Zuzanna SIWY University of Florida Department of Chemistry

Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL 32611 -7200 E-mail: zuzanna@chem. ufl. edu

Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL 32611 -7200 E-mail: zuzanna@chem. ufl. edu

Motivation What happens with ion transport when the dimensions of the pore become very, very small? How the pore’s structure influences its transport properties?

What does NATURE say? ~ 10 nm ~ 1 nm Science 175 (23) (1972) 720 The ion currents are rectified 20 p. A 60 m. V 0 20 ms BK channel (P. N. R. Usherwood) Y. Jiang, A. Lee, J. Chen, M. Cadene, B. T. Chait, R. Mac. Kinnon, Nature 417 (2002) 515. Ion current switches between discrete levels in a voltage-dependent manner Voltage-gated channels

Heavy ions are heavy atoms, which have been stripped of some of their outer electrons and are therefore positively charged. Irradiation animation e. g. Xe, Au, U (~2. 2 Ge. V i. e. ~ 15% c) Irradiation with heavy ions – formation of latent tracks “Development” of latent tracks Tailoring the size and shape of the pore by CHEMISTRY Diameter of pores: ~ nm range ÷ several m Number of pores: 1 pore/cm 2 ÷ 109 pores/cm 2 R. L. Fleischer, P. B. Price, R. M. Walker, Nuclear Tracks in Solids. Principles and Applications (Univ. of California Press, Berkeley, 1975).

Heavy ions damage Joint effect of many particles E. Loriot Linear accelerator UNILAC, GSI Darmstadt, Germany 1 ion 1 latent track 1 pore ! Single particle recording

A short glimpse at the "product" of track etching technique http: //www. Iontracktechnology. de

Why did we want to study asymmetric pores? Reducing the effective length of the pore A synthetic pore Asymmetric pores may offer new interesting transport properties For example voltage-gated biochannels Biological channel 10 m D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. Mac. Kinnon, Science 280 (1998) 69 -77

Nature likes asymmetry very much Y. Zhou, J. H. Morais-Cabral, R. Mac. Kinnon, Nature 414 (2001) 43 E. Perozo et al. Nature 418 (2002) 942 D. Lu, P. Grayson, K. Schulten, Biophys. J. 85 (2003) 2977

Polymer materials Polyethylene terephthlalate (PET), Hostaphan, RN 12 Polyimide (Kapton 50 HN, Du. Pont) n ETCHING – CHEMICAL “SMOOTHING” Formation of carboxylate groups COO- Carboxylate groups become a part of flexible “dangling ends” Carboxylate groups are attached to the rigid aromatic rings

Preparation of single-pore membranes U etchant stopping solution Current (p. A) I time (min) Z. Siwy et al. Nucl. Instr. Meth. B 208, 143 -148 (2003); Applied Physics A 76, 781 -785; Surface Science 532 -535, 1061 -1066 (2003).

Large opening of a pore in a PET membrane Large opening of a pore in a Kapton membrane 2 m D measured by SEM (or calculated on the basis of etching time and bulk-etch rate) D d d – estimated from the pore’s resistance R R=4 L/ Dd - specific conductivity of KCl L – length of the pore d 2 nm

Current-voltage characteristics of single conical pores Single PET pore I (n. A) - Single Kapton pore I (n. A) 12 4 p. H 7 + - p. H 7 + 2 400 4 p. H 5 p. H 3 -3 1 U (V) 3 -400 p. H 5 p. H 2 U (m. V) -4 Z. Siwy, Gu Y. , Spohr H. , Baur, D. , Wolf-Reber A. , Spohr, R. , Apel, P. , Korchev Y. E. Europhys. Lett. 60, 349 (2002). Z. Siwy, Apel P. Baur D. , Dobrev, D. D. , Korchev Y. E. , Neumann R. , Spohr R. , Trautmann, R. , Surface Science 532535, 1061 (2003)

Can cations be transported against the concentration gradient? AC voltage signal is applied across the membrane 0. 1 M KCl 0. 75 M KCl Diffusion flow Well, not yet. . . K+ still follow the diffusion flow

But now, when the higher amplitude of the AC signal is applied they can!! 0. 1 M KCl Diffusion flow 0. 75 M KCl Preferential direction of K+ flow in a conical pore Z. Siwy, A. Fulinski, Phys. Rev. Lett. 89, 158101 (2002)

Potassium ions are transported against the concentration gradient ! 0. 1 M/0. 1 M 0. 1/0. 25 M KCl 0. 1/0. 75 M KCl 0. 1/1. 0 M KCl Net ion current through a single nanofabricated conical pore <I> is an average of the signal recorded for applied voltage oscillations of various amplitudes and frequency of 0. 01 Hz. PUMPING ION P. Ball, Nature Materials THE SIMPLEST PUMP J. J. Minkel, Physical Review Focus TINY HOLE GUIDES ATOMS AGAINST TIDE Kim Patch, Research Technology News SYNTHETIC ION PUMP, E. Lerner, The Industrial Physicist

Which features are crucial for rectification and pumping? Asymmetric shape of the pore The pore has to be charged The diameter of the pore has to be very small !

