Ion Transport through Nanometric Pores: Rectification, Pumping, and Voltage-Gating

1. Zuzanna SIWYUniversity of FloridaDepartment of ChemistryCenter for Research at the Bio/Nano Interface Gainesville, FL 32611-7200 Rectification, pumping and voltage-gating in ion currents through nanometric pores E-mail: zuzanna@chem.ufl.edu

2. Zuzanna SIWYUniversity of FloridaDepartment of ChemistryCenter for Research at the Bio/Nano Interface Gainesville, FL 32611-7200 Rectification, pumping and voltage-gating in ion currents through nanometric pores E-mail: zuzanna@chem.ufl.edu

3. 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

4. The ion currents are rectified pA 20 60 mV 0 20 ms BK channel (P.N.R. Usherwood) Ion current switches between discrete levels in a voltage-dependent manner Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait, R. MacKinnon, Nature417 (2002) 515. Voltage-gated channels What does NATURE say? ~ 10 nm Science175 (23) (1972) 720 ~ 1 nm

5. Heavy ions as a working tool Irradiation animation Heavy ions are heavy atoms, which have been stripped of some of their outer electrons and are therefore positively charged. e.g. Xe, Au, U (~2.2 GeV 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 R.L. Fleischer, P.B. Price, R.M. Walker, Nuclear Tracks in Solids. Principles and Applications (Univ. of California Press, Berkeley, 1975). Diameter of pores: ~ nm range ÷ several mNumber of pores: 1 pore/cm2 ÷ 109pores/cm2

6. Joint effect of many particles Single particle recording What is unique about heavy ion irradiation? Heavy ions damage E. Loriot Linear accelerator UNILAC, GSI Darmstadt, Germany 1 ion  1 latent track  1 pore !

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

8. A synthetic pore Asymmetric pores may offer new interesting transport properties Biological channel For example voltage-gated biochannels 10 m D. A. Doyle, J. M. Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon,Science280 (1998) 69-77 Why did we want to study asymmetric pores? Reducing the effective length of the pore

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

10. Polymer materials Polyimide (Kapton 50HN, DuPont) Polyethylene terephthlalate(PET), Hostaphan, RN12 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

11. I Preparation of single-pore membranes U Current (pA) stopping solution etchant time (min) Z. Siwy et al. Nucl. Instr. Meth. B208, 143-148 (2003); Applied Physics A76, 781-785; Surface Science532-535, 1061-1066 (2003).

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

13. - - + + Current-voltage characteristics of single conical pores Single PET pore Single Kapton pore I(nA) 12 4 I(nA) pH 7 pH 7 2 400 4 pH 5 pH 5 pH 3 pH 2 -400 -3 3 U(mV) U(V) 1 -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 532-535, 1061 (2003)

14. 0.1 M KCl 0.75 M KCl Can cations be transported against the concentration gradient? AC voltage signal is applied across the membrane Diffusion flow Well, not yet... K+ still follow the diffusion flow

15. But now, when the higher amplitude of the AC signal is applied they can!! 0.1 MKCl 0.75 MKCl Diffusion flow Preferential direction of K+ flow in a conical pore Z. Siwy, A. Fulinski, Phys. Rev. Lett.89, 158101 (2002)

16. Potassium ions are transported against the concentration gradient ! 0.1 M/0.1 M 0.1/0.25M KCl 0.1/0.75M 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. TINY HOLE GUIDES ATOMS AGAINST TIDE Kim Patch, Research Technology News PUMPING IONP. Ball, Nature Materials THE SIMPLEST PUMPJ.J. Minkel, Physical Review Focus SYNTHETIC ION PUMP, E. Lerner, The Industrial Physicist

17. 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?

18. Two charges in vacuum separated by a distance r: q1 and q2 are in a dielectric medium  - screening length q1 and q2 are in a solution with other ions present D = 0.3 nm / [KCl]0.5 for 1:1 electrolytesD = 0.18 nm / [MgCl2]0.5 for 1:2 electrolytesD = 0.15 nm / [MgSO4]0.5 for 2:2 electrolytes r z 0 L J.N. Israelachvili Intermolecular and Surface Forces with Applications to Colloidal and Biological Systems (1985)

19. Rocking ratchet Home page of H. Linke http://www.uoregon.edu/~linke/ Why do asymmetric nanopores rectify? Asymmetry in electric potential inside the pore 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).

20. TRANSIENT transport properties of asymmetric pores A single pore in PET A single pore in Kapton pA 180 mV pA 5 s 180 mV 10 s 240 mV 5 s Z. Siwy et al. Surface Science532-535, 1061 (2003): Europhys. Lett.60, 349 (2002).

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

22. Power spectra pA2/Hz BK channel, 60 mV pA 20 0 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).

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

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

25. 1 Model pores - hemolysine pore a • 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.

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

27. 200 pA 10 000 ms 0 pA An asymmetric single-molecule detector Kapton 120 mV

28. 2 m I II 200 pA 10 ms An asymmetric single-molecule detector Kapton 200 pA 20 000 ms 0 pA d ~ 4 nm dsDNA, 284 and 4100 bp A. Mara, Z. Siwy, C. Trautmann, J. Wan, F. Kamme, Nano Letters, in press

29. Transmembrane Ion Current for an Applied Transmembrane Potential of 200 mV No a-hemolysin With nM a-hemolysin

30. 6.9 0.5 sec Long Short Current (nA) 6.7 Effect of the Applied Transmembrane Potential on the Number and Duration of Events Transmembrane Potential = 200 mV 350 mV

31. 1+2 Proof of principle: sensing streptavidin Biotin-SH Is the gold nanotube modified with biotin specific for streptavidin?

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

33. Sensing lysozyme and streptavidin pA Buffer: 1 M KCl -200 mV -200 10 000 ms -400 + 1 M KCl + 10-7 M lysozyme pA -40 mV 0 -100 10 000 ms -50 mV 0 -100 200 ms

34. Sensing streptavidin 1 M KCl, pH 9 + 2 10-9 M streptavidin pA 5 0 500 ms -5 Au tube modified with biotin and blocked by streptavidin nA mV Au tube modified with biotin Au tube

35. -S -S COO- -S -S A B C H-S Direct chemical modification Application of thiol monolayer on gold surface Engineered synthetic pore

36. There are still many things to do... 1. Studies of the origin of ion current fluctuations • “forcing” Kapton nanopores to fluctuate • finding the “critical” length of attached dangling ends, which bring about fluctuations • building an analogue of ligand-gating channel

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

38. „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“! U1 U2

39. 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. What to do next ?

40. How to build a quantitative model of rectification and pumping? Electro-diffusion, Smoluchowski equation

41. microneedles and nanowires microneedles and nanowires template methode template methode 10 µm 10 µm filled with aq. CuSO4 filled with aq. CuSO4 D. Dobrev, I. Schuchert, E. Toimil, J. Vetter