Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano...

Zuzanna SIWYUniversity of Florida

Department of ChemistryCenter for Research at the Bio/Nano Interface

Gainesville, FL 32611-7200

E-mail: [email protected]

What happens with ion transport when the dimensions of the pore become very, very small?

How the pore’s structure influences its transport properties?

MotivationMotivation

Science 175 (23) (1972) 720

Ion current switches between discrete levels in a voltage-dependent manner

20 ms0

20pA 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?What does NATURE say?

Irradiation with heavy ions – formation of latent tracks

“Development” of latent tracks

Diameter of pores: ~ nm range ÷ several mNumber of pores: 1 pore/cm2 ÷ 109pores/cm2

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 !

http://www. Iontracktechnology.de

A short glimpse at the "product" A short glimpse at the "product" of track etching techniqueof 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) 69-77

Asymmetric pores may offer new interesting transport properties

For example voltage-gated biochannels

Why did we want to study Why did we want to study asymmetric pores?asymmetric pores?

Center for Research at the Bio/Nano InterfaceCenter for Research at the Bio/Nano Interface

D. Lu, P. Grayson, K. Schulten, Biophys. J. 85 (2003) 2977

Nature likes asymmetry very muchNature likes asymmetry very much Center for Research at the Bio/Nano InterfaceCenter 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

ETCHING – CHEMICAL “SMOOTHING”

Formation of carboxylate groups COO-

Polyethylene terephthlalate(PET), Hostaphan, RN12

Polyimide (Kapton 50HN, DuPont)

n

Polymer materialsPolymer materials

Carboxylate groups become a part of flexible

“dangling ends”

Carboxylate groups are attached to the

rigid aromatic rings

I

U

etchantstopping solution

242 244 246 248 250 2520

50

100

150

200

curr

ent

(pA

)

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).

Preparation of single-pore Preparation of single-pore membranesmembranes

Cu

rren

t (p

A)

time (min)

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

- 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 532-535, 1061 (2003)

400

4

2

1

-3

3U (V)

I (nA)

pH 7

pH 5

pH 3

- + pH 7

pH 5

pH 2-400

U (mV)

I (nA)

-4

4

12

- +

Current-voltage characteristics of Current-voltage characteristics of single conical poressingle conical pores

Single PET pore Single 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

0 100 200 300 400-400

-200

0

200

400

current (pA)

time (s)0 100 200 300 400

-100

-50

0

50

100

voltage (mV)

time (s)

Can cations be transported against Can cations be transported against the concentration gradient?the concentration gradient?


0.75 MKCl

0.1 MKCl

Diffusion flow Preferential direction of K+ flow in a conical pore

But now, when the higher amplitude of the AC signal is applied they can!!

0 100 200 300 400 500-1000

-500

0

500

1000

current (pA)

time (s)0 100 200 300 400 500

-400

-200

0

200

400

voltage (mV)

time (s)

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

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.

Potassium ions are transported against the concentration gradient !

TINY HOLE GUIDES ATOMS AGAINST TIDE

Kim Patch, Research Technology News

PUMPING IONP. 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

0.0 0.2 0.4 0.6 0.8

0.0

0.2

0.4

0.6

c1 > c0c0< I >c1 > c0c0< I >

< I

> (

nA)

U (V)

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 Which features are crucial for rectification and pumping?rectification and pumping? Center for Research at

the Bio/Nano InterfaceCenter for Research at the Bio/Nano Interface

Two charges in vacuum separated by a distance r:

r

qkqrU 21

q1 and q2 are in a dielectric

medium

r

qkqrU

21

q1 and q2 are in a solution

with other ions present

/21

're

r

qqkrU

- screening length

5.0

24

i

ii

BD zcke

Tk

D = 0.3 nm / [KCl]0.5 for 1:1 electrolytesD = 0.18 nm / [MgCl2]0.5 for 1:2 electrolytesD = 0.15 nm / [MgSO4]0.5 for 2:2 electrolytes

z0 L

J.N. Israelachvili Intermolecular and Surface Forces with Applications to Colloidal and

Biological Systems (1985)

r

Siwy Z., Fulinski A. Phys. Rev. Lett. 89, 198103 (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 http://www.uoregon.edu/~linke/

• Rocking ratchet

Why do asymmetric nanopores rectify?Why do asymmetric nanopores rectify?

