Ionic Transport Through Nanopores: From Living Cells to Ionic Diodes and Transistors Zuzanna S. Siwy Department of Physics and Astronomy University of California, Irvine

Our main object of studies is a single nanopore in a polymer film We study ionic transport through single conical nanopores + - Main Object of Our Studies Several nanometers, typically 2-6 nm ~ 1  m 12  m

Outline 1.Motivation for studies of single nanopores 2.Fabrication of single nanopores by the track-etching technique. 3.Motivation for studying conically shaped nanopores. 4.Preparation of ionic devices controlling transport of ions in water solutions:  Preparation of ionic unipolar rectifiers.  Preparation of an ionic bipolar diode and transistor (BJT); similarities and differences to semiconductor devices.  On the way to make a field effect transistor for ions.  Ionic diodes as biosensors. 5. Nanoprecipitation in nanopores and electrochemical oscillations. 6. Conclusions. heavy ion polymer foil

Impermeable lipid bilayer membrane Membrane-Bound Transport Proteins Allow for highly selective transport of ions, sugars, amino acids, etc. across the lipid bilayer membrane Lessons from Nature Transport Proteins are Nature’s Nanotubes

Biological Pores are Smart “Holes” – Very Selective Transport of Millions of Ions per 1 s Potassium selective channel with four K + in the selectivity filter (right panel). R. MacKinnon, P. Agre 2003 < 1 nm E. Gouaux, R. MacKinnon, Science 310, 1461 (2005). S. Berneche, B.Roux, Nature 414, 73 (2001). A potassium selective channel is a very important player in the nerve signaling.

W. Nonner, D. Gillespie, D. Henderson, B. Eisenberg, J. Phys. Chem. 105, 6427 (2001); E.W. McCleskey, J. Gen. Physiol. 113, 765 (1999) [Ca 2+ ] << [Na + ]Ca 2+ and Na + have basically the same diameter. Selectivity of L-Type Calcium Channels (Heart Muscle Regulation) Negative groups COO -

Preparation of the Simplest Calcium Channel/Pore PHYSICS approach Gillespie, D., Boda, D., He Y. Apel, P., Siwy, Z.S. (2008) Synthetic Nanopores as a Test Case for Ion Channel Theories: The Anomalous Mole Fraction Effect. Biophysical Journal 95, Our synthetic analogue (a synthetic hole) is indeed Ca 2+ selective! Theoretical predictions: highly charged lining of the pore and small pore volume lead to Ca 2+ selectivity. COO - ~1 e/nm 2 e = electron charge COO - = carboxyl group with charge -e

Diode - Like Characteristics of Biological Channels I [pA] V [mV] T. Baukrowitz et al. EMBO 18, 847 (1999)Y. Jiang et al. Nature 417, 515 (2002) Many biological channels are switches for ions What are the Physical Requirements for Making Ionic Diodes and Transistors? Perhaps a Basis for Ionic Electronics? PHYSICS approach A diode perfectly rectifies currents so that it flows in one direction rectifier diode

Nanopores – Studying Interactions at the Nanoscale __________ ________ Nanopores give a unique possibility to control transport of ions and charged molecules in water-based solutions. Nanopores have very large surface!

Nanopores as Basis for Biosensors Sub-femtoliter volume! Very few molecules actually fit there! Basis for single molecule detection!

Preparation of various components of IONIC CIRCUITS for ions and molecules in a water solution: urgent need for systems that operate in water. For that we need: TEMPLATE - robust single nanopores with tunable geometry and surface chemistry i.e. tunable electrochemical potential. I Will Talk About..

Outline heavy ion polymer foil 1.Motivation for studies of single nanopores. 2.Fabrication of single nanopores by the track-etching technique. 3.Motivation for studying conically shaped nanopores. 4.Preparation of ionic devices controlling transport of ions in water solutions:  Preparation of ionic unipolar rectifiers.  Preparation of an ionic bipolar diode and transistor (BJT); similarities and differences to semiconductor devices.  On the way to make a field effect transistor for ions.  Ionic diodes as biosensors. 5. Nanoprecipitation in nanopores and electrochemical oscillations. 6. Conclusions.

