules and the pore wall will undoubtedly result in a decreased current pulse frequency. Furthermore, non specific interactions between proteins and the pore wall have been implicated in increased current pulse duration and the occurrence of ion current rec tification. 95 It is believed that

PAGE 150

150 both phenomena adversely impact the analytical utility of nanoscale resistive pulse sensing. In this work, amine modified poly(ethylene glycol) was directly coupled to single, conical shaped nanopores in PET. The non speci fic adsorption of three model proteins, bovine serum album, fibrinogen, and lysozyme, was studied via ionic pore conductance (i.e., current voltage curves) and X ray photoelectron spectroscopy. Experimental Materials Poly(ethylene terephthalate) (PET) mem branes (3 cm diameter, 12 m thick) that were irradiated with a swift heavy ion to produce a single damage track through the membrane were obtained from Gesellschaft fuer Schwerionenforschung (GSI, Darmstadt, Germany). Amine modified poly(ethylene glycol) (PEG amine), MW 550 Da (PEG 550), was obtained from Laysan Bio ( Huntsville AL). The 1 ethyl 3 [3 dimethylaminopropyl]carboiimide hydrochloride (EDC), N hydroxysulfosuccinimide (sulfo NHS), and 2 ( N morpholino)ethanesulfonic acid buffered saline (MES) were obtained from Pierce (IL). Bovine serum albumin (BSA), lysozyme, and fibrinogen were obtained from Sigma (MO). All other chemicals were reagent grade or better and used as distilled water through a Barnstead E pure water purification system) was used to prepare all solutions. Fabrication of Conical Nanopore s Single, conical shaped nanopores were etched into single ion tracked PET membranes by anisotropic and subsequent isotropic chemical etching, also referred to as the two step etch method. 63 This process entails mounting the irradiated PET membrane betwee n two halves of a U tube cell. An etch solution (9 M NaOH) was placed in one half cell and a stop, or neutralizing, solution (1 M formic acid with 1 M KCl) was placed

PAGE 151

151 in the other half cell. To monitor the etching process, a platinum wire electrode was pla ced into both half cells with the anode in the etch solution and cathode in the stop solution. A transmembrane potential difference of 1 V was applied, and the resulting ion current measured using a Keithley 6487 voltage source/picoammeter (Keithley Instru ments, Cleveland, OH). Initially, the ion current was zero, and upon breakthrough, the ion current suddenly increased. Breakthrough generally occurred after 60 90 minutes from the start of etching. As etching proceeds, the latent ion induced damage track i s preferentially etched in anisotropic fashion from the PET surface in contact with the etch solution to the PET surface contacting the stop solution. This anisotropic etch step was continued for 2 hours. Previous studies have shown that this process produ ces a base opening diameter of ~520 nm in PET. 63 The pore was then etched in isotropic fashion by placing 1 M NaOH on each side of the single pore PET membrane. 63 A transmembrane potential difference of 1 V was again applied by placing the platinum wire a node in the etch solution located at the base opening and the cathode at the tip opening of the pore. This second etch step was monitored by measuring the transmembrane ion current flowing through the etchant filled conical nanopore. As reported by Wharton et al., this provides a means of monitoring the increasing tip diameter in real time because the tip opening diameter correlates to the value of the ion current. 63 Typically, this fabrication step was stopped at an ion current value of ~25 nA which provi ded a tip opening diameter of ~40 50 nm as determined electrochemically via current voltage curves. The conical nanopore was then rinsed with and stored in water.

PAGE 152

152 Ionic Pore Conductance Measurements Two approaches were utilized in tandem to study non s pecific adsorption of three model proteins, fibrinogen, BSA, and lysozyme, on the pore surface and impact on tip diameter. For each protein, 3 single, conical shaped nanopores in track etched PET were fabricated having a tip opening diameter of ~45 55 nm. As will be discussed below, an electrochemical method was used to measure the tip size in 1 M KCl. 59,63,75,89,90,95 The conical pores were then exposed overnight to a solution containing 100 nM protein in pH 7.4 10 mM phosphate that contained 100 mM KCl. T he protein solution was then completely discarded and the pore thoroughly rinsed with water. The tip diameter was re measured after protein exposure using the same electrochemical method based on ionic pore conductance. All pore conductance measurements we re obtained with the nanopore containing membrane mounted in a U tube cell. After measuring the tip diameter, the membrane area around the pore was analyzed via X ray photoelectron spectroscopy ( vide infra ). To confirm PEG amine attachment and evaluate t he behavior of PEG amine modified nanopores towards protein adsorption, single, conical shaped nanopores were fabricated in track etched PET having a tip opening diameter of ~45 55 nm. PEG amine was coupled via amide bond formation between the amine group on the PEG chains and the free carboxylates on the pore surface via well established EDC/sulfo NHS coupling chemistry. 173 The tip diameter was measured via current voltage curves before and after PEG amine modification. XPS was used to confirm PEG amine at tachment. In experiments analogous to the protein exposure experiments describe above, the PEG amine coated nanopores were exposed overnight to a solution containing 100 nM protein in pH 7.4 10 mM phosphate that contained 100 mM KCl. The protein solution

PAGE 153

153 was then completely discarded and the pore thoroughly rinsed with water. The tip diameter was then re measured to evaluate the impact of the PEG layer on protein adsorption. As a control experiment, 3 comparable PET pores were fabricated and treated in si milar fashion as the protein exposed pores but were not exposed to protein. The tip opening diameters for these nanopores were monitored for a period of several days. X Ray Photoelectron Spectroscopy A PHI 5100 XPS (Perkin Elmer, Waltham, MA) was used to investigate non specific protein adsorption on single, conical shaped nanopores in track etched PET. More specifically, XPS was used for two reasons. First, XPS was used to verify attachment of the PEG amine to the PET surface by comparing the N 1s signal of unmodified PET to that for PEG amine modified PET. Secondly, XPS was utilized to verify that any change observed in the tip opening diameter (i.e., in unmodified PET) was due to protein adsorption by again comparing the N 1s signal for nanopores exposed to protein to that for pores not exposed to protein. EDC/Sulfo NHS Coupling of Amine Modified Poly(ethylene glycol) to PET The track etch process of fabricating conical nanopores in PET membranes yields free carboxylate groups along the pore walls and me mbrane surfaces. 62 A commonly used conjugation technique, using EDC chemistry, 173 was used to cross link amine modified PEGs to these free carboxylates via formation of amide bonds. This entailed first equilibrating the single pore contai ning membrane in 2 0 mL of pH 5.5 0.1 MES buffer for 1 hour. To this solution, 10 mL of both 10 mM sulfo NHS and 4 mM EDC in pH 5.5 0.1 MES buffer were added. The membrane was immersed into this activating solution for 1 hour Then, the membrane was removed, briefly immers ed into pH 7.4 10

PAGE 154

154 mM phosphate buffer for rinsing, and subsequently immersed in fresh pH 7.4 10 mM phosphate buffer which contained 100 mM PEG amine The coupling reaction was allowed to proceed for 2 hours at room temperature with gentle stirring. Then, th e membrane was removed, rinsed with phosphate buffer, and stored in purified water overnight. Results and Discussion Nanopore Characterization Conical shaped nanopores have both a large diameter, or base, opening and a small diameter, or tip, opening. The base opening diameter for these studies was ~520 nm based on the two step etch method for reproducibly etching conical pores in PET reported by Wharton, et al. 63 Knowing the base diameter allows the tip opening diameter to be determined using a electroch emical method based on the ionic conductance of the electrolyte filled nanopore. 59,63,75,89,90,95 For conical pores, the tip diameter (d tip ) is related to the base diameter (d base ), pore length (L, membrane thickness), conductivity of the 0.5 11.5 S/m for 1 M KCl), and ionic conductance of the pore (G) via the following equation: 59,63,75,89,90,95 (Eq. 5.1) Th e value for G was experimentally determined via current voltage curves obtained by scanning the applied transmembrane potential from 1 V to + 1 V. That is, the pore containing membrane was mounted into a U tube cell, and 1 M KCl was placed into both half cells along with Ag/AgCl electrodes (Bioanalytical Systems/BASi, West Lafayette, IN). A Keithley 6487 voltage source/picoammeter (Keithley Instruments, Cleveland, OH)

PAGE 155

155 was used to obtain the current voltage curves. This pore characterization procedure was u tilized throughout this study on non specific protein adsorption. Tip Diameter Stability of Unmodified Conical Pores in PET As a control experiment, the stability of the tip opening diameter was evaluated by monitoring the tip size for 3 track etched sin gle pores in PET for a period of 4 days. That is, 3 single ion tracked PET membranes were etched to tip diameters of ~38 41 nm. All 3 pore containing membranes were left mounted in comparable U tube cells for the duration of the stability evaluation. Curre nt voltage curves were obtained for each membrane for 4 consecutive days in 1 M KCl to measure the tip diameter. Figure 5 1 to 5 3 show composite current voltage curves for all 4 days for each membrane. Table 5 1 summarizes the tip size data and shows that the tip diameter for each pore changed very little (i.e., the greatest change was 3 % r.s.d.) over the 4 day time interval. Thus, single pores were deemed stable for the duration of these experiments. XPS of Unmodified PET Membranes As a control experime nt for XPS studies, 6 PET membranes were analyzed via XPS. Figures 5 4 to 5 9 shows XPS surveys for each membrane. The unmodified PET membranes show strong signals for carbon (C 1s) and oxygen (O 1s). This is to be expected since the atomic percentages of carbon and oxygen in the PET monomer, C 10 O 4 H 8 are 71.4% and 28.6%, respectively (XPS does not detect hydrogen). Table 5 2 summarizes the XPS data for all 6 PET membranes. Excellent reproducibility in the XPS data was observed amongst the 6 membranes. The percent relative standard deviations for the C 1s and O 1s signals were 1.1% and 2.8%, respectively. One PET membrane exhibited weak signals for Si 2s and Si 2p while all other membranes showed none. These peaks were attributed to some sample contamination during handling. Auger peaks

PAGE 156

156 for oxygen and carbon were observed at 720 eV and 990 eV, respectively. Furthermore, the XPS revealed the absence of any detectable nitrogen (N 1s) signal in all 6 membranes. This is to be expected since unmodified PET does no t contain any nitrogen. No sodium (Na 1s) signal was observed for any of the 6 PET membranes because the membranes were not chemically etched. Kececi, et al. observed a Na 1s XPS signal for PET as a consequence of etching with NaOH. 90 That is, the positi vely charged sodium from NaOH binds to the negatively charged carboxylate groups generated along the pore wall during track etching. Aside from this, the C 1s and O 1s data obtained for all 6 membranes were consistent with the XPS results obtained by Kecec i, et al. for unmodified PET. 90 Impact of Non Specific Protein Adsorption on Tip Diameter in Unmodified PET Three model proteins, BSA, fibrinogen, and lysozyme, were used to evaluate the impact of non specific protein adsorption on the tip opening diamete r of single, conical shaped nanopores in track etched PET. These 3 proteins were selected in an effort to expose a variety of different protein interactions to the nanopore surface. BSA is a 66 kDa protein (isoelectric point (pI) ~4.8) from the albumin fam ily that is widely used in many biochemical applications (e.g., ELISA). 239 Fibrinogen is a 340 kDa protein (pI ~6.0) involved in blood clotting 223,244 and was chosen for its propensity to stick to surfaces in a manner akin to sticky serum proteins. Lysozym e is a 14 kDa enzyme (pI ~12.0) that damages the cell wall of bacteria and was chosen as a model cationic protein. 223,244 Single, conical shaped nanopores were track etched in PET using the two step method 63 to tip opening diameters of ~38 45 nm as measur ed via current voltage curves in 1 M KCl. Each pore was then thoroughly rinsed with water. Then, the pore was

PAGE 157

157 immersed overnight in 100 nM protein in pH 7.4, 10 mM phosphate buffer that also contained 100 mM KCl. The pore was then removed from protein solu tion, rinsed thoroughly with water, and the tip diameter re measured in 1 M KCl. Any change in tip diameter was attributed to non specific adsorption of the protein to the pore surface. Presence of the protein on the PET surface was verified via measuring the N 1s signal using XPS. Figures 5 10 to 5 12 show the results for BSA. Before BSA exposure, the tip opening diameters were 40 nm, 41 nm, and 39 nm for pore A (Figure 5 10), pore B (Figure 5 11), and pore C (Figure 5 12), respectively. After BSA exposu re, the tip diameters decreased to 27 nm (pore A), 29 nm (pore B), and 29 nm (pore C). After measuring the tip size, the single pore containing membranes were rinsed thoroughly with water, allowed to dry, and analyzed via XPS ( vide infra). The results for fibrinogen are shown in Figures 5 13 to 5 15. Before fibrinogen exposure, the tip opening diameters were 38 nm, 38 nm, and 39 nm for pore A (Figure 5 13) pore B (Figure 5 14), and pore C (Figure 5 15), respectively. After fibrinogen exposure, the tip diam eters decreased to 26 nm (pore A), 20 nm (pore B), and 23 nm (pore C). Again, after measuring the tip diameter, the pore containing PET membranes were rinsed with water, allowed to dry, and analyzed via XPS ( vide infra ). Figures 5 16 to 5 18 shows the res ults for lysozyme. Prior to lysozyme exposure, the tip diameters were 45 nm, 41 nm, and 42 nm for pore A (Figure 5 16), pore B (Figure 5 17), and pore C (Figure 5 18), respectively. Following lysozyme exposure, the tip opening diameters decreased to 36 nm (pore A), 34 nm (pore B), and 34 nm (pore C).

