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
pA
20
60 mV
0
20 ms
BK channel (P.N.R. Usherwood)
Y. Jiang, A. Lee, J. Chen, M. Cadene, B.T.
Chait, R. MacKinnon, 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 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/cm2 ÷ 109pores/cm2
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
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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
200
U
etchant
stopping
solution
current(pA)
(pA)
Current
I
150
100
50
0
242
244
246
248
250
252
time (min)
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
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=4L/Dd
- specific conductivity of KCl
L – length of the pore
d  2 nm
Current-voltage characteristics of
single conical pores
Single PET pore
I (nA)
-
Single Kapton pore
I (nA) 12
4
pH 7
+
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
-
pH 7
+
2
400
4
pH 5
pH 3
-3
1
pH 5
pH 2
-400
U (V) 3
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?
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
AC voltage signal is applied across the membrane
0.1 M
KCl
0.75 M
KCl
Diffusion flow
v o lta g e (m V )
c u rre n t (p A )
400
100
50
200
0
0
-5 0
-2 0 0
-1 0 0
0
100
200
300
400
-4 0 0
0
100
tim e (s )
Well, not yet... K+ still follow the diffusion flow
200
300
tim e (s )
400
But now, when the higher amplitude of the AC signal is
applied they can!!
0.1 M
KCl
0.75 M
KCl
Diffusion flow
v o lta g e (m V )
Preferential direction of K+
flow in a conical pore
c u rre n t (p A )
1000
400
200
500
0
0
-2 0 0
-5 0 0
-4 0 0
0
100
200
300
400
500
-1 0 0 0
tim e (s )
Z. Siwy, A. Fulinski, Phys. Rev. Lett. 89, 158101 (2002)
0
100
200
300
tim e (s )
400
500
Potassium ions are transported against the
concentration gradient !
0.6
< I > (nA)
0.1 M/0.1 M
< I >c > c
1
0
c0
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
0.6
0.8
U (V)
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?
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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
z
L
U r  
kq 1 q 2
r
q1 and q2 are in a dielectric
medium
kq 1 q 2
U r  
r
 - screening length
D


k BT

2
4

ke
 ci zi

i






0 .5
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
q1 and q2 are in a solution
with other ions present
U r  
'
k q1 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
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
A single pore in PET
20
pA
A single pore in Kapton
180 mV
10
pA
60
0
5s
30
60
0
10 s
30
0
180 mV
5s
240 mV
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
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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
pA
BK channel, 60 mV
Power spectra
0
pA2/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
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
e.g. building single-molecule sensors
Current
A
VBIAS
time
Current
time
Sensors based on single-pore
membranes
Changes in ion current signal in
time
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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 pA
120 mV
0 pA
10 000 ms
2 m
200 pA
Kapton
20 000 ms
0 pA
200 pA
I II
10 ms
d ~ 4 nm
dsDNA, 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 mV
C u rren t (n A )
4 .4
4 .3
5 se c
No hemolysin
4 .2
4 .1
C u rre n t (n A)
C u rre n t-T
- im e T ra n s ie n t n o  H L E a p p = 2 0 0 m V
3 .9
With nM hemolysin
3 .8
0 .5 s e c
-
Effect of the Applied Transmembrane Potential on
the Number and Duration of Events
C u rre n t (n A )
Transmembrane
Potential =
3.9
Long
3.8
Short
200 mV
0.5 sec
Current (nA)
6.9
0.5 sec
6.7
350 mV
1+2
Proof of principle: sensing
streptavidin
Biotin-SH
Is the gold nanotube modified with
biotin specific for streptavidin?
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
Pore modified only with biotin
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
nA
4
2
0
-1000
-500
0
-2
Au tube modified
with biotin
mV
-4
Au tube
-6
500
1000
Sensing lysozyme and
streptavidin
pA
-200
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
Buffer: 1 M KCl
-200 mV
10 000 ms
-400
1 M KCl + 10-7 M lysozyme
pA
-40 mV
0
-100
+
10 000 ms
-50 mV
Im scaled
(pA)
0
-100
-50
-100
200 ms
-150
1000
Sensing streptavidin
C e n te r fo r R e s e a r c h a t
th e B io /N a n o In te r fa c e
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
4
2
mV
0
-1000
-500
0
-2
Au tube modified
with biotin
-4
Au tube
-6
500
1000
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.
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“!
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.
Electro-diffusion, Smoluchowski equation
c z , t 
t


z
I z, t 
J  I z, t    D
J  D
c0e

 W 0 
L
0
dze
c z , t 
z
 cLe
 W
  Ftot  z c  z , t 
 W L 
 z  V  z  
template methode
10 µm
filled with aq. CuSO4
D. Dobrev, I. Schuchert, E. Toimil, J. Vetter