The profile of electrostatic potential V(z) inside an asymmetric pore

180 mV

240 mV

0

30

60

180 mV

10 s

A single pore in PET

0

10

20

5 s

pA

5 s0

30

60

pA

Z. Siwy et al. Surface Science 532-535, 1061 (2003): Europhys. Lett. 60, 349 (2002).

A single pore in Kapton

TRANSIENT transport properties of TRANSIENT transport properties of asymmetric poresasymmetric pores Center for Research at


The closer we look the more we see !

time

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.

fS

(f)

POWER SPECTRA

Fluctuations of ion current are self-Fluctuations of ion current are self-similar in timesimilar in time Center for Research at


Siwy Z., Fulinski A. Phys. Rev. Lett. 89, 158101 (2002): AIP Conference Proceedings Vol 665(1) pp. 273-282, 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

20pA BK channel, 60 mV

20 ms

1/f noise !! No 1/f noise !!

pA2/Hz

Power spectraPower spectra

AVBIAS

time

Current

time

Current

What are nanopores good for in What are nanopores good for in biotechnologybiotechnology

e.g. building single-molecule sensorse.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 Sensors based on single-pore membranesmembranes Center for Research at


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) 13770.

1

• Chemically modified pore

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

200

pA

0 pA10 000 ms

Kapton

An asymmetric single-molecule An asymmetric single-molecule detectordetector

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

pA20 000 ms

10 ms

200

pA

III

Transmembrane Ion Current for an Applied Transmembrane Potential of 200 mV

4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)

Current-Time Transient no HL Eapp = 200 mV

4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)

-

4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)


4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)

-

4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)


4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)

-

4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)


4.1

4.2

4.3

4.4

5 sec

Cu

rren

t (n

A)

-

0.5 sec3.8

Cur

rent

( nA

) 3.9

-

0.5 sec3.8

Cur

rent

( nA

) 3.9

0.5 sec3.8

Cur

rent

( nA

) 3.9

-

0.5 sec3.8

Cur

rent

( nA

) 3.9

No hemolysin

With nM hemolysin

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

6.7

Cur

rent

(nA

)

6.90.5 sec

0.5 sec3.8

Cur

rent

(nA

) 3.9

0.5 sec3.8

Cur

rent

(nA

) 3.9

Transmembrane Potential =

200 mV

350 mV

Long Short

Biotin-SH

Is the gold nanotube modified with biotin specific for streptavidin?

Proof of principle: sensing Proof of principle: sensing streptavidinstreptavidin Center for Research at


1+2

-6

-4

-2

0

2

4

-1000 -500 0 500 1000

nA

mV

Au tube

Au tube modified with biotin

Pore modified only with biotinPore modified only with biotin Center for Research at the Bio/Nano InterfaceCenter for Research at the Bio/Nano Interface

227000226800226600Time (ms )

Im s

ca

led

(pA

)

- 150

-100

-50

10

V

m

(mV

)

- 1000

0

1000

200 ms

Sensing lysozyme and streptavidin

10 000 ms

0

-100

1 M KCl + 10-7 M lysozyme

-40 mVpA

-50 mV0

-100

-400

-20010 000 ms

Buffer: 1 M KClpA-200 mV

+


-6

-4

-2

0

2

4

-1000 -500 0 500 1000

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

-5

5

0

mV

nA

500 ms

Au tube

Au tube modified with biotin

Au tube modified with biotin and blocked by

streptavidin

Center for Research at the Bio/Nano InterfaceCenter for Research at the Bio/Nano InterfaceSensing streptavidinSensing streptavidin

COO-

Direct chemical modification

A B

H-S

C

-S

-S

-S

-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 fluctuations1. Studies of the origin of ion current fluctuations

2. Studies of channel inactivation2. Studies of channel inactivation

The ball-chain model

3. Are the synthetic nanopores selective for ions?3. Are the synthetic nanopores selective for ions?

I-V for various mono and polyvalent ions

4. Do synthetic nanopores function as valves for 4. Do synthetic nanopores function as valves for uncharged molecules?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 controlled5. The degree and direction of rectification should be controlled. Introduction of well-defined and localized „gate“!

U1

U2

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.

tzIzt

tzc,

,

tzczFz

tzcDtzIJ tot ,

,,

Electro-diffusion, Smoluchowski equation

L zVzW

LWL

W

dze

ececDJ

0

0 0

template methode

10 µm

filled with aq. CuSO4

template methode

10 µm

filled with aq. CuSO4

D. Dobrev, I. Schuchert, E. Toimil, J. Vetter

Zuzanna SIWY University of Florida Department of Chemistry Center for Research at the Bio/Nano...

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