PAGE 158

158 After measuring the tip diameter, the membranes were rinsed with water, allowed to dry, and analyzed via XPS ( vide infra). Analysis of Non Specific Protein Adsorption to PET Membranes via XPS After measuring t he pore tip diameter before and after exposure to proteins, each membrane was analyzed via XPS. More specifically, the membrane face around the tip opening was analyzed for nitrogen (N 1s). Since nitrogen was not present in any of the unmodified PET membra nes, any protein present on the PET surface would be detected by a positive nitrogen signal. This nitrogen is largely due to the nitrogen containing peptide backbone of proteins. Figures 5 19 to 5 21 show the XPS results for the 3 pore containing PET membr anes exposed to BSA. The N 1s signals were 9.1%, 10.0%, and 8.6% for pore A (Figure 5 19), pore B (Figure 5 20), and pore C (Figure 5 21), respectively. Figures 5 22 to 5 24 show the XPS results for the 3 pore containing membranes exposed to fibrinogen. Th e N 1s signals were 14.6%, 13.3%, and 15.2% for pore A (Figure 5 22), pore B (Figure 5 23), and pore C (Figure 5 24), respectively. Some sodium (from etching), silicon (contaminant), and chlorine signal were observed for pores A and B but not pore C. Last ly, Figures 5 25 to 5 27 exhibit the XPS data obtained for the membranes exposed to lysozyme. The N 1s signal was 10.8%, 11.3%, and 9.6% for pore A (Figure 5 25), pore B (Figure 5 26), and pore C (Figure 5 27), respectively. Thus, each protein adsorbed to the PET surface. Table 5 3 summarizes these findings. XPS of PEG Amine Modified Single Pore Containing PET XPS was utilized to verify the attachment of PEG amine to the single, conical shaped nanopores in PET. For the non specific adsorption studies, a s mall PEG amine (MW 550 Da, PEG 550) was used. Conical pores comparable to those used above were

PAGE 159

159 fabricated in 2 PET membranes via the two step etch method. 63 Using the previously discussed EDC/sulfo NHS coupling method, PEG 550 was attached to the pore sur face and membrane faces. After PEG 550 attachment, the membrane was rinsed thoroughly with water, allowed to dry, and analyzed via XPS. Figures 5 28 and 5 29 show that after PEG 550 attachment, the nitrogen (N 1s) signal was detected at 1.7% and 1.4% for p ore A and pore B, respectively. This reflected the successful coupling of the PEG 550 to the PET surface. Impact of Non Specific Protein Adsorption on Tip Diameter in PEG amine modified PET To investigate the impact of non specific protein adsorption on p ore tip diameter in the PEG 550 coated pores, several sets of current voltage curves were obtained in 1 M KCl to measure the tip diameter. First, the tip diameters of the unmodified single pores were obtained. Three conical pores per protein were evaluated Then, the pores were modified with PEG 550 and the tip opening diameters were re measured. These PEG coated pores were then immersed overnight in 100 nM protein in pH 7.4, 10 mM phosphate that was also 100 mM in KCl. The pores were removed from the prote in solution, rinsed thoroughly with water, and the tip size re measured a second time to determine the extent to which the PEG 550 layer affected non specific adsorption. For BSA, Figure 5 30A shows the current voltage curves before and after PEG 550 modi fication of the pore and membrane surfaces. That is, the tip opening diameter before PEG 550 attachment was 54 nm. After attachment, the tip diameter decreased to 47 nm. This pore was then exposed to BSA. After BSA exposure, the tip diameter was re measure d to be 46 nm (Figure 5 additional conical pores, pores 2 and 3, were treated in a similar manner. Pore 2 (Figure

PAGE 160

160 5 31A) showed pre and post PEG 550 modification tip sizes of 45 nm and 36 nm, respectively. After BSA exposure, the tip diameter of pore 2 was 34 nm (Figure 5 31B). Pore 3 (Figure 5 32A) exhibited pre and post PEG 550 modification tip diameters of 45 nm and 37 nm, respectively. After BSA exposure, the tip size of pore 3 was 38 nm (Figure 5 32B) Since there is ~10% error in the tip size, this change is negligible Similarly, for fibrinogen, Figure 5 33A shows the current voltage curves obtained before and after PEG 550 modification of the pore and PET membrane surfaces (pore 1). The tip openin g diameter before and after PEG 550 attachment was 41 nm and 25 nm, respectively. After exposure of the PEG 550 coated pore to fibrinogen, the tip size was 18 nm (Figure 5 33B). Pore 2 (Figure 5 34A) had pre and post PEG 550 modification tip sizes of 44 n m and 36 nm, respectively. After fibrinogen exposure, the tip diameter of pore 2 was 27 nm (Figure 5 34B). Pore 3 (Figure 5 35A) had pre and post PEG 550 modification tip diameters of 40 nm and 26 nm, respectively. After exposure to fibrinogen, the tip si ze was re measured to be 20 nm (Figure 5 35B). Lastly, lysozyme adsorption onto PEG 550 coated single pores was studied. Figure 5 36A shows the current voltages curves obtained before and after PEG 550 modification of the pore and membrane surfaces (pore 1). The tip opening diameter before and after PEG 550 attachment was 42 nm and 34 nm, respectively. After lysozyme exposure to the PEG 550 coated pore, the tip diameter was 30 nm (Figure 5 36B). Pore 2 (Figure 5 37A) had pre and post PEG 550 modification tip sizes of 45 nm and 35 nm, respectively. After lysozyme exposure, the tip size of pore 2 was 32 nm (Figure 5 37B). Pore 3 (Figure 5 38A) had pre and post PEG 550 modification tip diameters of 48 nm and 41 nm, respectively. After exposure to lysozyme, the tip size was

PAGE 161

161 measured to be 36 nm (Figure 5 38B). Table 5 4 summarizes the data for BSA, fibrinogen, and lysozyme. By attaching PEG 550 to the pore wall and membrane faces, non specific protein adsorption was reduced but not eliminated. Although the ad sorption of all 3 proteins was reduced in the PEG 550 coated pores, the PEG 550 layer was more efficient at blocking BSA and lysozyme adsorption than that of fibrinogen (Tables 5 4 and 5 5). It is important to discuss why PEG is used and the impact of no n specific protein adsorption on the pore wall for developing resistive pulse sensors. Such non specific binding is generally due to different types of interactions between the proteins and the pore surface. 222,223 First, proteins tend to adhere more readi ly to hydrophobic surfaces than hydrophilic surfaces. 222 PEG reduces this tendency by making the surface more hydrophilic. Secondly, electrostatic interactions between charged proteins and the charged pore wall can also lead to non specific adsorption. 222 PEG decreases the ability of proteins to interact with the pore surface in this manner because the PEG molecules reduce the surface charge of the pore wall. Consequently, PEG can also impact the electrical double layer. 222 Bentzen, et al. studied the eff ect of PEG length on non specific adsorption of quantum dots in live cell assays. 222 They discovered that the length of the PEG chain was not important until it was shorter than 14 units. Thus, PEG 350 and PEG 550 conjugates were able to reduce non specifi c binding by 70% and 60%, respectively. Longer PEG chains, such as PEG 2000, decreased binding even further (i.e., 90% reduction). Steric hindrance was not a problem until the PEG chain length was increased to MW 2000 Da or greater. As a consequence, Bentz en, et al. observed a inverse relationship between the

PAGE 162

1 62 number of PEG chains bound and chain length. 222 That is, as chain length increased, steric hindrance increased, thereby effectively reducing the number of cross linked PEG chains. While this work dealt with quantum dots, it does suggest that PEG chain length, steric hindrance, and PEG coverage are key factors to consider in eliminating non specific protein adsorption in conical nanopores. During resistive pulse sensing with conical nanopores, it is im portant that the cross sectional area of the tip opening remain constant. Without a fixed tip diameter, the electrophoretic f lux through the nanopore varies and, in some cases, may decrease due to a decrease in pore size caused by non specific adsorption o f proteins to the pore wall. For instance, Yu, et al. observed a decrease in the electrophoretic flux of proteins through an unmodified, multipore membrane due to non specific adsorption. 217 The electrophoretic flux (J, mol s 1 cm 2 ) through the tip openin g is directly proportional to analyte concentration via the following equation: 89,95 (Eq. 5.2) where z is charge on the analyte, D t is the diffusion coefficient of the analyte through the tip opening, C is analyte concentration, and E is the electric field strength in the tip. F, R, and T have their usual meanings. Eq. 5.2 can be converted to current pulse frequency (f p ) by multiplyi ng both sides of Eq. 5.2 with the cross 2 ) 89,95 (Eq. 5.3) Eq. 5.3 shows that the current pulse frequency, which reflects the analyte concentration, is proportional to the cross sectional area of the tip opening. If non specific protein

PAGE 163

163 adsorption occurs, the current pulse frequency is a dversely impacted. For instance, Sexton, et al. reported a tip size dependence of the current pulse frequency for BSA. 95 It is believed that this can lead to other problems such a poor pore to pore reproducibility, and wide distributions in current pulse d uration. Thus, non specific interactions must be minimized. An obvious question is why discard the previous PEGylation method of coating the pore surface with gold and subsequently modifying the gold layer with thiol modified PEG? 95,117,217 Eq. 5.3 shows that the current pulse frequency is proportional to the electric field strength. To obtain improved limits of detection, higher curre nt pulse frequencies are needed. One way to achieve this is by increasing the electric field strength by applying higher tr ansmembrane potentials. The problem is the gold layer delaminates at increased potentials. In Figure 5 39, a single, conical shaped nanopore in track etched PET was fabricated with a tip opening diameter of 42 nm. Electroless gold deposition 163 was used to coat the pore surface with a thin gold film, thereby decreasing the tip diameter for 4 nm. In Figure 5 40, an applied transmembrane potential of 10 V was applied and the impact on the tip diameter measured as a function of time. After 2 minutes, the tip o pening diameter increased from 4 nm to 12 nm. The tip size continued to increase with time while the high transmembrane potential was applied. This was attributed to a delamination of the gold layer due to resistive heating. 64 To mitigate this problem, an alternative approach for directly modifying the pore surface with PEG, based on EDC/sulfo offer any conclusive evidence of the stability of PEG coated pores via EDC/sulfo NHS at

PAGE 164

164 such hi gh potentials, it does indicate that gold coated PET pores at higher transmembrane potentials are not stable. Conclusions Non specific protein adsorption is problematic in resistive pulse sensing due to the dependence of the cross sectional area of the t ip opening on the current pulse frequency. 89,95 Three model proteins, lysozyme, BSA, and fibrinogen, were found to absorb to the pore surface and decrease the tip opening diameter. In previous studies, tip size was experimentally found to impact current pu lse frequency, in agreement with theory. 95 Furthermore, a surface adsorption phenomenon has been previously implicated in longer current pulse durations. 95,239 This is believed to adversely impact selectivity. Thus, PEG is attached to the pore wall to help mitigate these adsorption problems. Taking advantage of the free carboxylates generated along the pore wall as a result of chemical etching, 62 a direct coupling method using EDC/sulfo NHS chemistry 173,174,175 was presented to modify the pore surface wit h amine modified PEG (MW 550 Da). Successful modification was verified via XPS. The ability of such PEG amine PET pores to resist non specific adsorption of BSA, lysozyme, and fibrinogen was evaluated. The PEG amine layer reduced the adsorption of all 3 pr oteins to varying extents. The previous method of coating conical pores in PET with PEG by first depositing a gold surface film and subsequently attaching thiol modified PEG suffers from instability at higher potentials. This approach also suffers from p oor reproducibility. It is believed that the direct cross linking of PEG amine to the pore wall via an amide bond provides a more stable and reproducible PEG coating of the PET surface. Further improvements in the biocompatibility of the pore surface may b e gained by considering

PAGE 165

165 PEG chains of different lengths, surface layers comprised of mixed PEG lengths, and PEG coverage density.

PAGE 166

166 Figure 5 1. Current voltage curves obtained for 4 consecutive days in pH 6, 1 M KCl (pore 1). Curves for days 0, 1, 2, 3, and 4 are overlaid. Figure 5 2. Current voltage curves obtained for 4 consecutive days in pH 6, 1 M KCl (pore 2). Curves for days 0, 1, 2, 3, and 4 are overlaid.

PAGE 167

167 Figure 5 3. Current voltage curves obtained for 4 consecutive day s in pH 6, 1 M KCl (pore 3). Curves for days 0, 1, 2, 3, and 4 are overlaid. Figure 5 4. XPS spectra of unmodified PET membrane 1.

PAGE 168

168 Figure 5 5. XPS spectra of unmodified PET membrane 2. Figure 5 6. XPS spectra of unmodified PET membrane 3.

PAGE 169

169 Figure 5 7. XPS spectra of unmodified PET membrane 4. Figure 5 8. XPS spectra of unmodified PET membrane 5.

PAGE 170

170 Figure 5 9. XPS spectra of unmodified PET membrane 6. Figure 5 10. Current voltage curves obtained in 1 M KCl for pore A befo re exposure (blue trace, d tip = 40 nm) and after exposure (red trace, d tip = 27 nm) to BSA.

PAGE 171

171 Figure 5 11. Current voltage curves obtained in 1 M KCl for pore B before exposure (blue trace, d tip = 41 nm) and after exposure (red trace, d tip = 29 nm) t o BSA. Figure 5 12. Current voltage curves obtained in 1 M KCl for pore C before exposure (blue trace, d tip =39 nm) and after exposure (red trace, d tip = 29 nm) to BSA.

PAGE 172

172 Figure 5 13. Current voltage curves obtained in 1 M KCl for pore A before e xposure (blue trace, d tip = 38 nm) and after exposure (red trace, d tip = 26 nm) to fibrinogen. Figure 5 14. Current voltage curves obtained in 1 M KCl for pore B before exposure (blue trace, d tip = 38 nm) and after exposure (red trace, d tip = 20 nm) to fibrinogen.

PAGE 173

173 Figure 5 15. Current voltage curves obtained in 1 M KCl for pore C before exposure (blue trace, d tip = 39 nm) and after exposure (red trace, d tip = 23 nm) to fibrinogen. Figure 5 16. Current voltage curves obtained in 1 M KCl for p ore A before exposure (blue trace, d tip = 45 nm) and after exposure (red trace, d tip = 36 nm) to lysozyme.

PAGE 174

174 Figure 5 17. Current voltage curves obtained in 1 M KCl for pore B before exposure (blue trace, d tip = 41 nm) and after exposure (red trace, d tip = 34 nm) to lysozyme. Figure 5 18. Current voltage curves obtained in 1 M KCl for pore C before exposure (blue trace, d tip = 42 nm) and after exposure (red trace, d tip = 34 nm) to lysozyme.

PAGE 175

175 Figure 5 19. XPS spectra of chemically etched PET about a single nanopore exposed to BSA (pore A). Figure 5 20. XPS spectra of chemically etched PET about a single nanopore exposed to BSA (pore B).

PAGE 176

176 Figure 5 21. XPS spectra of chemically etched PET about a single nanopore exposed to BSA (pore C). Figure 5 22. XPS spectra of chemically etched PET about a single nanopore exposed to fibrinogen (pore A).

