Voltage-Gating in Synthetic Nanopores Induced
by Cobalt Ions
Michael Sullivan
Undergraduate Student, Department of Electrical and Computer
Engineering
George Mason University
Zuzanna S. Siwy
Professor and Mentor, Department of Physics and Astronomy
Univserity of California, Irvine
Matthew R. Powell
Graduate Student, Department of Physics and Astronomy
Univserity of California, Irvine
Eric Kalman
Graduate Student, Department of Physics and Astronomy
Univserity of California, Irvine
1
Abstract
The topic of ion transport through synthetic nanopores has attracted the interest of
researchers from various scientific fields. Nanopores have many potential uses, such as
serving as the basis for sensors of a variety of biological molecules. Also, nanopores and
channels that exist in biological cells are fundamental to several physiological processes
found in living organisms. The purpose of this study was to investigate the transport
properties of a single nanopore in a polymer film with the presence of sub-milimolar
concentrations of cobalt ions. These nanopores were produced by the track-etching
technique whereby a polymer film (in our case polyethylene terephthtalate) is irradiated
with energetic heavy ions and the resulting tracks are etched with various chemicals to
form an asymmetric nanopore. We worked with single conical nanopores obtained by
etching one side of the irradiated films with 9 M NaOH. Once the pores were etched,
current-voltage (I-V) curves and signals of ion current over time were recorded in the
range of voltages from -1V to +1V. The results show that cobalt acetate in sub-milimolar
concentrations consistently produces characteristic ion current fluctuations and
oscillations in certain voltage ranges. These ion current fluctuations are voltagedependent, and resemble those found in biological voltage-gated channels. The result of
cobalt producing controlled current instabilities is important, because it indicates a
possible potential for single polymer nanopores to be used as sensors for different
molecules in the future.
2
Key Terms
Ion Current, Current-voltage curve, Nanopore, Fluctuations, Rectification, VoltageGating
Introduction
In recent years, there has been great interest in synthetic nanopores as an area of scientific
study [1,2]. This is partly due to the very small size of the pores, which accentuates the
influence of the pore walls on translocating ions and molecules, such that experiments
with nanopores can offer insight into physics on the nanoscale. Also, recent findings
suggest that nanopores and biological channels play fundamental roles in many
physiological processes in living organisms [3]. It is hoped that due to their structural and
behavioral similarities, the study of synthetic nanopores may expand our knowledge of
these biological channels and pores. In addition, research indicates that there may be a
potential for synthetic nanopores to be used in building single molecule sensors in the
future [e.g. 4,5]. We have shown previously that conical nanopores in a poly(ethylene
terephthalate) (PET) substrate are cation selective and rectify ion current, meaning that
cations flow more easily through the pore in the direction of the narrow opening toward
wide opening than visa versa [2,6,7]. It has also been shown that certain divalent cations,
such as calcium and manganese, induce voltage-gating in our nanopores, whereby
voltage-dependent fluctuations of ion current occur when these molecules are found in
the electrolyte solution [8,9]. In this study, we show that cobalt ions also induce voltagegating behavior in single conical nanopores in PET.
3
Methods and Materials
(A) Preparation of Nanopores.
In order to prepare the nanopores, 12 µm thick PET films are irradiated by single heavy
ions (e.g. Au, U) at the linear accelerator at the Gesellschaft fuer Schwerionenforshung,
Darmstadt, Germany [10]. After the PET films are irradiated, the tracks left by heavy
ions are developed by chemical etching into pores. Since the tracks are cylindrical in
shape, the shape and size of the pores can be tailored by applying various etching
conditions. We have worked with conical
I
nanopores for which the etching process is
U
as follows: a 9 M NaOH etchant is applied
to one side of the film in a conductivity
cell, with a 1 M KCl + 1 M HCOOH
9 M NaOH
1 M KCl + 1 M
HCOOH
stopping solution on the other side [11]. A
Fig. 1: Diagram of the Etching Process Leading
to the Preparation of Conical Nanopores.
potential difference is applied across the
film using platinum electrodes and the
Keithley
6487
The etchant (9 M NaOH) is applied to one side of
the PET film. Once it reaches the other side of the
film, the basic etchant reacts with the acidic
stopping solution (1 M KCl + 1 M HCOOH)
placed on the other side of the membrane, and
stops the etching process. A voltage (U) is applied
across the film to further retard pore enlargement
once the etchant breaks through the film. The
current across the film (I) is monitored in order to
determine the time it takes for the pore to etch.
picoammeter/voltage
source. At the beginning of the etching
process the current is zero; only after the
pore breaks through does significant
current flow through the conductivity cell.
