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Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL 32611-7200 E-mail: [email protected]

Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano Interface Gainesville, FL 32611-7200 E-mail: [email protected]

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 The ion currents are rectified

Science 175 (23) (1972) 720 20

pA 60 mV

Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T. Chait, R. MacKinnon, Nature 417 (2002) 515.

0 20 ms

BK channel (P.N.R. Usherwood) 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

Irradiation anim ation

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

Diameter of pores: ~ nm range ÷ several  m Number of pores: 1 pore/cm 2 ÷ 10 9 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).

Linear accelerator UNILAC, GSI Darmstadt, Germany

E. Loriot Heavy ions dam age

Joint effect of many particles Single particle recording 1 ion

1 latent track

1 pore !

A short glimpse at the "product" of track etching technique

http://www. Iontracktechnology.de

Why did we want to study asymmetric pores?

Center for Research at the Bio/Nano Interface 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. MacKinnon

, Science 280 (1998) 69-77

Nature likes asymmetry very much

Center for Research at the Bio/Nano Interface 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

Polymer materials

Polyethylene terephthlalate (PET), Hostaphan, RN12 Polyimide (Kapton 50HN, DuPont)

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

I U

etchant stopping solution

200 150 100 50 0 242 244 246 248 250 252 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

D

2  m 2  m

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

Current-voltage characteristics of single conical pores

Center for Research at the Bio/Nano Interface -

-3

Single PET pore

I

(nA) 4

+ pH 7

2

pH 5 pH 3

1

U

(V) 3

-

-400

Single Kapton pore

I

(nA) 12

+

4

pH 7 pH 5 pH 2

400

U

(mV) -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)

Can cations be transported against the concentration gradient?

AC voltage signal is applied across the membrane 0.1 M KCl Diffusion flow 0.75 M KCl Center for Research at the Bio/Nano Interface voltage (mV)

100 50 0 -50 -100 0 100 200

time (s)

300 400

current (pA)

400 200 0 -200 -400 0 100 200

time (s)

300 400

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 voltage (mV)

400 200 0 -200 -400 0 100 200 300

time (s)

400 500

current (pA)

1000 500 0 -500 -1000 0 100 200 300

time (s)

400 500 Z. Siwy, A. Fulinski, Phys. Rev. Lett. 89 , 158101 (2002)

Potassium ions are transported against the concentration gradient !

0.6

c 0 < I > c 1 > c 0 0.1 M/0.1 M

0.4

0.1/0.25M KCl 0.1/0.75M KCl

0.2

0.1/1.0 M KCl

0.0

0.0

0.2

0.4

U

(V) 0.6

0.8

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.

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?

Center for Research at the Bio/Nano Interface Asymmetric shape of the pore The pore has to be charged The diameter of the pore has to be very small !

0 r L z Two charges in vacuum separated by a distance r:

U

kq

1

q

2

r

q 1

and q

2

are in a dielectric medium

U

kq

1 

r q

2 

- screening length

0 .

5 

D

   4  

k ke

2

B T

i c i z i

  

D

D

D = 0.3 nm / [KCl] 0.5

= 0.18 nm / [MgCl electrolytes 2 = 0.15 nm / [MgSO for 1:1 electrolytes ] 4 0.5

] 0.5

for 1:2 electrolytes for 2:2

q 1

and q

2

are in a solution with other ions present

U

k

'

q

1

q

2 

r e

r

/ 

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

20

pA A single pore in PET 180 mV

10 0

5 s

60 30 0

5 s 240 mV pA

60 30 0

A single pore in Kapton 10 s Center for Research at the Bio/Nano Interface 180 mV

Z. Siwy et al. Surface Science 532-535 , 1061 (2003):

Europhys.

Lett. 60 , 349 (2002).

Fluctuations of ion current are self similar in time The closer we look the more we see !

Center for Research at the Bio/Nano Interface POWER SPECTRA Studies of the origin of 1/f

noise in membrane channels currents current

t

time

t/n

time L.S. Liebovitch, Fractals and Chaos Simplified for the Life Sciences, Oxford University Press, New York, 1998

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.

20

pA

0

BK channel, 60 mV

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)

pA 2 /Hz Power spectra 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

Current

V BIAS A Center for Research at the Bio/Nano Interface

time Current time

Sensors based on single-pore membranes Changes in ion current signal in time Center for Research at the Bio/Nano Interface Changes in current-voltage characteristics

Current

without DNA I

time Current

with DNA

time

Yes/No sensor

V

• •

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.

1

Chemically modified pore

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

An asymmetric single-molecule detector

Kapton 120 mV

10 000 ms 0 pA

Kapton 2

m

20 000 ms 0 pA

I II d ~ 4 nm dsDNA, 284 and 4100 bp

10 ms

A. Mara, Z. Siwy, C. Trautmann, J. Wan, F. Kamme, Nano Letters, in press

Transmembrane Ion Current for an Applied Transmembrane Potential of 200 mV No



hemolysin

Current-Time Transient no

HL E app

With nM



hemolysin

-

Effect of the Applied Transmembrane Potential on the Number and Duration of Events Transmembrane Potential =

Long

Short

200 mV

6.9

0.5 sec 6.7

350 mV

1+2

Proof of principle: sensing streptavidin

Center for Research at the Bio/Nano Interface Biotin-SH Is the gold nanotube modified with biotin specific for streptavidin?

Pore modified only with biotin

Au tube modified with biotin nA

4 2 -1000 -500 0 0 -2

Au tube

-4 -6 500 1000

mV Center for Research at the Bio/Nano Interface

pA

-200

Sensing lysozyme and streptavidin

Buffer: 1 M KCl

10 000 ms -400

1 M KCl + 10 -7 M lysozyme + pA

0 -100 10 000 ms 0 -50 -100 -100 -150 1000 0 -1000 200 ms 226600 226800 Time (ms) 227000

Center for Research at the Bio/Nano Interface -200 mV -40 mV -50 mV

Sensing streptavidin

pA

5

1 M KCl, pH 9 + 2 10 -9 M streptavidin

0 -5

Au tube modified with biotin and blocked by streptavidin nA

4 2 500 ms

Au tube modified with biotin

-1000 -500 0 0 -2 -4

Au tube

-6 500 1000

mV Center for Research at the Bio/Nano Interface

A B

H-S

C

-S -S -S -S

Application of thiol monolayer on gold surface

COO -

Direct chemical modification

1. Studies of the origin of ion current fluctuations a) b) c) “forcing” Kapton nanopores to fluctuate finding the “critical” length of attached dangling ends, which bring about fluctuations 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. 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?

„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

c

  

t

   

z I J

I

 

D

c

z

 

F tot J

 

D c

0

e

 0

L

W dze

  

W c L e

W

  

V

D. Dobrev, I. Schuchert, E. Toimil, J. Vetter filled with aq. CuSO 4