PAGE 177

177 Figure 5 23. XPS spectra of chemically etched PET about a single nanopore exposed to fibrinogen (pore B). Figure 5 24. XPS spectra of c hem ically etched PET about a single nanopore exposed to fibrinogen (pore C).

PAGE 178

178 Figure 5 25. XPS spectra of chemically etched PET about a single nanopore exposed to lysozyme (pore A). Figure 5 26. XPS spectra of chemically etched PET about a single na nopore exposed to lysozyme (pore B).

PAGE 179

179 Figure 5 27. XPS spectra of chemically etched PET about a single nanopore exposed to lysozyme (pore C). Figure 5 28. XPS spectra of chemically etched PET about a single nanopore modified with PEG 550 amine (po re A).

PAGE 180

180 Figure 5 29. XPS spectra of chemically etched PET about a single nanopore modified with PEG 550 amine (pore B). Figure 5 30. Current voltage curves of pore 1 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 54 nm), am ine PEG 550 modified nanopore (red trace, d tip = 47 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 47 nm) before BSA exposure and after BSA exposure (grey trace, d tip = 46 nm).

PAGE 181

181 Figure 5 31. Current voltage curves of pore 2 obtai ned in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 45 nm), amine PEG 550 modified nanopore (red trace, d tip = 36 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 36 nm) before BSA exposure and after BSA exposure (grey trace, d tip = 34 nm). Figure 5 32. Current voltage curves of pore 3 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 45 nm), amine PEG 550 modified nanopore (red trace, d tip = 37 nm) and (B) the same PEG 550 modified nanopore (red trace d tip = 37 nm) before BSA exposure and after BSA exposure (grey trace, d tip = 38 nm).

PAGE 182

182 Figure 5 33. Current voltage curves of pore 1 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 41 nm), amine PEG 550 modified nanopore (red tr ace, d tip = 25 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 25 nm) before fibrinogen exposure and after fibrinogen exposure (grey trace, d tip = 18 nm). Figure 5 34. Current voltage curves of pore 2 obtained in 1 M KCl for (A) an un modified nanopore (blue trace, d tip = 44 nm), amine PEG 550 modified nanopore (red trace, d tip = 36 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 36 nm) before fibrinogen exposure and after fibrinogen exposure (grey trace, d tip = 27 nm)

PAGE 183

183 Figure 5 35. Current voltage curves of pore 3 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 40 nm), amine PEG 550 modified nanopore (red trace, d tip = 26 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 26 n m) before fibrinogen exposure and after fibrinogen exposure (grey trace, d tip = 20 nm). Figure 5 36. Current voltage curves of pore 1 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 42 nm), amine PEG 550 modified nanopore (red tr ace, d tip = 34 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 34 nm) before lysozyme exposure and after lysozyme exposure (grey trace, d tip = 30 nm).

PAGE 184

184 Figure 5 37. Current voltage curves of pore 2 obtained in 1 M KCl for (A) an unm odified nanopore (blue trace, d tip = 45 nm), amine PEG 550 modified nanopore (red trace, d tip = 35 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 35 nm) before lysozyme exposure and after lysozyme exposure (grey trace, d tip = 32 nm). Figure 5 38. Current voltage curves of pore 3 obtained in 1 M KCl for (A) an unmodified nanopore (blue trace, d tip = 48 nm), amine PEG 550 modified nanopore (red trace, d tip = 41 nm) and (B) the same PEG 550 modified nanopore (red trace, d tip = 41 nm) bef ore lysozyme exposure and after lysozyme exposure (grey trace, d tip = 36 nm).

PAGE 185

185 Figure 5 39. Current voltage curves obtained in 1 M KCl for a single, conical shaped nanopore fabricated in track etched PET having tip diameters of 42 nm after etching (blu e trace) and 4 nm after electroless gold deposition (red trace). Figure 5 40. Current voltage curves obtained in 1 M KCl for a single, conical shaped nanopore fabricated in track etched PET. The initial tip opening diameter was 4 nm after electroless g old deposition (red trace). As an applied transmembrane potential of 10 V was applied, the tip diameter increased from 4 to 22 nm.

PAGE 186

186 Table 5 1. Summary of the tip opening diameter stability for 3 single, conical shaped nanopores in track etched PET over a period of 4 days. Table 5 2. Summary of XPS spectra for 6 unmodified PET membranes.

PAGE 187

187 Table 5 3. Summary of XPS spectra of chemically etched PET about the nanopore exposed to BSA, fibrinogen, and lysozyme (3 single pore containing me mbranes per protein). Table 5 4. Summary of the impact of non specific adsorption of BSA, fibrinogen, and lysozyme on single, conical shaped nanopores in track etched PET (3 single pore containing membranes per protein).

PAGE 188

188 Table 5 5. Summary of the e ffect of amine PEG 550 modification on the tip opening diameter and non specific adsorption of BSA, fibrinogen, and lysozyme.

PAGE 189

189 CHAPTER 6 FABRICATION OF LARGE R CONE ANG LE NANOPORES IN PET FOR STUDIES ON THE A FFECT OF CONE ANGLE ON ELECT RIC FIELD S TRENGTH, IONIC PORE CONDUCTANCE, AN D ION CURRENT RECTIF ICATION Introduction There is increasing interest in utilizing artificial nanopores as the sensing element in resistive pulse sensors for analyzing a wide variety of target analytes includi ng small molecules, 75 nucleic acids, 26,31 36,41,89,90 proteins, 38,39,42,95 and particles. 48,96 Resistive pulse sensing entails placing a membrane containing a single nanopore having a limiting diameter comparable to the size of the analyte between two elec trolyte solutions. 89,95 A potential difference is then applied across the nanopore, thereby generating an ion current flowing through the electrolyte filled pore. By measuring this ion current, analytes are detected when they are driven through nanopore. T hat is, when an analyte enters and translocates the nanopore, the analyte transiently blocks the ion current (i.e., increases the pore resistance), resulting in a downward current pulse. 75,89,90,95 The frequency of these current pulses is proportional to a nalyte concentration. 89,95 Analyte identity, or selectivity, is encoded in the current pulse signature, which is comprised of both the average current pulse duration and magnitude. 50,143,234,235 Single, conical shaped nanopores in track etched polymeric membranes are of particular interest because of the control and reproducibility of pore diameter, 63 cost effective fabrication and materials, controllable surface chemistry, 90,95,109,117,171,172,197,200,210 218,228,229 and the increased stability of polym eric membranes compared to lipid bilayer embedded pores under a variety of conditions (e.g., increased transmembrane potentials). These nanopores have a larger opening, or base, diameter at one face of the membrane and a smaller opening, or tip, diameter a t the other

PAGE 190

190 face. 63,89,95 The conical shape of such nanopores makes them ideally suited for resistive pulse sensing. 95,96 For instance, Lee, et al. used the finite element simulation method to simulate the magnitude and distribution of the electric field s trength within an electrolyte filled conical nanopore. 96 Their simulation showed that the electric field strength is highly focused at the tip opening and is on the order of 10 6 V/m. As a result, a sensing zone is formed at the tip opening that is highly s ensitive to the presence of any molecular species that enters the tip. 75,89,95,96 Thus, conical shaped pores are better suited for developing resistive pulse sensors than cylindrical shaped pores. The electric field strength is a key factor that governs current pulse frequency and current pulse frequency determines analyte concentration over the dynamic range for any respective analyte. 89,95 One strategy for increasing the current pulse frequency is to simply increase the electric field strength by increa sing the value of the applied transmembrane potential. In fact, Sexton used this approach to increase the current pulse frequency of BSA. 172 An alternative strategy presented here is based on a nanopore shape mediated approach. In other words, increasing the cone angle (i.e., increasing the base opening diameter at fixed tip diameter) was found, via finite element simulations, to have a dramatic impact on the magnitude of the electric field strength. These simulations also showed that the magnitude of the electric field strength was more sensitive to increasing cone angle than increasing transmembrane potential. Interestingly, very few, if any, studies have been reported on studying the impact of cone angle on conical nanopore based transport, particularly resistive pulse sensing. However, this strategy is ahead of its

PAGE 191

191 time because fabrication methods for controlling and reproducing larger cone angle nanopores must first be developed and validated. Harrell, et al. utilized very high transmembrane potentia ls during chemical etching of conical pores in multiple ion tracked polycarbonate to vary the base diameter. 64 Some control over cone angle was afforded by this approach but ideal pore asymmetry was arguably degraded. Furthermore, polycarbonate membranes a re often difficult to handle and, without chemical treatment ( e.g. with PVP), are highly hydrophobic. 246 Scopece, et al. studied the impact of etch solutions comprised of varying ethanol to water ratios on the cone angle in track etched poly(ethylene tere phthalate). 65 They discovered that larger cone angles were obtained by increasing the ethanol to water ratio. In this work, a non aqueous etchant was used to fabricate conical shaped nanopores in ion tracked PET membranes having cone angles that are larg er than such pores produced via the aqueous two step etch method. 63 Ionic pore conductance and ion current rectification were evaluated in single, conical shaped pores having comparable tip open diameters but different base diameters. An efficient approach for finding and imaging single nanopores in single ion irradiated membranes is also presented. Experimental Materials Poly(ethylene terephthalate) (PET, 3 cm diameter, 12 m thick) membranes containing single ion and multiple ion induced damage tracks were obtained from GSI (Darmstadt, Germany). Single ion irradiated PET was used for the ionic pore conductance, ion current rectification, and pore imaging work. Multiple ion irradiated PET (10 6 ions/cm 2 ) was used for the pore fabrication studies of base d iameter and cone

PAGE 192

192 height of gold nanocones. A ion track density of 10 5 ions/cm 2 was used for the high potential etching of PET membranes. HFIP (1,1,1,3,3,3 hexafluoroisopropanol) was obtained from Sigma (St. Louis, MO) and used as received. All other chemic als were of reagent grade and used as received. Purified water (obtained by sending house distilled water through a Barnstead E pure water purification system) was used to prepare all solutions. Fabrication of the Conical Nanopores and Gold Nanocones Seve ral different etch methods were used for this work. For the work dealing with the use of increased transmembrane potentials during etching, conical nanopores were fabricated in multiple ion irradiated PET membranes (10 5 ion tracks/cm 2 ) by mounting the memb rane in between two half cells of a U tube cell. One half cell was filled with etch solution (9 M NaOH) and the other half cell filled with stop solution (1 M formic acid and 1 M KCl). A platinum wire electrode was placed into each solution and a transmemb rane potential difference was applied during the entire etching process. In this case, anisotropic etching was allowed to proceed for 2 hours. The membrane was then rinsed briefly with stop solution, followed by water. The resulting nanopores were complete ly filled with gold by electroless gold deposition overnight (~15 hours) using a previously described electroless gold plating method. 163 This process also deposited gold on both faces of the membranes. These gold layers were removed via swabbing the membr ane faces with cotton swabs wetted with ethanol. 65,95,114 The PET membrane was then completely dissolved using HFIP. 65 The liberated gold nanocones were collected onto an alumina membrane (Anodisc Whatman) via filtration. For the work dealing with the c ontrol of base diameter and nanocone height, a non aqueous etch method was used. 114 An etching cell setup comparable to that used

PAGE 193

193 above was used except that the etch solution was 5 M KOH in 100% methanol and the stop solution used was the same (i.e., 1 M f ormic acid and 1 M KCl). Anisotropic etching was allowed to proceed as a function of etch time. No transmembrane potential was applied. After each etching time interval, the conical pores in the PET membrane were completely filled with gold by electroless gold deposition overnight (~15 hours) using the previously described electroless gold plating method. 163 Instead of liberating the resulting gold nanocones from the membrane, only the gold surface film on the membrane face at the tip side was removed via e thanolic swabbing. 65,114 As a result, the gold surface film on the membrane face at the base side was attached to a piece of double sided copper tape and mounted onto a scanning electron microscope (SEM) stub. The PET membrane was then carefully dissolved away via dropwise addition of HFIP. As a result, a randomly distributed array of gold nanocones standing on a gold surface film was obtained. 114 For the ionic pore conductance, pore imaging, and ion current rectification work, single, conical shaped nano pores were fabricated in single ion irradiated PET membranes. An etching cell setup comparable to that used above was used but the etch and stop solutions used were varied for the larger and smaller base opening diameters (i.e., larger and smaller cone ang les) for pores having comparable tip opening diameters. For the conical pores having the larger base diameters, a non aqueous etch method was used. 114 The etch solution was 5 M KOH in 100% methanol and the stop solution was 1 M formic acid and 1 M KCl. A p latinum wire electrode was placed into each half cell (i.e., anode immersed in the etch solution and cathode in the stop solution) and a transmembrane potential difference of 1 V was applied using a Keithley 6487 voltage source/picoammeter (Keithley Instru ments, Cleveland, OH). This provided a means of

PAGE 194

194 monitoring the etching process and determining when to stop etching by monitoring the ion current. Initially, the ion current was zero. When etch solution broke through to the stop solution, a sudden increase in the ion current was observed signaling breakthrough. In this case, etching was stopped when the ion current reached a value of ~50 150 pA. The pore was then briefly rinsed with stop solution followed by water. For the conical pore having the smaller base diameter, the previously described two step etch method was used. 63 The etch solution was 9 M NaOH and the stop solution was 1 M formic acid and 1 M KCl. A platinum wire electrode was placed into each half cell (i.e., anode placed in the etch solution and cathode in the stop solution) and a transmembrane potential difference of 1 V was applied. This anisotropic etching process was allowed to proceed for 2 hours. Then, the isotropic etching step of the two step etch method was used to tailor the tip ope ning diameter to match that obtained for the larger base diameter pores. Etch solutions of 1 M NaOH were placed on each side of the pore containing membrane during this step and an applied potential of 1 V was used. The platinum electrodes were configured such that the anode was immersed in the solution facing the base opening and the cathode was in the solution facing the tip opening. Field Emission Scanning Electron Microscopy A JEOL 6335F (JEOL, Ltd.) FE SEM was used to measure the base opening diamete rs of the single nanopores using a method presented below for finding single pores. FE SEM (Hitachi S 4000) was also used to the measure the base diameter of multi pore membranes for the high potential etching work and for measuring the base diameter and h eight of gold nanocones. The fabrication of these gold structures is described above.