1. Irradiation with e.g. Xe, Au, U (~2.2 GeV i.e. ~ 15% c) 2. Chemical etching Linear accelerator UNILAC, GSI Darmstadt, Germany E. Loriot 1 ion  1 latent track  1 pore ! Heavy Ions as a Working Tool Latent tracks R.L. Fleischer, P.B. Price, R.M. Walker (1975)

1. Irradiation with e.g. Xe, Au, U (~2.2 GeV i.e. ~ 15% c) 2. Chemical etching Linear accelerator UNILAC, GSI Darmstadt, Germany E. Loriot 1 ion  1 latent track  1 pore ! Heavy Ions as a Working Tool R.L. Fleischer, P.B. Price, R.M. Walker (1975)

Tuning the Pore Shape during Etching VbVb VtVt V b – Rate of non-specific etching the so-called bulk etching V t - Rate of etching along the latent track Recipes for cylindrical and conical nanopores: Cylindrical pores: high V t and low V b ; for PET 0.5 M NaOH in 70 ºC Conical pores: low V t and high V b ; for PET 9 M NaOH, RT

Why Do We Want to Work with Asymmetric Pores? Cylindrical pore Tapered cone dd D L >> d=1 nm results in current of 3.9 pA. d=1 nm, D=2  m, results in current of ~740 pA. Example for 0.5 V, 1 M KCl, L = 10  m

I U NaOH acidic solution Current (pA) time (min) Conical Pores are Obtained by Putting Etch Solution on One Side of Membrane and Stop Solution of the Other Z. Siwy et al. Nucl. Instr. Meth. B 208, (2003); Applied Physics A 76, ; Surface Science , (2003). Single ion irradiation

Gold Replica of a Single Conical Pore P. Scopece et al. Nanotechnology 17, 3951 (2006) ~ 2 – 10 nm

Etch solution 9 M NaOH CathodeAnode For polyethylene terephthalate Electro-Stopping Technique to Prepare Double-Conical Pores Etch solution 9 M NaOH

P. Apel, Dubna Cross – Section of Membranes with Double-Conical Nanopores

Hydrolysis of Ester Bonds with NaOH in PET Causes Formation of COOH Groups OH - The surface density of COOH groups was estimated to be ~ 1.0 per nm 2 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _

Outline heavy ion polymer foil 1.Motivation for studies of single nanopores 2.Fabrication of single nanopores by the track-etching technique. 3.Motivation for studying conically shaped nanopores. 4.Preparation of ionic devices controlling transport of ions in water solutions:  Preparation of ionic unipolar rectifiers.  Preparation of an ionic bipolar diode and transistor (BJT); similarities and differences to semiconductor devices.  On the way to make a field effect transistor for ions.  Ionic diodes as biosensors. 5. Nanoprecipitation in nanopores and electrochemical oscillations. 6. Conclusions.

Transport Properties of Conical Nanopores I U 0.1 M KCl

Z. Siwy et al. Europhys. Lett. 60, 349 (2002); Z. Siwy et al. Surface Science , 1061 (2003) Single Conical Nanopores Rectify Ion Current Current (nA) Voltage (mV) VtVt VbVb V b - V t ~ 3 nm ~ 600 nm 0.1 M KCl, pH M KCl, pH 3 COO - COOH

t + ~ 0.80 PET and Kapton pores are selective for positive ions (cations) I U Which Ions Are Transported? Z. Siwy, A Fulinski, Phys. Rev. Lett. 89, (2002); Am. J. Phys. 72, 567 (2004). Siwy Z., Adv. Funct. Mat.16, 735 (2006). UNIPOLAR DEVICE – mainly pass through _ _ _ _ _ _ _ _ _ _ _ _ __ _ _ _

Why do Asymmetric and Charged Pores Rectify Siwy Z., Fulinski A. Phys. Rev. Lett. 89, (2002); Siwy Z., Fulinski A. The American Journal of Physics 74 (2004) 567; Siwy Z., Adv. Funct. Mat.16, 735 (2006). The profile of electric potential V(z) of a cation in an asymmetric nanopore z Cervera, J., Schiedt, B., Ramirez, P. Europhys. Lett. 71, (2005).

