Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL

Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL

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

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

Irradiation with heavy ions – formation of latent tracks “Development” of latent tracks Diameter of pores: ~ nm range ÷ several  m Number of pores: 1 pore/cm 2 ÷ 10 9 pores/cm 2 Tailoring the size and shape of the pore by CHEMISTRY e.g. Xe, Au, U (~2.2 GeV i.e. ~ 15% c) Irradiation animation Heavy ions are heavy atoms, which have been stripped of some of their outer electrons and are therefore positively charged. R.L. Fleischer, P.B. Price, R.M. Walker, Nuclear Tracks in Solids. Principles and Applications (Univ. of California Press, Berkeley, 1975).

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

Iontracktechnology.de A short glimpse at the "product" of track etching technique

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

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

ETCHING – CHEMICAL “SMOOTHING” Formation of carboxylate groups COO - Polyethylene terephthlalate (PET), Hostaphan, RN12 Polyimide (Kapton 50HN, DuPont) n Polymer materials Carboxylate groups become a part of flexible “dangling ends” Carboxylate groups are attached to the rigid aromatic rings

I U etchant stopping solution Z. Siwy et al. Nucl. Instr. Meth. B 208, (2003); Applied Physics A 76, ; Surface Science , (2003). Preparation of single-pore membranes Current (pA) time (min)

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

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 , 1061 (2003) U (V) I (nA) pH 7 pH 5 pH 3 -+ pH 7 pH 5 pH U (mV) I (nA) Current-voltage characteristics of single conical pores Single PET poreSingle Kapton pore

0.75 M KCl 0.1 M KCl AC voltage signal is applied across the membrane Diffusion flow Well, not yet... K + still follow the diffusion flow Can cations be transported against the concentration gradient?

0.75 M KCl 0.1 M KCl Diffusion flowPreferential direction of K + flow in a conical pore But now, when the higher amplitude of the AC signal is applied they can!! Z. Siwy, A. Fulinski, Phys. Rev. Lett. 89, (2002)

Net ion current through a single nanofabricated conical pore is an average of the signal recorded for applied voltage oscillations of various amplitudes and frequency of 0.01 Hz. Potassium ions are transported against the concentration gradient ! TINY HOLE GUIDES ATOMS AGAINST TIDE Kim Patch, Research Technology News PUMPING ION P. Ball, Nature Materials THE SIMPLEST PUMP J.J. Minkel, Physical Review Focus SYNTHETIC ION PUMP, E. Lerner, The Industrial Physicist 0.1 M/0.1 M 0.1/0.25M KCl 0.1/0.75M KCl 0.1/1.0 M KCl

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

Two charges in vacuum separated by a distance r: q 1 and q 2 are in a dielectric medium q 1 and q 2 are in a solution with other ions present - screening length D = 0.3 nm / [KCl] 0.5 for 1:1 electrolytes D = 0.18 nm / [MgCl 2 ] 0.5 for 1:2 electrolytes D = 0.15 nm / [MgSO 4 ] 0.5 for 2:2 electrolytes z 0L J.N. Israelachvili Intermolecular and Surface Forces with Applications to Colloidal and Biological Systems (1985) r

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

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

The closer we look the more we see ! time current t t/n L.S. Liebovitch, Fractals and Chaos Simplified for the Life Sciences, Oxford University Press, New York, 1998 Studies of the origin of 1/f  noise in membrane channels currents 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. f S (f) POWER SPECTRA Fluctuations of ion current are self- similar in time

Siwy Z., Fulinski A. Phys. Rev. Lett. 89, (2002): AIP Conference Proceedings Vol 665(1) pp , May 28, (2003). 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) 0 20 pA BK channel, 60 mV 20 ms 1/f noise !!No 1/f noise !! pA 2 /Hz Power spectra

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

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

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)

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

200 pA 0 pA ms Kapton An asymmetric single-molecule detector 120 mV

2  m d ~ 4 nm dsDNA, 284 and 4100 bp Kapton A. Mara, Z. Siwy, C. Trautmann, J. Wan, F. Kamme, Nano Letters, in press 0 pA 200 pA ms 10 ms 200 pA III

Transmembrane Ion Current for an Applied Transmembrane Potential of 200 mV No  hemolysin With nM  hemolysin

Effect of the Applied Transmembrane Potential on the Number and Duration of Events 6.7 Current (nA) sec Transmembrane Potential = 200 mV 350 mV Long Short

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

nA mV Au tube Au tube modified with biotin Pore modified only with biotin

200 ms Sensing lysozyme and streptavidin ms M KCl M lysozyme -40 mV pA -50 mV ms Buffer: 1 M KCl pA -200 mV +

1 M KCl, pH M streptavidin pA mV nA 500 ms Au tube Au tube modified with biotin Au tube modified with biotin and blocked by streptavidin Sensing streptavidin

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

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 1. Studies of the origin of ion current fluctuations

2. Studies of channel inactivation The ball-chain model 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? B. Hille Ion Channels of Excitable Membranes, Sinauer Associates Inc. Sunderland2001

„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 5. The degree and direction of rectification should be controlled. Introduction of well-defined and localized „gate“! U1U1 U2U2

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