PAGE 195

195 Finite Element Simulations The electric field strength at the tip of the electrolyte filled, conical nanopore was simulated using COMSOL Multiphysics v.3.3a software (CO MSOL, Inc.). This program was run using a Dell Optiplex GX520 (Pentium D CPU, 3.2 GHz, 2 GB RAM) (Dell, Inc.) computer. This program has been previously described in simulations of the electric field strength magnitude and distribution in conical nanopores 95,96 The COMSOL software utilizes the finite element method to solve partial differential equations that 2 95 An electrolyte layer (600 m thick) on both sides of the membrane was included in the simulation. 95 The electrolyte filled, conical nanopore was positioned in between these layers. The conical pore l ength was assumed to be 12 m long (equivalent to the membrane thickness). Two conical nanopores were simulated. The first pore had a base diameter of 5000 nm and a tip diameter of 6 nm. The second pore had a base diameter of 520 nm and a tip diameter also of 6 nm. The electric field strength was simulated for each of these pores using applied transmembrane potentials ranging from 1 20 V. Each nanopore was divided in two along its long axis (i.e., axis of symmetry), and the simulation was done for only one of the halves. 95 By doing so, a larger number of elements could be used, which improved accuracy. The number of elements used to compute each result ranged between 150,000 and 165,000.

PAGE 196

196 Results and Discussion Finite Element Simulations: The Impact of Cone Angle on the Electric Field Strength As previously described, the main determinant of analyte concentration is the current pulse frequency. 89,95 The current pulse frequency (f p ) can be described by the following equation: 89,95 (Eq. 6 .1) in which z represents the charge on the analyte, D t is the diffusion coefficient associated with analyte transport through the tip opening, C is the analyte concentration, E is the electric tip 2 is the cross sectional area of the tip opening, and A is number. F, R, and T have their usual meanings. Eq. 6 .1 shows that the current pulse frequency is directly proportional to the electric field strength. The electric field strengths in two conical nanopores having different base diameters ( i.e. 5000 nm and 520 nm) and identical tip diameters of 6 nm were simulated using the finite element method. 95,96 The electric field strength for each of these pores was simulated at applied transmembrane potentials ranging from 1 20 V. This was done to simultaneously evaluate the impact of increasing the applied potential and increasing the cone angle of the electric field strength. Figure 6 1 shows the results of the simulations These simulations show that a much smaller increase in the magnitude of the electric field strength occurs in the smaller cone angle nanopores compared to that of the larger cone angles pore as the applied potential increases from 1 to 20 V. In other wor ds, for any change in the value of the transmembrane potential within this range, the electric field strength is more sensitive to

PAGE 197

197 changes in cone angle than potential. For instance, for the pore having the smaller cone angle (i.e., smaller base diameter), the magnitude of the field strength at 20 V is slightly less than the field strength of the pore having the larger cone angle (i.e., larger base diameter) at 3 V. Thus, dramatic gains in the magnitude of the electric field strength can be achieved at lowe r applied transmembrane potentials if a larger cone angle pore is used. One caveat of using this approach in resistive pulse sensing is that as the electric field strength is increased, the current pulse duration decreases correspondingly. 89,95 Thus, it i s believed that higher sampling frequencies may needed to operate at higher current pulse frequencies. However, before this approach can be experimentally evaluated in resistive pulse sensing, methods for fabricating and reproducibly controlling larger con e angle nanopores must be developed and validated. Nanopore Characterization For experiments involving multiple ion tracked PET membranes, SEM was used for characterization of gold replicas of the nanopores. For single, conical shaped nanopores in single ion irradiated PET membranes, base opening diameter for the smaller cone angle pores was 520 nm as described previously for the two step etch method. 63 The base diameter for the larger cone angle pores was determined via SEM using the imaging approach de scribed below. Knowing the base opening diameter allows the tip opening diameter to be determined via an electrochemical method based on the ionic conductance of the electrolyte filled nanopore. 59,63,75,89,90,95 That is, the ionic conductance (G) of the na nopore is related to the tip opening diameter (d tip ), base opening diameter (d base 11.5 S/m for 1 M KCl), and the length of the conical nanopore (L) via the following equation: 59,63,75,89,90,95

PAGE 198

198 (Eq. 6 .2) The value for G was obtained by first mounting the conical nanopore containing membrane between two halves of a U tube cell and filling each half cell with electrolyte. A Ag/AgCl electrode (Bioanalytical Systems/BASi, West Lafayette, IN) was then placed into each half cell and an applied transmembrane potential was scanned linearly from 1 V to +1 V while measuring the ion current flowing through the electrolyte filled nanopore at each potential step. The slope of the resulting current voltage curve is G (in amp/volt). With this value of G, all other values in Eq 6 .2 are known, and d tip can be calculated. Etching Multiple Ion Track ed PET at High Potentials To fabricate single, conical shaped nanopores with large cone angles in ion tracked PET membranes, a method similar to that reported by Harrell, et al. was first used. 64 Multiple ion irradiated PET membranes (10 5 ion tracks/cm 2 ) were etched using 9 M NaOH as the etch solution and 1 M formic and 1 M KCl as the stop solution. A platinum wire electrode was immersed into the etch solution (anode) and in the stop solution (cathode). Transmembrane potentials of 5, 10, 15, and 20 V were applied for the duration of the etching process which took 2 hours. FE SEM was used to characterize the pore diameter and gold replicas were used to evaluate the pore shape. Figure 6 2 shows a plot of the base diameters obtained for PET membranes etched at 5, 10, 15, and 20 V. The excellent linearity observed by Harrell, et al. for a similar plot of base opening diameter versus etching potential for 50 ion tracks/cm 2 polycarbonate was not observed here with PET. 64 Instead, some degradation of the PET membra ne was observed particular at 20 V and the PET membrane melted at 30 V.

PAGE 199

199 When the anode is placed in the etch solution, the hydroxide ions are electrophoretically retracted from the tip opening. 59,64 As a result, the local concentration of hydroxide ions i n the tip region becomes depleted. Thus, etching proceeds more slowly in the tip region. When the value of the applied transmembrane potential is increased, a higher ionic current flows through the nanopore during etching. Harrell, et al. proposed that thi s increased ionic current causes resistive heating of the solution inside the nanopore. 64 Since the etch rate is known to increase with temperature, the resistive heating causes the etch rate at the base opening side of the membrane to increase. Thus, a li nearly increase in base opening diameter (for polycarbonate) was observed with increasing etching potential (i.e., 0 30 V). However, the extent to which this occurs in track density of the PET was 4 orders of magnitude larger. Thus the membrane resistance (R membrane ) of the PET membrane was much lower than that of the polycarbonate membrane used by Harrell, et al. 64 With a much lower R membrane the ionic current flowing through the nanopore is undoubtedly much higher. Harrell, et a l. described an equation for calculating the heat (q) dissipated through the membrane during etching using the following equation: 64 (Eq. 6 .3) where U represents the applied transmembrane potential, t is etch time, c is the specific heat of the NaOH (c = 0.88 cal K 1 g 1 or 3.68 J K 1 g 1 ), and m is the mass of the NaOH solution obtained from an etch solution volume of 3.5 mL and an etch s olution density of 1.3 g mL 1 Since current voltage curves were not obtained for the PET membranes,

PAGE 200

200 stop solutions were used for the polycarbonate experiments by Harr ell et al. and the PET experiments here, an approximate comparison can be made with regards to q. Since R membrane is undoubtedly much lower (i.e., due to the higher pore density) with the PET membranes used here than the polycarbonate membranes, q is high er for the PET membranes. This is likely the reason why high pore den sity PET membranes are not amen able to etching at very high transmembrane potentials (i.e., E > 20 V). This was what was observed experimentally. Thus, lower track density PET membrane mu st be used or an alternative approach to etching with high transmembrane potentials. Eq. 6 .3 does not take into account the heat released from the neutralization reaction that occurs when the etch and stop solutions react at the tip opening. Figure 6 3 sho ws a representative SEM image of a gold replica of a pore fabricated in the PET membrane using 20 V during etching. This shows a much less than ideal conical shape and a truly conical shape is needed to accurately calculate the tip opening diameter using E q. 6 .2. Etching Multiple Ion Tracked PET With Non Aqueous Etchant Two etch rates govern the cone angle in conical nanopores. 59,60,62,99 The bulk, or radial, etch rate ( B ) describes the rate at which bulk material is etched away. This determines the etching rate of the base diameter. The track etch rate ( T ) is the rate at which the latent damage track resulting from ion irradiation is etched. This determines how fast t he etch solution penetrates the membrane. Thus, a small track etch ratio ( T / B ) is needed to construct large cone angle nanopores. Decreases in this ratio result in increases in the cone angle. Since very limited success was observed etching with the hi gh transmembrane potential approach, an alternative approach using a 100% non aqueous etchant was

PAGE 201

201 studied. That is, conical nanopores were etched in PET membranes (10 6 ion tracks/cm 2 ) using 5 M KOH in 100% methanol as the etch solution and 1 M formic acid with 1 M KCl as the stop solution. 114 Etching was performed at ambient temperature as a function of etch time using etch times of 50 s, 100 s, 175 s, 250 s, and 500 s. No transmembrane potential was applied. The resulting conical nanopores were completely filled with gold to create gold replicas of the pores (Figure 6 4). As described in more detail previously, dissolution of the PET membrane left a randomized array of gold nanocones standing up on the base end on top of a gold surface film. 114 Figure 6 5 shows that the base opening diameter varied linearly with etch time. From this relationship, a bulk etch rate of 4.7 0.1 nm s 1 was obtained. At an etch time of 500 s, a base diameter of ~2.3 m was obtained which is 4 times larger than the base diameter of 520 nm typically obtained via the aqueous, two step etch method. 63 Furthermore, the gold replicas showed that the pore shape was truly conical. Thus, larger cone angle nanopores were fabrica ted using the non aqueous method. The cone height of the gold nanocones was also observed to vary linearly over the etch times studied (Figure 6 6). From this relationship, a track etch rate of 21.2 + 0.4 nm s 1 was obtained. The gold nanocones are shown i n Figure 6 7. Thus, the track etch rate was much faster than the bulk etch rate using the non aqueous etching approach. Similar etching rate behavior was observed using aqueous etching conditions in prior etching studies. 59,60,62,99 Despite not obtaining t he large base opening diameter used in the finite element simulation, larger base diameters than those typically observed were obtained by using the non aqueous etch approach. Thus, a variation of this method was transferred to single ion irradiated

PAGE 202

202 PET me mbranes for evaluating the impact of cone angle on ionic pore conductance and ion current rectification. Impact of Increased Cone Angle on Ionic Pore Conductance in PET Single, conical shaped nanopores having 3 different base diameters were fabricated us ing the non aqueous etch method (for single pores) described in detail above. The base opening diameters of these nanopores were determined via FE SEM to be 1541 nm (pore A), 1475 nm (pore B), and 1370 nm (pore C) (Figure 6 8). The SEM images were obtained using an approach for finding single pores described in detail below. The ionic conductance of each electrolyte filled pore was used to determine the tip opening diameters for each pore. Tip diameters of 10 nm, 17 nm, and 19 nm were determined for pores A B, and C, respectively. Single, conical nanopores having comparable tip opening diameters to those obtained for pores A, B, and C but base diameters of 520 nm were fabricated using the two step etch method. Wharton, et al. reported that this method rep roducibly produces base opening diameters of 520 nm in single ion irradiated PET membranes. 63 Figure 6 9 compares the ionic pore conductance (G from Eq. 6 .2) for pore A (d base = 1541 nm, d tip = 10 nm) to that of the pore having base and tip diameters of 52 0 nm and 10 nm, respectively. Larger ionic pore conductance (i.e., higher ion currents) were obtained for the pore having the larger base diameter (i.e., larger cone angle). Similar results were observed for pores B and C. Figure 6 10 compares the ionic po re conductance for pore B (d base = 1475 nm, d tip = 17 nm) to that of the pore having a base diameter of 520 nm and a tip diameter of 18 nm. Figure 6 11 compares the ionic pore conductance for pore C (d base = 1370 nm, d tip = 19 nm) to that for the pore havi ng base and tip sizes of 520 nm and 18

PAGE 203

203 nm, respectively. Both Figures 6 10 and 6 11 showed comparable results to that of Figure 6 9. That is, ionic pore conductance increased with increasing cone angle. One explanation for this observation is that the hi gher electric field produced as a result of increasing the cone angle, as shown by the simulation, produces an increased ion current. Another way of explaining this is in terms of pore resistance. Taking the inverse of the ionic conductance of the electrol yte filled nanopore, G (Eq. 6 .2), gives pore resistance (R pore ) described by the following equation: 59,63,64,75,89,90,95 (Eq. 6 .4) wher e each variable has the same meaning as in Eq. 6 .2. From Eq. 6 .4, the value for R pore decreases as the product of the tip and base diameters is increased. Thus, by increasing the base opening diameter and maintain in g a constant tip diameter, the pore resis tance decreases. As a result, the ion current increases and that was what was observed experimentally. Finding Single Nanopores in Single Ion Irradiated PET Membranes for Imaging Imaging nanopores via SEM is typically reserved for high pore density membra nes because the pores of single pore containing membranes are challenging to find. Harrell, et al. introduced an approach for finding, isolating, and imaging single pores in polycarbonate membrane having a pore density of 50 pores/cm 2 165 This approach ent ails sputter coating the surface of the porous membrane with a thin metallic coating. A small drop of fluorescent dye is then placed onto a glass slide. The metal coated membrane is then applied (i.e., with metal side facing upward) onto this drop of dye a nd the dye wicks up through the pores. Because the dye expands, or blossoms, above the pore, the pores can be found and isolated using fluorescence microscopy. Once

PAGE 204

204 found, the vicinity of the pore is inscribed using a Sharpie pen. A mask (Scotch tape) co ntaining a 3 mm diameter hole is placed around the inscribed area for isolation. Thus, a 3 mm search area exists for imaging. Because the pore density is very small, single pores can be isolated. An alternative approach is presented here for single pore c ontaining membranes that circumvents the need for fluorescence microscopy and reduces the search area from 3 mm to 200 m. Figure 6 12 shows a detailed schematic of this process. First, a single pore containing membrane is sputter coated with a thin metallic coating on the membrane face that is to be imaged. A metallic mask, having a 200 m diameter hole in its center, is placed beneath the metal coated membrane with the metal coated surface facing upwards. This metallic mask is an exact replica of the filter aperture mask used during the single swift heavy ion irradiation of the membrane. 57,58 In other words, the ion has t o pass through such a 200 m diameter aperture during the ion irradiation process. Therefore, the pore has to be within this area of the membrane. Once the mask is lined up perfectly beneath the membrane, an ultrafine tip Sharpie pen is then used to inscribe a 200 m diameter circ le on the metal coated surface. The process enables a very efficient search of the membrane surface for finding the pores in single pore containing membranes for imaging. Impact of Increased Cone Angle on Ion Current Rectification in PET Two of the single conical shaped nanopores having the larger cone angles were utilized to evaluate the effect of increasing cone angle on ion current rectification at electrolyte concentrations of 0.01 M KCl, 0.1 M KCl, and 1 M KCl. Several models have been proposed to ex plain ion current rectification. According to the model proposed by