PROBLEM: Degree of Rectification of Conical Nanopores U (V) I (nA) Ideally, from application stand point one wants a SWITCH i.e. basically zero leakage current.

How to Make an Ionic Switch? H. Daiguji, P. Yang, A. Majumdar, NanoLett., 4, 137 (2005). I. Vlassiouk, Z.S. Siwy, Nano Lett. 7, 553 (2007) __________ ________ Depletion zone

__________ ________ HIGH Conductance State of Nanopore BIPOLAR DEVICE – current carried by both Eric Kalman

Targeted Modification of the Tip GOAL!, The negative groups (COO - ) at the narrow opening have to be changed into groups with positive charges, e.g. NH 3 +

Steady-State Solution of Diffusion Problem Steady-State Solution of Diffusion Problem Distribution of concentration of a reagent introduced only on the tip side of the membrane C0C0 C L =0 Targeted modification of the tip Only the region of the pore close to the tip with high enough EDC and amines concentration will be modified! x

Modification Chemistry Ethylene diamine + EDC, 0.1 M KCl, pH 5.5 Ethylenediamine + EDC Succinide anhydride + EDC _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ 0.1 M KCl, pH 5.5

An Ionic Diode Made From a Nanopore with a Positive Tip I. Vlassiouk, Z.S. Siwy, Nano Lett. 7, 553 (2007) 0.1 M KCl, pH 5.5

Positively Charged Nanopore M KCl, pH 5.5

I. Vlassiouk, Z.S. Siwy, Nano Lett. 7, 553 (2007) 0.1 M KCl, pH 5.5 An Ionic Diode Made From a Nanopore with a Negative Tip

Tuning Rectification We can measure ion rectification degree in situ during the modification! I. Vlassiouk, Z.S. Siwy, Nano Lett. 7, 553 (2007)

Miedema, H.; Vrouenraets, M.; Wierenga, J.; Meijberg, W.; Robillard, G.; Eisenberg, B. A Biological Porin Engineered into a Molecular, Nanofluidic Diode. Nano Letters 7 (2007) Diode Pattern Realized in a Bacterial Biopore WITHOUT charges WITH charges

Unipolar Diodes Were Also Prepared R. Karnik, C. Duan, K. Castelino, H. Daiguji, A. Majumdar Nano Letters 7, (2007). I. Vlassiouk, S. Smirnov, Z. Siwy, ACS Nano 2, 1589 (2008) Voltage (V) 10 mM KCl

Poisson-Nernst-Planck Modeling of Ionic Diodes C i – concentration of positive and negative ions  - electric potential  - dielectric constant J i – flux of an ion i with charge z i Density of charge carriers is described by the Boltzmann statistics

A Semiconductor Diode Vs an Ionic Diode Carrier concentration p-dopedn-doped electrons ( - ) holes ( + ) Voltage 1  m long, 0.5 e/nm 2, 0.1 M KCl Numerical solutions of PNP

doping 1-D Analytical Approximations for Diodes a – pore radius  - surface charge density Depletion zone I. Vlassiouk, S. Smirnov, Z. Siwy, ACS Nano 2, 1589 (2008) Current Voltage N.W. Ashcroft, N.D. Mermin, Solid State Physics, Thomas Learning, 1976 Current Voltage

__________ ________ Depletion zone Depletion Zone in LONG Pores I. Vlassiouk, S. Smirnov, ACS Nano 2, 1589 (2008)

Depletion Zone in SHORT Pores __ _ _______ ________ The depletion zone fills the whole pore, which can be treated as a neutral pore I. Vlassiouk, S. Smirnov, ACS Nano 2, 1589 (2008)

Opening of Short Diodes C bulk = 0.1 M KCl, charge density 0.5 e/nm 2, radius 4 nm (V)

Preparation of Ionic Bipolar Junction: Transistor Cl - K+K+ P. Apel, Dubna I V diode P-N junctions

Step-by-Step Modifications E. Kalman, I. Vlassiouk, Z. Siwy, Advanced Materials 20, 293 (2008).