The potential difference is applied such that the OH- ions are drawn back from the pore
4
after the breakthrough occurs and are replaced by chloride ions. Also, once the pore
breaks through, the basic etchant and acidic stopping solution neutralize in the following
reaction, stopping the etching process: HCOOH + OH-  HCOO- + H2O. This
procedure assures preparation of nanopores with very small openings of < 10 nm. The
current across the film is monitored with a piccoammeter in order to provide an indicator
of when the etching pore breaks through the PET substrate. Currents greater than
~0.15 nA are taken to indicate the formation of a newly formed pore. The etching process
generally takes anywhere from 1 to 4 hours, though the time required to etch a pore can
be highly variable even between different PET films in the same batch from the linear
accelerator. After etching, the pores are soaked in deionized water overnight to clean out
any residual etchant or stopping solution which may be trapped in the pore. It is
important to note that the chemical etching
PET Film
of PET in NaOH causes the formation of
COOH groups on the pore walls, which
makes the surface of the pore negatively
charged.
Working
Electrode
Ground
(B) Recording Ion Current Through a
Fig. 2 The Conductivity Cell Used for Ion Current
Measurements.
Single PET Conical Nanopore
The PET foil sits between two chambers of the
conductivity cell. The chambers are tightly pressed
together, which assures a seal resistance larger
than 100G. The entire system is placed inside of
a Faraday cage, and grounded. Electrodes are
placed on either side of the foil in order to apply a
potential difference and to measure the resulting
current through the pore.
In order to study the transport properties of
the nanopores, we record signals of ion
current in time as well as current-voltage
5
(I-V) characteristics. An I-V curve shows how the average current through the pore
changes with the applied voltage. It is important to note that the current that we measure
is an ionic current due to the movement of ions in a water solution under an applied
electric field. The procedure for collecting these measurements is as follows: the two
compartments of the conductivity cell shown in Fig. 1 are filled with KCl and platinum
electrodes are exchanged with Ag/AgCl electrodes. The grounded electrode is placed on
the small side of the nanopore. The potential difference between the electrodes is given to
be Vb  Vt , where Vb and Vt are the potentials of the electrodes on the big side and the tip
side
of
the
pore,
respectively.
In
this
is
written
as
i  g Vb  Vt  with g  0 .
Thus,
a
case,
Ohm’s
law
positive value of i means that cations
flow from base to tip. The conductivity
cell is placed within a Faraday cage and
then grounded, in order to minimize the
amount
of
noise
present
in
our
calculations. This is very important,
Fig. 3: The Data Acquisition Setup
because we measure currents as small
The Faraday cage containing the conductivity
cell, and the Axopatch 200B amplifier with the
Digidata 1322A analog-to-digital converter
(Molecular Devices, Inc) used to apply a
potential difference across the PET film and
record the resulting current through the pore.
as picoamperes (10-12 A). Recordings
of ion current over time are performed
with the Axopatch 200B and digitized
with the Digidata 1322A (Molecular Devices, Inc.). The signals are viewed on the
computer with the Clampex 9.2 program (Molecular Devices, Inc.). While conducting
this experiment, we would typically record 2 minute long series with 10 kHz sampling
6
frequency, filtered with a Bessel filter at 2 kHz. Time series of ion current were typically
recorded in the voltage range between <-1 V, +1V>.
The measurement of ion current through the nanopores over time gives us information on
their transient transport properties. In order to study their average transport properties as a
response to voltages from a given range, we looked at current-voltage curves of the data.
There are currently two ways that we record I-V curves for our nanopores. The first is to
take a collection of time series measurements throughout the range we study (usually -1V
to +1V), and to find the mean value of the ~120,000 samples typically taken per time
series, as well as the standard deviation of the distribution of currents over that time
range. The statistics on time series are found using the Clampfit 9.2 software package
(Molecular Devices, Inc.). The data is then displayed as a graph, with mean currents
corresponding to the current data, and the standard deviations corresponding to error bars
which show the variability of the data at any given point.