PAGE 205

205 Cervera, et al if a conical shaped pore has excess negative surface charge and a tip opening diameter that is sufficiently small (i.e., electrical double layer thickness > pore radius), then the tip region will preferentially transport cations and reject anions. 166 In other words, the fraction of the ion current carried by migrating cations, often represented by the cation transference number (t + ), is much greater than the fraction of the ion current carried by migrating anions (i.e., anion transference number, t ). The sum of the cation and anion transference numbers equals 1. As a result, asymmetric ion migration is observed as a non linear current When the electrode polarity is such that cathode is located at the base opening and the anode is located at the tip opening, migrating cations are transported from the a node (tip side) towards the cathode (base side). 166 Anion transport occurs in the opposite direction, from cathode towards the anode. However, electrostatic repulsion from the negatively charged pore wall at the tip region reduces, or prevents, anion trans port through the tip. As a result, the local concentration of anions in the tip increases. To maintain electroneutrality, an increased local concentration of cations are required in the tip region to balance the excess anionic charge. Consequently, the loc al electrolyte concentration in this region increases and membrane resistance decreases. Thus, an configured in this manner. Conversely, when the polarity of the electrodes is reversed such that anode is on the base side and the cathode is at the tip, migrating cation transport occurs in the base to tip direction in the conical nanopore. 166 Anions are retracted from the nanopore and

PAGE 206

206 into the bulk solution on the base side of the pore by the ion current. On the tip side of the pore, anion transport through the tip opening is inhibited due to electrostatic repulsion with the anionic surface charge of the pore wall. The net result is a local decrease in the electrolyte concentra tion in the tip region and increase in membrane resistance. As a However, this model does not take into account the surface conduction pathway available for migrating cations (i.e., for pores having excess negative surface charge such as PET 62 ). Thus, a brief discussion is included here. The tr ansference number for species i can be calculated using the following equation: 164 (Eq. 6 .5) where |z i | represents the magnitude o f the ion charge, u i is the mobility of the ion in an electric field, and C i is the concentration of the ion. charge. Thus, without surface charge, no electrical double layer forms. Assuming a simply electrolyte such as KCl is used (i.e., u K+ is comparable to u Cl |z K+ | = |z Cl | = 1) The transference numbers for the cations and anions can be approximated using the following equations: (Eq. 6 .6) (Eq. 6 .7)

PAGE 207

207 In the case of a neutral pore surface and this electrolyte, t + and t are both 0.5. Thus, a linear current voltage curve is expected since no ion current rectification occurs (i.e., t + = t ). charge, as is the case with pores fabricated in PET membranes. 62,100 103 Under low ionic strength electrolyte (e.g., dilute KCl) conditions, an electrical double layer that is greatly enriched with cations forms along the pore surface. 62,100 103,164,166 In an electric field, these d ouble layer cations migrate from the anode towards the cathode. As a result, a pore surface conduction pathway, based on these migrating double layer cations, is available for cations but not anions. Thus, for Case II, the cation and anion transference num bers can be approximated using the following equations: (Eq. 6 .8) (Eq. 6 .9) As long as the contribution of cations (in the electrical double layer) to t he ion current is significant relative to that carried by cations in bulk solution, the cation transference number will be greater than the anion transference number and ion current rectification will occur. Ion current rectification is typically quantif ied by determining the rectification ratio using the following equation: 104 (Eq. 6 .10)

PAGE 208

208 where i E = 1 V is the ion current value at an applied transmem brane potential of 1 V and i E = +1 V is the ion current value at an applied potential of +1 V. The rectification ratios for conical nanopores having base diameters of 1370 nm (Pore 1) and 1541 nm (Pore 2) were determined as a function of electrolyte conce ntration and compared to that obtained for pores have comparable tip sizes but a smaller base diameter of 520 nm. Pore 1 and pore 2 had tip diameters of 19 nm and 10 nm, respectively. Figures 6 13, 6 14, and 6 15 show the current voltage curves obtained fo r pore 1 compared to those obtained for a smaller base diameter pore at 0.01 M KCl, 0.1 M KCl, and 1 M KCl, respectively. Both pores had comparable tip diameters. Higher rectification ratios were observed for the smaller base diameter (i.e., smaller cone a ngle) pore. The rectification ratio for the small base diameter pore increased with decreasing electrolyte concentration; however, for the larger base diameter pore, a slightly larger rectification ratio of 1.44 was observed for 0.1 M KCl than the rectific ation ratio of 1.32 observed for 0.01 M KCl. This was unexpected since the thickness of the electrical double layer increases with decreasing ionic strength of the electrolyte. 164 Thus, for a fixed pore radius, greater rectification ratios (i.e., larger ion current rectification) are expected at lower electrolyte concentrations. Several factors could have contributed to this unexpected result. For instance, tip diameter is generally measured using 1 M KCl. By subsequently obtaining current voltage curves in 0.01 M KCl, salt carryover could have occurred even though the pore was rinsed with water in between electrolyte exchange. Another possible problem is salt leeching from the electrode. That is, each Ag/AgCl electrode is contained in a glass housing cont aining 3 M NaCl. A porous frit separates 3 M NaCl from 0.01 M

PAGE 209

209 KCl and some leeching of the highly concentrated salt solution from the electrode has been known to occur. This result was not observed for the second pore. Figures 6 16, 6 17, and 6 18 show th e current voltage curves obtained for pore 2 compared to those obtained for a smaller base diameter pore at similar concentrations of KCl. Both pores had comparable tip diameters. Again, higher rectification ratios were observed for the smaller base diamet er (i.e., smaller cone angle) pore at all electrolyte concentrations. The rectification ratio for both pores increased with decreasing electrolyte concentration. Table 6 1 summarizes the data obtained for all of the conical nanopores. Interestingly, a rect ification ration of 0.87 was observed for the larger cone angle pore using 1 M KCl. As mentioned previously, the pore resistance decreases with increasing base diameter for pores having comparable tip size. Thus, this could be attributed to scanning the v oltage under such reduced pore resistance conditions too quickly. In other words, a fraction of the ion current at one voltage step could carry over to the subsequent voltage step if the scan rate is too fast. This may be exacerbated at lower pore resistan ce observed with increasing cone angle because the ion current is higher. The ion current rectification model proposed by Cervera, et al. can be used to propose an explanation for the lower ion current rectification observed in the larger cone angle nanop ores. 166 This model assumes that the thickness of the electrical double layer (l DL ) is comparable to the radius of the pore (r pore ), l DL pore and ignores any contribution of surface conduction due to migrating double layer cations. Although the tip open ing diameters of both the larger and smaller base diameter nanopores were comparable, the region just inside the tip is changing. That is, as the cone angle is increased, r pore increases in the local region inside the tip towards the base opening. For a fi xed electrolyte

PAGE 210

210 concentration (i.e., fixed l DL ), increasing r pore with increasing cone angle means r pore > l DL As a result, the cation transference number, using the model proposed by Cervera, et al., decreases. As a result, ion current rectification decr eases. This is assuming that etching under 100% non aqueous conditions (i.e., 5 M KOH in 100% methanol) does not alter the surface charge on the conical nanopore. Conclusions Single, conical shaped nanopores having different cone angles were fabricated i n single ion irradiated PET membranes. The two step (aqueous) etch method was utilized to produce conical pores having base diameters of 520 nm. 63 A non aqueous etch method was presented and used to fabricate conical pores having base diameters that were ~ 3 times larger than those obtained via the two step etch method. With comparable tip opening diameters, the impact of increasing cone angle (i.e., larger base diameter) on ionic pore conductance and ion current rectification was investigated. The larger cone angle pores showed reduced ion current rectification which was attributed to an increase in the pore radius in the region just inside the tip continuing towards the base with increasing cone angle. As a consequence, the cation permselectivity was redu ced (i.e., t + decreased) and rectification decreased. Assuming the non suggests that some geometric control of ion current rectification in conical nanopores can be ach ieved b y varying the cone angle but further studies are needed in PET and other materials. The larger cone angle nanopores also exhibited higher ionic pore conductance which was attributed to lower pore resistance. Finite element simulations suggest that a highe r electric field strength also occurs in larger cone angle pores. Furthermore,

PAGE 211

211 simulations showed that the electric field strength, which is a main determinant of current pulse frequency in resistive pulse sensing, 89,95 is much more sensitive to increasing cone angle than increasing transmembrane potential. Fabricating large cone angle nanopores in PET using large ion track density membranes and high transmembrane potentials was not successful and is not a good strategy because the decreased membrane resi stance (due to the large pore density) generates a large degree of resistive heating. Such heating adversely impacted both the structural integrity of the polymer membrane as well as produced pores having less than ideal conical shape, particular at higher potentials. Heating can be reduced by using much lower ion track density, or even single ion tracked, PET membranes. For single ion irradiated PET membranes, the non aqueous etching approach provided a better process for fabricating larger cone angle sin gle pores. An approach for quickly finding and imaging single nanopores was successfully applied to single pores having base diameters less than 1600 nm. Furthermore, the non aqueous etching method provided a means for fabricating randomly distributed gold nanocone arrayed surfaces of controllable base diameter and cone height. It is believed that the non aqueous approach presented here, or a derivative thereof, provides a suitable approach for fabricating larger cone angle pores for developing resistive pu lse sensors capable of producing high current pulse frequencies.

PAGE 212

212 Figure 6 1. Plot of electric field strength in the tip opening of the conical nanopore obtained from finite element simulations versus applied transmembrane potential for nanopores havi ng a large base diameter of 5000 nm (red trace) and a smaller base diameter of 520 nm (blue trace). Identical tip diameters of 6 nm were used for each pore. Figure 6 2. Plot of the base diameter obtained for multiple ion tracked PET membranes etched at 5, 10, 15, and 20 V for 2 hours.

PAGE 213

213 Figure 6 3. SEM image of a gold replica of a pore fabricated in a multiple ion irradiated PET membrane at an applied transmembrane potential of 20 V applied during etching for 2 hours. Figure 6 4. SEM image of randomly distributed gold nanocone replicas of conical pores fabricated in multiple ion irradiated PET membrane using a non aqueous etch method.

PAGE 214

214 Figure 6 5. Plot of gold nanocone base diameter versu s etch time. Error bars encompass data obtained for three membranes prepared in an identical manner. Figure 6 6. Plot of gold nanocone height versus etch time. Error bars encompass data obtained for three membranes prepared in an identical manner.

PAGE 215

215 Figure 6 7. SEM images of randomly distributed arrays of gold nanocones obtained from pores etched at ambient temperature for (A) 30 s, (B) 100 s, (C) 175 s, (D) 250 s, and (E) 500 s.

PAGE 216

216 Figure 6 8. SEM images of the base opening diameters of single, conical shaped nanopores fabricated in single ion tracked PET. Base diameters of (A) 1541 nm (pore A), (B) 1475 nm (pore B), and (C) 1370 nm (pore C) were obtained using the filter aperture mask approach to finding pores for imaging. Larger base nanopore d tip = 10 nm, d base = 1541 nm A B C

PAGE 217

217 Figure 6 9. Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore A) having a base diameter of 1541 nm and tip diameter of 10 nm (red trace) and a second conical po re having a base diameter of 520 nm and a tip diameter of 10 nm (blue trace). Figure 6 10. Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore B) having a base diameter of 1475 nm and tip diameter of 17 nm (red trace) and a second c onical pore having a base diameter of 520 nm and a tip diameter of 18 nm (blue trace).

PAGE 218

218 Figure 6 11. Current voltage curves obtained in 1 M KCl for a conical nanopore (Pore C) having a base diameter of 1370 nm and tip diameter of 19 nm (red trace) and a second conical pore having a base diameter of 520 nm and a tip diameter of 18 nm (blue trace). Figure 6 12. Schematic detailing an approach for finding single nanopores in single ion irradiated membranes uses a replica of the filter aperture mask use d during single swift heavy ion irradiation.

PAGE 219

219 Figure 6 13. Current voltage curves obtained in 0.01 M KCl for pore 1 (red trace, d base = 1370 nm, d tip = 19 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 18 nm). Figure 6 14. Curr ent voltage curves obtained in 0.1 M KCl for pore 1 (red trace, d base = 1370 nm, d tip = 19 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 18 nm).

PAGE 220

220 Figure 6 15. Current voltage curves obtained in 1 M KCl for pore 1 (red trace, d b ase = 1370 nm, d tip = 19 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 18 nm). Figure 6 16. Current voltage curves obtained in 0.01 M KCl for pore 2 (red trace, d base = 1541 nm, d tip = 10 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 10 nm).

PAGE 221

221 Figure 6 17. Current voltage curves obtained in 0.1 M KCl for pore 2 (red trace, d base = 1541 nm, d tip = 10 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 10 nm). F igure 6 18. Current voltage curves obtained in 1 M KCl for pore 2 (red trace, d base = 1541 nm, d tip = 10 nm) and a smaller cone angle pore (blue trace, d base = 520 nm, d tip = 10 nm).

PAGE 222

222 Table 6 1. Tabulated s ummary of the effect of increasing base diameter in pores 1 and 2 relative to that of pores 3 and 4 with comparable tip diameters (pores 1 and 3, pores 2 and 4) and the effect of electrolyte concentration on the ion current rectification ratio.