Performance of Ionic BJT Salt concentration determines the potential in the pore and thus the leakage current level in BJT + _ _ _ _ _ _ _ M KCl

Performance of Ionic BJT – pH response “+ - +” junction “0 - 0” “+ 0 +” junction E. Kalman, I. Vlassiouk, Z. Siwy, Advanced Materials 20, 293 (2008).

Ionic Gated Channel with Electrically Addressable Gate – On the Way to Make FET 12  m PET Membrane Not to scale Au Gate Electrode SiO 2 Insulating Layer Ti Adhesion Layers

Gated Conical Nanopore Applying negative gate voltage to the gate causes suppression of ion currents 0 V -1.0 V

Gated Conical Nanopore E. Kalman, O. Sudre, I. Vlassiouk, Z. Siwy, Analytical and Bioanalytical Chemistry 394, 413 (2009)

1.Motivation for studies of single nanopores 2.Fabrication of single nanopores by the track-etching technique. 3.Motivation for studying conically shaped nanopores. 4.Preparation of ionic devices controlling transport of ions in water solutions:  Preparation of ionic unipolar rectifiers.  Preparation of an ionic bipolar diode and transistor (BJT); similarities and differences to semiconductor devices.  On the way to make a field effect transistor for ions.  Ionic Diodes as Biosensors 5. Nanoprecipitation in nanopores and electrochemical oscillations. 6. Conclusions.Outline

Summary: Tuning Current-Voltage Curves Of Nanopores by the Surface Charge I I I U U U Surface charge patternsCorresponding current-voltage curvesAND Changes of the surface pattern are induced upon binding of an analyte I Vlassiouk, T, Kozel, Z.S. Siwy, JACS 131,

Prototype of the Sensor for Avidin and Streptavidin + Avidin (+) _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ biotin Current Voltage KCl as the background electrolyte

Prototype of the Sensor for Avidin Voltage (V) Current (nA) Nanopore with the tip modified with biotin; 10 mM KCl, pH 7.0 With avidin 0.5  M, 2 h With biotin

Prototype of the Sensor for Streptavidin + Streptavidin, pI ~ 6 _ + pH < 6pH > 6 Current (nA) Voltage (V) 10 mM KCl biotin Rectification degree I(+2V)/I(-2) pH 8.0 pH 4.2 pH 5.8

GOAL Label-free sensor for antigens that are bioterrorism agents Prototype: Monitoring infection with Bacillus anthracis Capsule of poly-  -glutamic acid (  DPGA) thus it is heavily negatively charged Infection with Bacillus anthracis results in  DFGA in the blood at the levels that are higher than 20 ng/ml (~10 pM  DFGA). Bacillus anthracis

Sensor for a Real “Stuff” – pI of the mAb for  DPGA pH 4.8 pH 6.0 pH 8.0 _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ pH < pIpH ~ pIpH > pI Current (nA) Voltage (V) Monoclonal antibody for polyglutamic acid Prof. T. Kozel, University of Nevada (F2G26) I Vlassiouk, T, Kozel, Z.S. Siwy, JACS 131,

Sensing Signal pH 4.8 pH 6.0 Current (nA) Voltage (V) + polyglutamic acid Current (nA) Voltage (V) Rectification degree I(+5V)/I(-5) Before adding  DPGA After adding  DPGA pH 8.0 pH 6.0 pH 4.8 pH 8.0 Monoclonal antibody for polyglutamic acid

1.Motivation for studies of single nanopores 2.Fabrication of single nanopores by the track-etching technique. 3.Motivation for studying conically shaped nanopores. 4.Preparation of ionic devices controlling transport of ions in water solutions:  Preparation of ionic unipolar rectifiers.  Preparation of an ionic bipolar diode and transistor (BJT); similarities and differences to semiconductor devices.  On the way to make a field effect transistor for ions.  Ionic Diodes as Biosensors. 5. Nanoprecipitation in nanopores and electrochemical oscillations. 6. Conclusions.Outline