The second method to find I-V characteristics of the nanopores is to use a Keithley 6487
picoammeter/voltage source to measure the current over a varying voltage range, and
report the information as a set of current-voltage pairs. Readings are normally taken as
the mean current over a 2 second time period, starting from the bottom voltage, with a
given positive voltage step size. This method is quicker to compute than manually
averaging a set of time series data, but it does not report any information about the
variability of the readings. An example of an I-V curve generated via this method is given
in Figure 4. The ion current rectification of the conical pores in the PET nanopore can be
7
seen in this Figure, as negative applied voltages induce a significantly greater current
through the pore than their positive counterparts.
I-V Curve for KCl
1000
500
Current (pA)
0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-500
-1000
-1500
-2000
-2500
Voltage (V)
Fig. 4 Current-Voltage Curve of a Single Conical Nanopore with Opening Diameters d≈6nm,
D≈960nm
The I-V Curve was recorded from a 0.1 M solution of potassium chloride buffered to pH 8. Ion
current rectification can be seen, as the magnitude of currents at negative applied voltages is
larger than those at the corresponding positive voltages.
(C) Sizing the Nanopores.
Once the pores have been etched and cleaned, the size of the large diameter (D) and small
diameter (d) of the pore are found via the following process. The big opening D (in nm)
is calculated from the following formula using the so-called bulk etch rate which equals
2.13 nm/min at room temperature [11]:
 nM 
D (nm) = 2.13 
  2  Time to Etch (min)
 min 
(1)
The small opening can be calculated given that the resistance of a conical nanopore is
related to its geometry by:
8
R (Ω) =
4L
   d  D
 d (m) =
4L
   R  D
(2)
where R is the resistance of the pore, L is the thickness of the PET film, and  is the
specific conductivity of electrolyte that fills the nanopore. The length, L, of our samples
is 12 µm, and the specific conductivity of 1 M KCl,  , is equal to 10
S
.
m
The resistance of the nanopore can be found easily from conductivity measurements [11].
In order to do that, the nanopore is
Voltage vs. Ion Current
placed in a conductivity cell, and
current readings in a 1 M KCl
taken from -0.1 V to 0.1 V in
0.005 V
increments
using
Current (A)
solution (averaged over 2 s) are
0.000000001
R=V/I
5E-10
0
-0.15
the
-0.1
-0.05
0
0.05
0.1
0.15
Voltage (V)
Keithley 6487 picoammeter/voltage
source. The resulting I-V curve is
approximately linear in this voltage
range, and thus can used to calculate
R using Ohm’s Law. An example of
Fig. 5: Calculating the Resistivity of a Nanopore with
Opening Diameters d≈6nm, D≈960nm
The resistance of a nanopore can be closely approximated
by applying Ohm’s Law to the nearly-linear region of
current induced by an applied voltage between -0.1V and
0.1V.
this recording is shown in Fig. 5.
Typically, nanopores etched by the track etching process have a smaller opening <10 nm,
and a large opening <1 µm.
9
Results and Discussion
Figure 4 shows that single conically shaped nanopores in PET rectify ion current. It was
found in our previous studies that the pores are cation selective; therefore we can treat the
signal of ionic current as cation (in our case potassium ion) current [6,12]. The
preferential direction of potassium ion flow is from the small opening towards the big
opening of a conical nanopore.
Our recent studies also show that new, interesting transport properties are observed in the
nanopores when one introduces sub-milimolar concentrations of calcium or manganese
ions to the electrolyte when it is buffered to pH 8 [8,9]. Pore blockage and voltagedependent ion current fluctuations were found to be an effect of these two divalent
cations (at this pH value) on the transport properties of the nanopores. Buffering the
solution to a pH of 8 assures deprotonation of carboxyl groups that are present in PET
nanopores, without causing any further etching of the substrate material. This leaves the
film with an excess negative charge on the pore walls. Here, we show response of the
PET nanopores to micromolar and sub-micromolar concentrations of cobalt ions. Figure
6, below, shows the behavior of a nanopore in a KCl solution both with and without the
addition of 0.1 mM of cobalt ions. As shown in the Figure, ion currents recorded for
positive voltages, and for small values of negative voltages, do not respond to the
presence of cobalt ions--ion currents before and after adding the cobalt are essentially the
same in these voltage ranges. Only currents recorded for more negative voltages started
to ‘see’ cobalt. Qualitatively, the effects of adding cobalt to the solution are as follows.