PAGE 223

223 CHAPTER 7 CONCLUSION The objective of this research was to develop sensors based on the resistive pulse method using conical shaped nanopores in track etched polymeric membranes, investigate properties of such pores that impact their sensi ng capabilities, and investigate nanopore fabrication. Chapter 1 introduced an overview of pertinent background information for this dissertation, including nanopore materials, ion irradiation of polymer membranes, the track etch method, nanopore character ization, the resistive pulse method, previous resistive pulse sensing work with biological and artificial nanopores, and methods for controlling pore size and surface chemistry. In Chapter 2, resistive pulse sensing of a model protein, streptavidin, was ac hieved using a single, conical nanopore fabricated via the two step etch method in poly(ethylene terephth a late ) (PET). Thus, the two step etch method was indeed an effective way of tailoring the tip size to that of the analyte for constructing functional r esistive pulse sensors. The conical nanopore surface was first coated with a thin, conformal layer of gold via electroless gold deposition and subsequently functionalized, via chemisorption, with thiol modified poly(ethylene glycol) (PEG SH) of large molec ular weight (5 kDa). The PEG coated nanopore sensing element detected current pulses for 500 nM streptavidin. The frequency of these current pulses was found to follow an exponential dependence on the applied transmembrane potential. Such current pulses we re predominantly downward (i.e., decreasing below the baseline), and had a tailing peak shape which was attributed to protein position within the nanopore. Removal of the PEG and underlying gold layers changed the current pulses such that each pulse consis ted of

PAGE 224

224 both an upward (i.e., increasing above the baseline) and a downward pulse. Furthermore, the absence of the PEG and gold layers reduced the current pulse frequency obtained using comparable tip diameters, sensing buffers, protein concentration, and applied potentials by ~50%. This suggested that the threshold potential for the PEG coated sensing element was different (i.e., lower) than that for the unmodified sensing element. In Chapter 3, a model cationic analyte, poly L lysine conjugated gold nanop articles were detected via the resistive pulse method using a single, conical nanopore in track etched PET. This work represented a departure from previous resistive pulse studies using track etched PET for two reasons. First, an unmodified nanopore was us ed. Previous studies used either a PEG SH modified gold coated nanopore or an ethanolamine coated pore. Secondly, both the electrode polarity at the tip side of the membrane and the net surface charge of the analyte were opposite in polarity (i.e., positiv e) relative to the anionic pore wall. Particles were detected as transient, upward current pulses at nanomolar, picomolar, and femtomolar concentration levels although the current pulse frequency was really low at the femtomolar level. Such upward shaped, current pulses reflect the current enhancing effect of the counter ions accompanying each nanoparticle into and through the nanopore sensing zone A definition for the detection limit in resistive pulse sensing was proposed and discussed. A narrow current p ulse amplitude was observed which was attributed to the use of a hard to many biological molecules. A wide current pulse duration was observed which was attributed to an electrostat ic binding and release between the cationic particles and the anionic pore wall. It is believed that an effective surface passivation technique that (1)

PAGE 225

225 removes the negative surface charge of the pore surface and (2) maintains a high degree of hydrophilici ty will undoubtedly decrease the wide spread in current pulse duration. In Chapter 4, an alternative polymer type, ion tracked polyimide, was investigated for use in resistive pulse sensing. Carboxylated nanoparticles that were also fluorescent were detect ed using an unmodified, conical nanopore in polyimide. To fabricate the nanopore sensing element, a two step etch method for tailoring the tip diameter to a size comparable to that of the particles was presented. In all resistive pulse sensing work, contro lling the tip opening diameter during fabrication is absolutely critical to constructing functional resistive pulse sensors. The tip diameter was observed to scale linearly with the final value of the ion current during the two step etch. The extent of io n current rectification, as described by the rectification ratio, was inversely related to the tip opening diameter at the tip sizes evaluated. An often overlooked aspect of resistive pulse experimentation is technique. For instance, what is the best appro ach to properly and completely fill a single nanoscopic pore with aqueous electrolyte? This work suggests that by combining the use of a wetting agent, vacuum degassing, and perfusion, better filling of the nanopore with sensing buffer can be facilitated. Current pulses were exclusively upward and detected using much lower applied transmembrane potentials than those typically used for resistive pulse sensors housed in track etched PET. For instance, current pulses were detected with potentials as low 50 mV although the current pulse frequency was very low. This was attributed to the large cone angle and correspondingly lower pore resistance characteristic of conical pores fabricated in polyimide. A narrow distribution in current pulse amplitude was observed and attributed to the use of a hard sphere nanoparticle which does not possess the

PAGE 226

226 conformational flexibility akin to many biomolecules. The current pulse amplitude was observed to increase linearly over all potentials studied. The current pulse frequency increased with increasing transmembrane potential for the first three potentials used; however, the pulse frequency showed unexpected behavior at the higher three potentials used. The current pulse duration followed a similar trend by decreasing with incre ase potential over the lower three potentials and showing wide variability over the higher three potentials. It is believed that a de wetting of the pore may have occurred at higher transmembrane potentials. In Chapter 5, an alternative strategy to electr oless gold deposition for functionalizing the surfaces of single, conical nanopores with PEG based on EDC/sulfo NHS coupling chemistry was introduced. Minimizing non specific pore surface adsorption is absolutely critical in resistive pulse sensing for two reasons. First, since current pulse frequency, the primary determinant of analyte concentration, is governed in part by the cross sectional area of the tip opening, a constant tip size is essential to obtaining reproducible data and low limits of detectio n. Secondly, non specific interactions between the pore wall and translocating analyte molecules have been implicated as a key contributor to wide distributions in current pulse duration, a component of selectivity, observed in prior studies and those pres ented herein. Thus, by eliminating, or significantly reducing, non specific interactions between translocating proteins and the pore wall, it is believed that improvement in current pulse duration can be achieved. In this work, the stability of the tip op ening diameter of single conical pores in track etched PET membranes was demonstrated over a 4 day time interval. Three model

PAGE 227

227 proteins, BSA, fibrinogen, and lysozyme, were found to reduce the tip diameters of unmodified conical pores via non specific adsor ption. Such adsorption of the three proteins was verified via X ray photoelectron spectroscopy (XPS). Comparable conical nanopores were functionalized with PEG NH 2 using EDC/Sulfo NHS coupling chemistry which cross links the amine group on the PEG to the f ree carboxylates on the pore wall via an amide bond. Such cross linking was confirmed using XPS and current voltage curves.. The PEG modified conical pores were then exposed to the 3 model proteins and a reduction in non specific adsorption was observed to varying extents for each protein. Furthermore, the prior approach of coating single pores in PET with PEG by first depositing a gold surface film and subsequently attaching PEG SH was shown the suffer from instability at higher potentials. It is believed that the direct cross linking of PEG NH 2 to the pore wall via EDC/sulfo NHS coupling chemistry provides a more stable and reproducible PEG coating of the PET pore surface. Further improvements in the biocompatibility of the pore surface may be gained by co nsidering PEG chains of different lengths, surface layers comprised of mixed PEG lengths, and PEG coverage density. In Chapter 6, the electric field strength distributions inside two single, conical shaped nanopores having identical tip diameters but diffe rent base diameters (i.e., one large and one small) were evaluated via finite element simulations. These simulations show the electric field strength, which is directly proportional to current pulse frequency, increases more with increasing cone angle than with increasing transmembrane potential. Thus, this provides the impetus for fabricating larger cone angle nanopores. However,

PAGE 228

228 before doing resistive pulse sensing, methods for fabricating, controlling and reproducing large cone angle nanopores are needed A high voltage approach for fabricating large cone angle nanopores in high track heating. Thus, a non aqueous approach to fabricating single, conical nanopores having larger cone angles than pores typically produced via the aqueous two step etch method was presented. Using this approach, the effect of increasing cone angle on ionic pore conductance and ion current rectification was evaluated. Increased ionic pore conductance and decrease d ion current rectification were observed with the larger cone angle pores relative to those having a smaller cone angle. Although the field of molecular scale, resistive pulse sensing remains very much in its infancy, the research presented herein demons trates that such sensors can be constructed in track etched polymeric membranes. This work stresses the importance of controlling and optimizing the pore surface properties and pore geometry to truly realize the analytical utility that can potentially be d erived from resistive pulse devices based on conical nanopores. It is hoped that this work provides the impetus for continued research efforts in these critical areas. Furthermore, since no one material has yet emerged has the best material for fabricating conical nanopores for use in resistive pulse sensors, continued research in this area is also encouraged. Single molecule, resistive pulse sensors constitute a conceptually simple, label free detection paradigm that is very promising for developing a var iety of useful applications.

PAGE 229

229 LIST OF REFERENCES (1) Martin, C. R.; Mitchell, T. D. Electroanal. Chem. 1999 21 1 7. (2) Martin, C. R.; Mitchell, D. T. Anal. Chem. 1998 70 322A 327A. (3) Martin, C. R.; Kohli, P. Nat. Rev. Drug Dis cov. 2003 29 37. (4) Martin, C. R. Science (Washington, D. C.) 1994 266 1961 196 6. (5) Whitesides, G. M. Nat. Biotechnol. 2003 21 1161 1165. (6) Rosei, F. J. Phys.: Condens. Matter 2004 16 S1373 S1436. (7) Sotiropoulou, S.; Vamvakaki, V.; Chanio takis, N. A. Biosens. Bioelectron. 2007 22 1566. (8) Heo, K.; Yoon, J.; Jin, K. S.; Jin, S.; Ree, M. IEE Proc.: Nanobiotechnol. 2006 153 121 128. (9) Yianoulis, P.; Giannouli, M. J. Nano Res. 2008 2 49 60. (10) Esmaeili Rad, M. R.; Lee, H. J.; Saz onov, A.; Nathan, A. Int. J. High Speed Electron. Syst. 2008 18 1055 1068. (11) Raciukaitis, G. Proc. SPIE 2008 7142 714207 714207 15. (12) Espinosa, H. D.; Zhu, Y.; Peng, B.; Loh, O. Adv. Multiphys. Simul. Exp. Test. MEMS 2008 455 489. (13) Zhang, L.; Qiao, S. Z.; Yan, Z. F.; Zheng, H. J.; Li, L.; Ding, R. G.; Lu, G. Q. Chin. Sci. Bull. 2009 54 516 520. (14) Yoon, T. H. Appl. Spectrosc. Rev. 2009 44 91 122. (15) Zhang, M.; Hu, C.; Liu, H.; Xiong, Y.; Zhang, Z. Sens. Actuators, B 2009 136 12 8 132. (16) Andreescu, S.; Njagi, J.; Ispas, C.; Ravalli, M. T. J. Environ. Monit. 2008 11 27 40. (17) Hille, B. Ion Channels of Excitable Membranes ; 3rd ed.; Sinauer: Sunderland, MA, 2001. (18) Voet, D.; Voet, J. D. Biochemistry ; J. Wiley & Sons: New York, 2004. (19) DeBlois, R. W.; Bean, C. P. Rev. Sci. Instrum. 1970 41 909 916

PAGE 230

230 (20) DeBlois, R. W.; Bean, C. P.; Wesley, R. K. A. J. Colloid Interf. Sci. 1977 61 323 3 3 5. (21) DeBlois, R. W.; Uzgiris, E. E.; Cluxton, D. H.; Mazzone, H. M. Anal. Bi ochem. 1978 90 273 2 88. (22) DeBlois, R. W.; Wesley, R. K. J Virol 1977 23 227 2 33. (23) Li, J.; Gershow, M.; Stein, D.; Brandin, E.; Golovchenko, J. A. Nat. Mater. 2003 2 611 615. (24) Li, J.; Stein, D.; McMullan, C.; Branton, D.; Aziz, M. J.; Go lovchenko, J. A. Nature (London, U. K.) 2001 412 166 169. (25) Fologea, D.; Gershow, M.; Ledden, B.; McNabb, D. S.; Golovchenko, J. A.; Li, J. Nano Lett. 2005 5 1905 1909. (26) Chen, P.; Gu, J.; Brandin, E.; Kim, Y. R.; Wang, Q.; Branton, D. Nano Let t. 2004 4 2293 2298. (27) Chen, P.; Mitsui, T.; Farmer, D. B.; Golovchenko, J.; Gordon, R. G.; Branton, D. Nano Lett. 2004 4 1333 1337. (28) Storm, A. J.; Chen, J. H.; Ling, X. S.; Zandbergen, H. W.; Dekker, C. Nat. Mater. 2003 2 537 540. (29) Sto rm, A. J.; Chen, J. H.; Zandbergen, H. W.; Dekker, C. Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys. 2005 71 051903/1 051903/10. (30) Storm, A. J.; Storm, C.; Chen, J.; Zandbergen, H.; Joanny, J. F.; Dekker, C. Nano Lett. 2005 5 1193 1197. (31) He ng, J. B.; Aksimentiev, A.; Ho, C.; Marks, P.; Grinkova, Y. V.; Sligar, S.; Schulten, K.; Timp, G. Nano Lett. 2005 5 1883 1888. (32) Heng, J. B.; Aksimentiev, A.; Ho, C.; Marks, P.; Grinkova, Y. V.; Sligar, S.; Schulten, K.; Timp, G. Biophys. J. 2006 9 0 1098 1106. (33) Heng, J. B.; Ho, C.; Kim, T.; Timp, R.; Aksimentiev, A.; Grinkova, Y. V.; Sligar, S.; Schulten, K.; Timp, G. Biophys. J. 2004 87 2905 2911. (34) Ho, C.; Qiao, R.; Heng, J. B.; Chatterjee, A.; Timp, R. J.; Aluru, N. R.; Timp, G. Proc. Natl. Acad. Sci. U S A 2005 102 10445 10450. (35) Chang, H.; Kosari, F.; Andreadakis, G.; Alam, M. A.; Vasmatzis, G.; Bashir, R. Nano Lett. 2004 4 1551 1556.