Two charges in vacuum separated by a distance r: r 0 L z q 1 and q 2 are in a dielectric medium - screening length q 1 and q 2 are in a solution with other ions present D = 0. 3 nm / [KCl]0. 5 for 1: 1 electrolytes D = 0. 18 nm / [Mg. Cl 2]0. 5 for 1: 2 electrolytes D = 0. 15 nm / [Mg. SO 4]0. 5 for 2: 2 electrolytes J. N. Israelachvili Intermolecular and Surface Forces with Applications to Colloidal and Biological Systems (1985)

Why do asymmetric nanopores rectify? Asymmetry in electric potential inside the pore • Rocking ratchet The profile of electrostatic potential V(z) inside an asymmetric pore Siwy Z. , Fulinski A. Phys. Rev. Lett. 89, 198103 (2002) Siwy Z. , Fulinski A. The American Journal of Physics in press (2004). Home page of H. Linke http: //www. uoregon. edu/~linke/

TRANSIENT transport properties of asymmetric pores A single pore in PET A single pore in Kapton p. A 180 m. V p. A 5 s 180 m. V 10 s 5 s 240 m. V Z. Siwy et al. Surface Science 532 -535, 1061 (2003): Europhys. Lett. 60, 349 (2002).

Fluctuations of ion current are selfsimilar in time The closer we look the more we see ! current POWER SPECTRA Studies of the origin of 1/f noise in membrane channels currents time S (f) t f t/n time L. S. Liebovitch, Fractals and Chaos Simplified for the Life Sciences, Oxford University Press, New York, 1998 The spectral density through a single ion channel; S. M. Bezrukov, in Proc. First Int. Conf. on Unsolved Problems of Noise, Szeged 1996, edited by C. R. Doering, L. B. Kiss, and M. F. Schlesinger.

20 p. A BK channel, 60 m. V Power spectra 0 p. A 2/Hz 20 ms The 1/f noise “reflects the complex hierarchy of equilibrium protein dynamics that modulate channel conductance” (S. M. Bezrukov & M. Winterhalter, Phys. Rev. Lett. 85, 202 (2000) 1/f noise !! No 1/f noise !! Siwy Z. , Fulinski A. Phys. Rev. Lett. 89, 158101 (2002): AIP Conference Proceedings Vol 665(1) pp. 273 -282, May 28, (2003).

What are nanopores good for in biotechnology e. g. building single-molecule sensors A VBIAS Current time

Sensors based on single-pore membranes Changes in ion current signal in time Changes in current-voltage characteristics Current without DNA I time V Current with DNA time Yes/No sensor

1 • The ion currents through the pore are not rectified and do not fluctuate • Current blockage caused by the polymer translocation is easy detectable J. J. Kasianowicz, et al. , Proc. Natl. Academ. Sci. USA 93 (1996) 13770.

• Chemically modified pore S. Howorka, S. Cheley, H. Bayley, Nature Biotech. 19 (2001) 636.

An asymmetric single-molecule detector Kapton 200 p. A 120 m. V 0 p. A 10 000 ms

2 m 200 p. A Kapton 20 000 ms 0 p. A 200 p. A I II 10 ms d ~ 4 nm ds. DNA, 284 and 4100 bp A. Mara, Z. Siwy, C. Trautmann, J. Wan, F. Kamme, Nano Letters, in press

Transmembrane Ion Current for an Applied Transmembrane Potential of 200 m. V No -hemolysin With n. M -hemolysin

Effect of the Applied Transmembrane Potential on the Number and Duration of Events Transmembrane Potential = Long Short 200 m. V Current (n. A) 6. 9 0. 5 sec 6. 7 350 m. V

1+2 Proof of principle: sensing streptavidin Biotin-SH Is the gold nanotube modified with biotin specific for streptavidin?

Pore modified only with biotin n. A m. V Au tube modified with biotin Au tube

Sensing lysozyme and streptavidin p. A -200 Buffer: 1 M KCl -200 m. V 10 000 ms -400 1 M KCl + 10 -7 M lysozyme p. A -40 m. V 0 -100 + 10 000 ms -50 m. V 0 -100 200 ms

Sensing streptavidin 1 M KCl, p. H 9 + 2 10 -9 M streptavidin p. A 5 0 500 ms -5 Au tube modified with biotin and blocked by streptavidin n. A m. V Au tube modified with biotin Au tube

A B H-S C -S -S COO- -S -S Application of thiol monolayer on gold surface Direct chemical modification

1. Studies of the origin of ion current fluctuations a) “forcing” Kapton nanopores to fluctuate b) finding the “critical” length of attached dangling ends, which bring about fluctuations c) building an analogue of ligand-gating channel

2. Studies of channel inactivation The ball-chain model B. Hille Ion Channels of Excitable Membranes, Sinauer Associates Inc. Sunderland 2001 3. Are the synthetic nanopores selective for ions? I-V for various mono and polyvalent ions 4. Do synthetic nanopores function as valves for uncharged molecules?

„Your pores are in fact boring – they rectify but you cannot change the direction of rectification, you have no switch!“ 5. The degree and direction of rectification should be controlled. Introduction of well-defined and localized „gate“! U 1 U 2

6. Optimalization of the ion pump functioning: - We have to make it work faster - The seperation of ions should be realized 7. Physical modeling of rectification and pumping processes. Mathematical treatment of ion current time series.

Electro-diffusion, Smoluchowski equation

template methode 10 µm filled with aq. Cu. SO 4 D. Dobrev, I. Schuchert, E. Toimil, J. Vetter