I U 0.1 M KCl + Ca 2+ Conductivity Cell Used for Recording Current-Voltage Curves [Ca 2+ ] << [K + ] or [Mg 2+ ] << [K + ] or [Co 2+ ] << [K + ]

_ Mg(OH) 2 [Mg 2+ ] [OH - ] 2 <K sp = [Mg 2+ ] [OH - ] 2 >> K sp Precipitation in a Nanopore 0.1 M KCl – background electrolyte A ‘plug’ can be created inside a nanopore!! Ionic concentrations inside a nanopore depend on the surface charge and applied voltage Concentration of cations in a negatively charged pore can be much higher than in the bulk.

20 s Current (pA) 40 s Current (pA) KCl 0.5 mM Mg mM Mg  M Mg 2+ A A B B Evidence for the Precipitation, Mg(OH) 2 Mg(OH) 2 K sp = 5.61· M. Powell et al. Nature Nanotechn. 3, 51 (2008)

Modeling by the Poisson-Nernst-Planck Equations Products of ionic activities at -1 V are above the solubility product for Mg(OH) 2 Products of ionic activities at +1 V are below the solubility product

Modeling by the Poisson-Nernst-Planck Equations Products of ionic activities are very strongly voltage-dependent!

Voltage (V) Current (nA) 0.1 M KCl 0.1 M KCl mM CaCl M KCl mM CaCl M KCl mM CaCl 2 Z. Siwy et al. Nano Lett. 6 (2006) Evidence for the Precipitation (I) CaHPO 4 pH 8, 2 mM PBS Pore opening 5 nm CaHPO 4 K sp  2 ·10 -7

Current (pA) 400 ms Current (pA) 400 ms C B 10 s Current (pA) A B C KCl 0.1 mM Ca mM Ca mM Ca 2+ A Evidence for the Precipitation 2 mM PBS 20 s Current (pA) 2 s Current (pA) 1 s D E F D E F G 0.1 mM PBS 1.0 mM PBS 5.0 mM PBS 0.2 mM PBS 0.2 mM Ca 2+

Evidence for the Precipitation (II ), CoHPO 4 A B 0.01 mM Co mM Co 2+ KCl 20 s 4 s Current (pA) CoHPO 4 K sp  1 ·10 -7 M. Powell et al. Nature Nanotechn. 3, 51 (2008)

Singing of Divalent Cations pA 20 s 0.1 M KCl mM Co 2+ pA 1 s 0.1 M KCl mM Ca 2+ Co 2+ Ca 2+

Application of the System with Calcium/Cobalt to Build Stochastic Sensors? Detecting Neomycin

Detecting Spermine

Conclusions We have a lot of fun doing research with nanopores! 1. Unipolar and Bipolar ionic diodes were prepared on the basis of conical nanopores with tailored surface chemistry. 2. The principle of operation of the bipolar diode is analogous to that of a bipolar semiconductor diode.

SIWY GROUP Eric Kalman Matt Powell Dr. Dragos Constantin Alumni Dr. Ivan Vlassiouk Graduate students Laura Inees – IM-SURE and UROP Fellow Matt Davenport Catherine Smith Gael Nguyen Mike Chiang, MCSB student

Acknowledgments UC Irvine Prof. Clare Yu Prof. Craig Martens Prof. Reg Penner Prof. Thorsten Ritz Prof. Ken Shea Prof. Vicente Aguillella Prof. Robert S. Eisenberg, Rush Medical College, Chicago Gesellschaft fuer Schwerionenforschung (GSI), Darmstadt, Germany Dr. Christina Trautmann, GSI, Germany Dr. Olivier Sudre, Teledyne & Imaging, Thousand Oaks Prof. S. Smirnov, New Mexico State University A.P. Sloan Foundation RCE Pacific Southwest ACS Petroleum Research Fund Institute for Complex Adaptive Matter Institute for Surface and Interface Science TEMPO group (Prof. Steve White, Prof. Doug Tobias Prof. Thomas Kozel, University of Nevada