PAGE 231

231 (36) Iqbal, S. M.; Akin, D.; Bashir, R. Nat. Nanotechnol. 2007 2 243 248. (37) Han, A.; Creus, M.; Schuermann, G.; Linder, V.; Ward, T. R.; de Rooij, N. F.; Staufer, U. Anal. Chem. 2008 80 4651 4658. (38) Han, A.; Schurmann, G.; Mondin, G.; Bitterli, R. A.; Hegelbach, N. G.; de Rooij, N. F.; Staufer, U. Appl. Phys. Lett. 2006 88 093901/ 1 093901/3. (39) Carbonaro, A.; Sohn, L. L. Lab Chip 2005 5 1155 1160. (40) Saleh, O. A.; Sohn, L. L. Nano Microsens. Chem. Biol. Terrorism Surveill. 2008 60 81. (41) Saleh, O. A.; Sohn, L. L. BioMEMS Biomed. Nanotechnol. 2006 4 35 53. (42) Saleh, O. A.; Sohn, L. L. Proc. Natl. Acad. Sci. U S A 2003 100 820 824. (43) Saleh, O. A.; Sohn, L. L. Nano Lett. 2003 3 37 38. (44) Saleh, O. A.; Sohn, L. L. Rev. Sci. Instrum. 2001 72 4449 4451. (45) Ito, T.; Sun, L.; Crooks, R. M. Chem. Commun. (Cam bridge, U. K.) 2003 1482 1483. (46) Ito, T.; Sun, L.; Crooks, R. M. Anal. Chem. 2003 75 2399 2406. (47) Ito, T.; Sun, L.; Henriquez, R. R.; Crooks, R. M. Acc. Chem. Res. 2004 37 937 945. (48) Sun, L.; Crooks, R. M. J. Am. Chem. Soc. 2000 122 1234 0 12345. (49) Sun, L.; Crooks, R. M. Langmuir 1999 15 738 741. (50) Henriquez, R. R.; Ito, T.; Sun, L.; Crooks, R. M. Analyst (Cambridge, U. K.) 2004 129 478 482. (51) Uram, J. D.; Ke, K.; Hunt, A. J.; Mayer, M. Small 2006 2 967 972. (52) Uram, J D.; Ke, K.; Hunt, A. J.; Mayer, M. Angew. Chem., Int. Ed. 2006 45 2281 2285. (53) Uram, J. D.; Mayer, M. Biosens. Bioelectron. 2007 22 1556 1560. (54) Park, S. R.; Peng, H.; Ling, X. S. Small 2007 3 116 119.

PAGE 232

232 (55) Spohr, R. Ions Tracks and Microt echnology Principles and Application ; Friedr. Vieweg&Sohn: Verlegsgschaft: Braunschweig, 1990. (56) Apel, P. Nucl. Instrum. Methods Phys. Res., Sect. B 2003 208 11 20. (57) Spohr, R.; Patent, German. DE 2 951 376 C2 1983. (58) Spohr, R.; Patent, U.S 4 369 370, 1983. (59) Apel, P. Y.; Korchev, Y. E.; Siwy, Z.; Spohr, R.; Yoshida, M. Nucl. Instrum. Methods Phys. Res., Sect. B 2001 184 337 346. (60) Trautmann, C.; Bouffard, S.; Spohr, R. Nucl. Instrum. Methods Phys. Res., Sect. B 1996 116 429 43 3. (61) Trautmann, C.; Bruechle, W.; Spohr, R.; Vetter, J.; Angert, N. Nucl. Instrum. Methods Phys. Res., Sect. B 1996 111 70 74. (62) Siwy, Z.; Apel, P.; Baur, D.; Dobrev, D. D.; Korchev, Y. E.; Neumann, R.; Spohr, R.; Trautmann, C.; Voss, K. O. Surf. Sci. 2003 532 535 1061 1066. (63) Wharton, J. E.; Jin, P.; Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Mino, W. K.; Martin, C. R. Small 2007 3 1424 1430. (64) Harrell, C. C.; Siwy, Z. S.; Martin, C. R. Small 2006 2 194 198. (65) Scopece, P.; Ba ker, L. A.; Ugo, P.; Martin, C. R. Nanotechnology 2006 17 3951 3956. (66) Braha, O.; Gu, L. Q.; Zhou, L.; Lu, X.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2000 18 1005 100 7. (67) Kasianowicz, J. J.; Burden, D. L.; Han, L. C.; Cheley, S.; Bayley, H. Bi ophys. J. 1999 76 837 845. (68) Kang, X. F.; Cheley, S.; Guan, X.; Bayley, H. J. Am. Chem. Soc. 2006 128 10684 10685. (69) Guan, X.; Gu, L. Q.; Cheley, S.; Braha, O.; Bayley, H. ChemBioChem 2005 6 1875 1881. (70) Gu, L. Q.; Braha, O.; Conlan, S.; Cheley, S.; Bayley, H. Nature 1999 398 686 6 90. (71) Gu, L. Q.; Bayley, H. Biophys. J. 2000 79 1967 1975.

PAGE 233

233 (72) Gu, L. Q.; Cheley, S.; Bayley, H. Proc. Natl. Acad. Sci. U. S. A. 2003 100 15498 15503. (73) Gu, L. Q.; Cheley, S.; Bayley, H. Science 2 001 291 636 640. (74) Heins, E. A.; Baker, L. A.; Siwy, Z. S.; Mota, M. O.; Martin, C. R. J. Phys. Chem. B 2005 109 18400 18407. (75) Heins, E. A.; Siwy, Z. S.; Baker, L. A.; Martin, C. R. Nano Lett. 2005 5 1824 1829. (76) Kobayashi, Y.; Martin, C R. J. Electroanal. Chem. 1997 431 29 33. (77) Kobayashi, Y.; Martin, C. R. Anal. Chem. 1999 71 3665 3672. (78) Howorka, S.; Cheley, S.; Bayley, H. Nat. Biotechnol. 2001 19 636 63 9. (79) Astier, Y.; Braha, O.; Bayley, H. J. Am. Chem. Soc. 2006 1 28 1705 1710 (80) Ashkenasy, N.; Sanchez Quesada, J.; Bayley, H.; Ghadiri, M. R. Angew. Chem. Int. Ed. 2005 44 1401 1404. (81) Mathe, J.; Askimentiev, A.; Nelson, D. R.; Schulten, K.; Meller, A. Proc. Natl. Acad. Sci. U. S. A. 2005 102 12377 12382. (82) Kasianowicz, J. J.; Henrickson, S. E.; Weetall, H. H.; Robertson, B. Anal. Chem. 2001 73 2268 2272. (83) Henrickson, S. E.; Misakian, M.; Robertson, B.; Kasianowicz, J. J. Phys. Rev. Lett. 2000 85 3057 3060. (84) Kasianowicz, J. J.; Brandin, E. ; Branton, D.; Deamer, D. Proc. Natl. Acad. Sci. U S A 1996 93 13770 13773. (85) Meller, A.; Branton, D. Electrophoresis 2002 23 2583 2591. (86) Meller, A.; Nivon, L.; Brandin, E.; Golovchenko, J.; Branton, D. Proc. Natl. Acad. Sci. U S A 2000 97 1079 1084. (87) Meller, A.; Nivon, L.; Branton, D. Phys. Rev. Lett. 2001 86 3435 3438. (88) Deamer, D. W.; Branton, D. Acc. Chem. Res. 2002 35 817 825. (89) Harrell, C. C.; Choi, Y.; Horne, L. P.; Baker, L. A.; Siwy, Z. S.; Martin, C. R. Langmuir 20 06 22 10837 10843.

PAGE 234

234 (90) Kececi, K.; Sexton, L. T.; Buyukserin, F.; Martin, C. R. Nanomedicine (London, U. K.) 2008 3 787 796. (91) Mara, A.; Siwy, Z.; Trautmann, C.; Wan, J.; Kamme, F. Nano Lett. 2004 4 497 501. (92) Kasianowicz, J. J.; Henrickson S. E.; Weetall, H. H.; Robertson, B. Anal. Chem. 2001 73 2268 2272. (93) Howorka, S.; Nam, J.; Bayley, H.; Kahne, D. Angew. Chem., Int. Ed. 2004 43 842 846. (94) Movileanu, L.; Howorka, S.; Braha, O.; Bayley, H. Nat. Biotechnol. 2000 18 1091 109 5 (95) Sexton, L. T.; Horne, L. P.; Sherrill, S. A.; Bishop, G. W.; Baker, L. A.; Martin, C. R. J. Am. Chem. Soc. 2007 129 13144 13152. (96) Lee, S.; Zhang, Y.; White, H. S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004 76 6108 6115. (97) Harrell, C. C., Ph.D. Dissertation, University of Florida, 2004. (98) Baker, L.; Jin, P.; Martin, C. Crit. Rev. Solid State Mater. Sci. 2005 30 183 205. (99) Siwy, Z.; Dobrev, D.; Neumann, R.; Trautmann, C.; Voss, K. Appl. Phys. A: Mater. Sci. Process. 2003 7 6 781 785. (100) Siwy, Z.; Gu, Y.; Spohr, H. A.; Baur, D.; Wolf Reber, A.; Spohr, R.; Apel, P.; Korchev, Y. E. Europhys. Lett. 2002 60 349 355. (101) Siwy, Z.; Apel, P.; Dobrev, D.; Neumann, R.; Spohr, R.; Trautmann, C.; Voss, K. Nucl. Instrum. Method s Phys. Res., Sect. B 2003 208 143 148. (102) Siwy, Z.; Fulinski, A. Am. J. Phys. 2004 72 567 574. (103) Siwy, Z.; Heins, E.; Harrell, C. C.; Kohli, P.; Martin, C. R. J. Am. Chem. Soc. 2004 126 10850 10851. (104) Harrell, C. C.; Kohli, P.; Siwy, Z .; Martin, C. R. J. Am. Chem. Soc. 2004 126 15646 15647. (105) Siwy, Z. S.; Powell, M. R.; Petrov, A.; Kalman, E.; Trautmann, C.; Eisenberg, R. S. Nano Lett. 2006 6 1729 1734. (106) Wharton, J. E., Ph.D. Dissertation, University of Florida, 2007.

PAGE 235

235 (1 07) Wang, J., Ph.D. Dissertation, University of Florida, 2008. (108) Caicedo, H. M., Ph.D. Dissertation, University of Florida, 2008. (109) Li, N.; Yu, S.; Harrell, C. C.; Martin, C. R. Anal. Chem. 2004 76 2025 2030. (110) Wolf, A.; Reber, N.; Apel, P Y.; Fischer, B. E.; Spohr, R. Nucl. Instrum. Methods Phys. Res., Sect. B 1995 105 291 293. (111) Baker, L. A.; Choi, Y.; Martin, C. R. Curr. Nanosci. 2006 2 243 255. (112) Kohli, P.; Martin, C. R. Handb. Membr. Sep. 2009 693 708. (113) Xu, F.; Wh arton, J. E.; Martin, C. R. Small 2007 3 1718 1722. (114) Mukaibo, H.; Horne, L. P.; Park, D.; Martin, C. R. Small 2009 5(21) 2474 2479. (115) Beckman Coulter (http://www.beckmancoulter.com) (116) Wallace H. Coulter Foundation (http://www.whcf.org ) (117) Yu, S.; Lee, S. B.; Martin, C. R. Anal. Chem. 2003 75 1239 1244. (118) Bayley, H.; Braha, O.; Gu, L. Adv. Mater. 2000 12 139 142. (119) Bayley, H.; Cremer, P. S. Nature 2001 413 226 2 30. (120) Cheley, S.; Gu, L. Q.; Bayley, H. Chem. Bio l. 2002 9 829 838. (121) Shin, S. H.; Bayley, H. J. Am. Chem. Soc. 2005 127 10462 10463. (122) Bayley, H.; Jayasinghe, L. Mol. Membr. Biol. 2004 21 209 220. (123) Gu, L. Q.; Cheley, S.; Bayley, H. J. Gen. Physiol. 2001 118 481 493. (124) Bezruk ov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Macromolecules 1996 29 8517 8522. (125) Kasianowicz, J. J.; Bezrukov, S. M. Biophys. J. 1995 69 94 105 (126) Halverson, K. M.; Panchal, R. G.; Nguyen, T. L.; Gussio, R.; Little, S. F.; Misak ian, M.; Bavari, S.; Kasianowicz, J. J. J. Biol. Chem. 2005 280 34056 34062. (127) Sigworth, F. J. Nature 2003 423 21 22.

PAGE 236

236 (128) Preston, G. M.; Carroll, T. P.; Guggino, W. B.; Agre, P. Science 1992 256 385 387. (129) Walz, T.; Hirai, T.; Murata, K .; Heymann, J. B.; Mitsuoka, K.; Fujiyoshi, Y.; Smith, B. L.; Agre, P.; Engel, A. Nature 1997 387 624 626. (130) Yasui, M.; Hazama, A.; Kwon, T H.; Nielsen, S.; Guginno, W. B.; Agre, P. Nature 1999 402 187 187. (131) Murata, K.; Mitsuoka, K.; Hirai, T.; Walz, T.; Agre, P.; Heymann, J. B.; Engel, A.; Fujiyoshi, Y. Nature 2000 407 599 605. (132) Jiang, Y.; Lee, A.; Chen, J.; Cadene, M.; Chait, B. T.; MacKinnon, R. Nature 2002 417 523 526 (133) Jiang, Y.; Lee, A.; Chen, J.; Cadene, M.; Chait, B. T.; MacKinnon, R. Nature 2002 417 515 522 (134) MacKinnon, R. Science 2004 306 1304 1305 (135) Dutzler, R.; Campbell, E. B.; MacKinnon, R. Science 2003 300 108 112 (136) Doyle, D. A.; Cabral, J. M.; Pfuetzner, R. A.; Kuo, A.; Gulbis, J. M.; Coh en, S. L.; Chait, B. T.; MacKinnon, R. Science 1998 280 69 77 (137) Long, S. B.; Campbell, E. B.; MacKinnon, R. Science 2005 309 903 908 (138) Long, S. B.; Campbell, E. B.; MacKinnon, R. Science 2005 309 897 903 (139) Bezrukov, S. M.; Vodyanoy, I.; Brutyan, R. A.; Kasianowicz, J. J. Macromolecules 1996 29 8517 8522 (140) Chandler, E. L.; Smith, A. L.; Burden, L. M.; Kasianowicz, J. J.; Burden, D. L. Langmuir 2004 20 898 905 (141) Howorka, S.; Movileanu, L.; Braha, O.; Bayley, H. Proc. N atl. Acad. Sci. U. S. A. 2001 98 12996 13001. (142) Braha, O.; Walker, B.; Cheley, S.; Kasiaowicz, J. J.; Song, L.; Gouaux, J. E.; Bayley, H. Chem. Biol. 1997 4 497 505. (143) Schmidt, J. J. Mater. Chem. 2005 15 831 840. (144) Mayer, M.; Kriebel, J. K.; Tosteson, M. T.; Whitesides, G. M. Biophys. J. 2003 85 2684 2695.

PAGE 237

237 (145) Nilsson, J.; Lee, J. R. I.; Ratto, T. V.; Letant, S. E. Adv. Materials 2006 18 427 431. (146) Jin, P., Ph.D. Dissertation, University of Florida, 2009. (147) GE Osmonics, http://www.osmolabstore.com (148) Hanks, P. L.; Forschner, C. A.; Lloyd, D. R. J. Membr. Sci. 2008 322 91 97. (149) Hwang, K. J.; Liao, C. Y.; Tung, K. L. Desalination 2008 234 16 23. (150) Molla, S.; Bhattacharjee, S. Langmuir 2008 24 5659 5662. (151) Vijay, Y. K. Int. J. Hydrogen Energy 2008 33 340 345. (152) Brumlik, C. J.; Menon, V. P.; Martin, C. R. J. Mater. Res. 1994 9 1174 11 83. (153) Cepak, V. M.; Martin, C. R. Chem. Mater. 1999 11 1363 1367. (154) Ali, M.; Yameen, B.; Neumann, R.; Ensinger, W.; Knoll, W.; Azzaroni, O. J. Am. Chem. Soc. 2008 130 16351 16357. (155) White, H. S.; Bund, A. Langmuir 2008 24 2212 2218. (156) Jin, P.; Mukaibo, H.; Horne, L. P.; Bishop, G. W.; Martin, C. R. J. Am. Chem. Soc. 2010 132 2118 2119. (157) Siwy, Z.; Trofin, L.; Kohli, P.; Baker, L. A.; Trautmann, C.; Martin, C. R. J. Am. Chem. Soc. 2005 127 5000 5001. (158) Wang, J.; Martin, C. R. Nanomedicine (London, U. K.) 2008 3 13 20. (159) Apel, P. Y.; Blonskaya, I. V.; Orelovitch, O. L.; Root, D.; Vutsadakis, V.; Dmitriev, S. N. Nuclear Instruments and Methods in Physics Research Section B: Beam Interactions with Materials and Atoms 2003 209 329 334. (160) Apel, P. Y.; Blonskaya, I. V.; Dmitriev, S. N.; Orelovitch, O. L.; Presz, A; Sarto wska, B. A. Nanotechnology 2007 18(30) 305302. (161) Apel, P. Y.; Blonskaya, I. V.; Dmitriev, S. N.; Mamonova, T. I.; Orelovitch, O. L.; Sartowska, B.; Yamauchi, Y. Radiation Measurements 2008 43 S552 S557. (162) Vlassiouk, I.; Apel, P. Y.; Dmitriev, S. N.; Healy, K.; Siwy, Z. S. PNAS 2009 106 21039 21044.

PAGE 238

238 (163) Menon, V. P.; Martin, C. R. Anal. Chem. 1995 67 1920 192 8. (164) Bard, A. J.; Faulkner, L. R. Electrochemical Methods ; 2nd ed.; John Wiley & Sons: New York, 2001. (165) Harrell, C. C.; Lee, S. B.; Martin, C. R. Anal. Chem. 2003 75 6861 6867. (166) Cervera, J.; Schiedt, B.; Ramirez, P. Europhys. Letters 2005 71 35 41. (167) Kovarik, M. L.; Zhou, K.; Jacobson, S. C. J. Phys. Chem. B 2009 113 15960 15966. (168) Yusko, E. C.; An, R. ; Mayer, M. ACS Nano 2010 4(1) 477 487. (169) Umehara, S.; Pourmand, N.; Webb, C. D.; Davis, R. W.; Yasuda, K.; Karhanek, M. Nano Letters 2006 6(11) 2486 2492. (170) Fu, Y.; Tokuhisa, H.; Baker, L. A. Chem. Commun. 2009 4877 4879. (171) Vlassiouk, I.; Kozel, T. R.; Siwy, Z. S. J. Am. Chem. Soc. 2009 131 8211 8220. (172) Sexton, L. T. Ph.D. Dissertation, University of Florida, 2009. (173) Hermanson, G. T. Bioconjugate Techniques ; Academic Press: San Diego, 1996. (174) de Crombrugghe, A.; Yunus, S.; Bertrand, P. Surf. Interface Anal. 2008 40 404 407. (175) De Wael, K.; Buschop, H.; De Smet, L.; Adriaens, A. Talanta 2008 76 309 313. (176) Brynaert, J. M.; Deldime, M.; Dupont, I.; Dewez, J. L.; Schneider, N. Y. J. Colloid Interface Sci. 1995 173 236 244. (177) Ali, M.; Schiedt, B.; Healy, K.; Neumann, R.; Ensinger, W. Nanotechnology 2008 19 085713. (178) Grabarek, Z.; Gergely, J. Anal. Biochem. 1989 185 131 135. (179) Hillebrenner, H.; Buyukserin, F.; Kang, M.; Mota, M. O.; Stewart, J. D.; Martin, C. R. J. Am. Chem. Soc. 2006 128 4236 4237. (180) Buyukserin, F.; Kang, M.; Martin, C. R. Small 2007 3 106 110. (181) Buyukserin, F.; Medley, C. D.; Mota, M. O.; Kececi, K.; Rogers, R. R.; Tan, W.; Martin, C. R. Nanomedicine 2008 3(3) 283 292.

PAGE 239

239 (182) Cepak, V. M.; Hulteen, J. C.; Che, G.; Jirage, K. B.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R. J. Mater. Res. 1998 13 3070 3080. (183) Cepak, V. M.; Hulteen, J. C.; Che, G.; Jirage, K. B.; Lakshmi, B. B.; Fisher, E. R.; Martin, C. R. ; Yoneyama, H. Chem. Mater. 1997 9 1065 1067. (184) Cepak, V. M.; Martin, C. R. J. Phys. Chem. B 1998 102 9985 9990. (185) Che, G.; Jirage, K. B.; Fisher, E. R.; Martin, C. R. J. Electrochem. Soc. 1997 144 4296 4302. (186) Foss, C. A., Jr.; Hornya k, G. L.; Stockert, J. A.; Martin, C. R. J. Phys. Chem. 1994 98 2963 29 71. (187) Hou, S.; Wang, J.; Martin, C. R. J. Am. Chem. Soc. 2005 127 8586 8587. (188) Hou, S.; Wang, J.; Martin, C. R. Nano Lett. 2005 5 231 234. (189) Hulteen, J. C.; Martin, C. R. Nanopart. Nanostruct. Films 1998 235 262. (190) Hulteen, J. C.; Martin, C. R. J. Mater. Chem. 1997 7 1075 1087. (191) Lakshmi, B. B.; Dorhout, P. K.; Martin, C. R. Chem. Mater. 1997 9 857 862. (192) Lakshmi, B. B.; Patrissi, C. J.; Martin, C R. Chem. Mater. 1997 9 2544 2550. (193) Martin, C. R. Acc. Chem. Res. 1995 28 61 68. (194) Martin, C. R.; Mitchell, D. T. Electroanal. Chem. 1999 21 1 74. (195) Nishizawa, M.; Mukai, K.; Kuwabata, S.; Martin, C. R.; Yoneyama, H. J. Electrochem. Soc. 1997 144 1923 1927. (196) Parthasarathy, R. V.; Phani, K. L. N.; Martin, C. R. Adv. Mater. (Weinheim, Ger.) 1995 7 896 89 7. (197) Sapp, S. A.; Lakshmi, B. B.; Martin, C. R. Adv. Mater. (Weinheim, Ger.) 1999 11 402 404. (198) Sapp, S. A.; Mart in, C. R. Book of Abstracts, 217th ACS National Meeting, Anaheim, Calif., March 21 25 1999 (199) Sapp, S. A.; Mitchell, D. T.; Martin, C. R. Chem. Mater. 1999 11 1183 1185. (200) Wirtz, M.; Parker, M.; Kobayashi, Y.; Martin, C. R. Chem. -Eur. J. 2002 8 3572 3578.

PAGE 240

240 (201) Wirtz, M.; Yu, S.; Martin, C. R. Analyst (Cambridge, U. K.) 2002 127 871 879. (202) Prime, K. L.; Whitesides, G. M. Science 1991 252 1164 1167. (203) Li, L. Y.; Chen, S. F.; Zheng, J.; Ratner, B. D.; Jiang, S. Y. J. Phys. Chem. B 2005 109 2934 2941. (204) Herrwerth, S.; Eck, W.; Reinhardt, S.; Grunze, M. J. Am. Chem. Soc. 2003 125 9359 9366. (205) Harder, P.; Grunze, M.; Dahint, R.; Whitesides, G. M.; Laibinis, P. E. J. Phys. Chem. B 1998 102 426 436. (206) Mirkin, C. A. ; Letsinger, R. L.; Mucic, R. C.; Storhoff, J. J. Nature 1996 382 607 609. (207) Taton, T. A.; Mirkin, C. A.; Letsinger, R. L. Science 2000 289 1757 1760. (208) Cao, Y. W. C.; Jin, R. C.; Mirkin, C. A. Science 2002 297 1536 1540. (209) Park, S. J .; Taton, T. A.; Mirkin, C. A. Science 2002 295 1503 1506. (210) Lee, S. B.; Martin, C. R. Anal. Chem. 2001 73 768 775 (211) Martin, C. R.; Nishizawa, M.; Jirage, K.; Kang, M. J. Phys. Chem. B 2001 105 1925 1934. (212) Kang, M.; Martin, C. R. Lan gmuir 2001 17 2753 2759 (213) Hulteen, J. C.; Jirage, K. B.; Martin, C. R. J. Am. Chem. Soc. 1998 120 6603 6604 (214) Jirage, K. B.; Hulteen, J. C.; Martin, C. R. Anal. Chem. 1999 71 4913 4918 (215) Kohli, P.; Wirtz, M.; Martin, C. R. Electroan alysis 2004 16 9 18 (216) Buyukserin, F.; Kohli, P.; Wirtz, M. O.; Martin, C. R. Small 2007 3 266 270 (217) Yu, S.; Lee, S. B.; Kang, M.; Martin, C. R. Nano Lett. 2001 1 495 498. (218) Jirage, K.; Hulteen, J. C.; Martin, C. R. Science 1997 278 655 658 (219) Lu, H. B.; Campbell, C. T.; Castner, D. G. Langmuir 2000 16 1711 1718. (220) Finklea, H. O.; Avery, S.; Lynch, M.; Furtsch, T. Langmuir 1987 3 409 413.

PAGE 241

241 (221) Yang, Z.; Galloway, J. A.; Yu, H. Langmuir 1999 15 8405 8411. (222) Bent zen, E. L.; Tomlinson, I. D.; Mason, J.; Gresch, P.; Warnement, M. R.; Wright, D.; Sanders Bush, E.; Blakely, R.; Rosenthal, S. J. Bioconjugate Chem. 2005 16 1488 1494. (223) Lahiri, J.; Isaacs, L.; Tien, J.; Whitesides, G. M. Anal. Chem. 1999 71 777 790. (224) Staros, J. V.; Wright, R. W.; Swingle, D. M. Analytical Biochemistry 1986 156 220 222. (225) Nakajima, N.; Ikada, Y. Bioconjugate Chem. 1995 6 123 130. (226) Sam, S.; Touahir, L.; Andresa, J. S.; Allongue, P.; Chazalviel, J. N.; Gouget L aemmel, A. C.; Villeneuve, C. H.; Moraillon, A.; Ozanam, F.; Gabouze, N.; Djebbar, S. Langmuir 2010 26(2) 809 814. (227) Gilles, M. A.; Hudson, A. Q.; Borders, Jr., C. L. Analytical Biochemistry 1990 184 244 248. (228) Vlassiouk, I.; Siwy, Z. S. Nano Letters 2007 7(3) 552 556. (229) Ali, M.; Bayer, V.; Schiedt, B.; Neumann, R.; Ensinger, W. Nanotechnology 2008 19 485711. (230) Kovtyukhoua, N. I.; Mallouk, T. E.; Mayer, T. S. Adv. Materials 2003 15 780 785. (231) Bezrukov, S. M.; Kullman, L.; Winterhalter, M. FEBS Lett. 2000 476 224 228. (232) Kullman, L.; Winterhalter, M.; Bezrukov, S. M. Biophys. J. 2002 82 803 812. (233) Branton, D.; Meller, A. NATO Sci. Ser., 3, 2002 87 177 185. (234) Choi, Y.; Baker, L. A.; Hillebrenner, H.; Mart in, C. R. Phys. Chem. Chem. Phys. 2006 8 4976 4988. (235) Bayley, H.; Martin, C. R. Chem. Rev. 2000 100 2575 2594. (236) Smeets, R. M. M.; Keyser, U. F.; Krapf, D.; Wu, M. Y.; Dekker, N. H.; Dekker, C. Nano Lett. 2006 6 89 95. (237) Sexton, L. T. ; Horne, L. P.; Martin, C. R. Mol. BioSyst. 2007 3 667 685.

PAGE 242

242 (238) Haeuptle, M. T.; Aubert, M. L.; Djiane, J.; Kraehenbuhl, J. P. J. Biol.Chem. 1983 258 305 314. (239) Sexton, L. T.; Mukaibo, H.; Katira, P.; Hess, H.; Sherrill, S. A.; Horne, L. P.; Ma rtin, C. R. J. Am. Chem. Soc. 2010 ASAP online article. (240) Mathew, C. K.; Van Holde, K. E. Biochemistry ; Benjamin/Cummings: California, 1996. (241) Ted Pella, Inc. (California), http://www.tedpella.com (242) Bangs Laboratories, Inc. (Indiana), http: //www.bangslabs.com (243) Schiedt, B.; Healy, K.; Morrison, A. P.; Neumann, R.; Siwy, Z. Nuclear Instruments and Methods in Physics Research B 2005 236 109 116. (244) Nelson, D. L.; Cox, M. M. Lehninger Principles of Biochemistry 3 rd ed. ; Worth: New York, 2000. (245) He, Y.; Gillespie, D.; Boda, D.; Vlassiouk, I.; Eisenberg, R. S.; Siwy, Z. S. J. Am. Chem. Soc. 2009 131 5194 5202. (246) Spohr, R. European Research Training Network 2001 pages 1 9.

PAGE 243

243 BIOGRAPHICAL SKETCH Lloyd Peyton Ho rne, Jr. was born in Durham, NC. For his undergraduate studies, he attended the University of North Carolina at Chapel Hill and earned a B.S. in chemistry in the spring of 1997. He then spent 7 years working in the pharmaceutical industry in the areas of a nalytical research and development, technology transfer, manufacturing process optimization, and drug product commercialization. Lloyd optimization of pulsed amperometric w aveforms in HPLC for the detection of biomolecules and earned a M.S. certificate in 2002. In January of 2005, Lloyd started the doctoral program in analytical chemistry at the University of Florida, and joined the research group of Prof. Charles R. Martin. He completed his research in the spring of 2010, and obta ined a Doctor of Philosophy in c hemistry