10
Following the addition of cobalt ions to the electrolytic solution, the values of the current
through the nanopore become much smaller than they were prior to the presence of the
Co2+ ions. Additionally, at some negative voltage (~ -600 mV in Figure 6), the current
starts to fluctuate between two states: one close to 0 pA, and the other one several
hundreds of pA. This switching pattern clearly depends on the applied voltage across the
film, which is observed as ion current oscillations in certain voltage ranges. The shape of
these oscillations typically manifests itself as a blocked pore, with open states looking
like a sharp downward spike in current often followed by a short secondary open state
where the pore is partially blocked, followed by a closed state to complete the cycle.
Fluctuations tend to be more drastic at more negative voltages, and have larger
amplitudes as the voltage decreases.
The frequency of oscillations of the current through the pore under the influence of cobalt
ions is much lower than that seen with calcium ions in previous studies. However,
depending on the size of the pore, the timescale of fluctuations with cobalt changes, as
can be seen by comparing Figure 6 to Figure 7. There are quantitative differences in the
timescale and amplitude of ion current fluctuations with the same solution in different
pores, perhaps owing to the size differences between every pore. However, the
characteristic form of the cobalt fluctuations is preserved.
11
Voltage (V)
0.1 M KCl + 0.1 mM Co2+
0.1 M KCl
+500 mV
-100 mV
-600 mV
-1000 mV
Figure 6: Voltage-Gating in Cobalt Ion Current Time Series through a Single Conical Nanopore with
Opening Diameters d≈3nm, D≈270nm
The time series to the left show ion current recordings in a KCl solution, pH 8. Under the influence of 0.1
mM of cobalt ions, the system is driven out of equilibrium and oscillates in certain voltage ranges. It can
be seen from the -600 mV and -1000 mV readings that the system opens and closes in a manner
dependent on the applied voltage.
12
Voltage (V)
0.1 M KCl + 0.1 mM Co2+
0.1 M KCl
+500 mV
-100 mV
-600 mV
-1000 mV
Fig. 7 Voltage-Gating in Cobalt Ion Current Time Series through a Single Conical Nanopore with
Opening Diameters d≈2.8nm, D≈360nm
It can be seen from comparisons between Figures 6 and 7 that different size pores react differently to the
same concentration of cobalt ions. Although the I-t curves are quantitatively different in the same voltage
ranges for the two pores, qualitatively the same characteristic shape of the current response is seen in
both figures.
13
In addition to being voltage dependent, the response of our system to cobalt ions is also
concentration dependent. Typically, as the concentration increases, the system starts
fluctuating at a less negative voltage. These findings can be seen in Figure 8, which
shows the I-V curves of KCl without the addition of cobalt ions, and also with six
different concentrations of cobalt in solution. The Figure shows that sub-milimolar
concentrations of cobalt can change the behavior of the pore. Already at 30 µM, the
cobalt causes a change in the I-V curve of this conical nanopore, which corresponds to a
closing of the pore (judging from the mean values), and a notable increase in the
variability of the readings (which can be seen from the increase in the size of the error
bars). For this particular pore at a cobalt concentration of 30 µM, we see voltage-gating
in the range of voltages between -600mV and -1000mV. As the concentration of cobalt
ions in solution increases, we see current fluctuations in a wider voltage range—for 0.1
mM Co2+, the response was seen for voltages ≤ -400 mV, while for 0.3 mM Co2+ the
response was seen already at voltages of -100 mV and below. We also see the pore
become more and more blocked as the concentration of cobalt ions increases, where the
mean tends to shrink towards zero, and the variability shrinks, indicating a majority of
time being spent in the closed state.
14
200
Current (pA)
100
0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
-100
-200
0.1 M KCl
1 uM Co
3 uM Co
10 uM Co
30 uM Co
0.1 mM Co
0.3 mM Co
-300
-400
Voltage (V)
Fig. 8 I-V Curves for a Single Conical Nanopore with Opening Diameters d≈3nm, D≈270nm
The electrolyte solution for this I-V curve was 0.1 M KCl with the addition of various cobalt
concentrations as indicated in the Figure. The solutions were buffered to pH 8.
In order to investigate the origin of the response of our nanopores to Co2+, we checked
how surface charge of the pore influences these ion current instabilities. No response to
the presence of cobalt ions was seen in solutions with pH below 8, as demonstrated by
Figure 9. It is thought that this is because at higher concentrations of H+ ions, more
COOH groups on the pore walls are protonated, which causes the net surface charge to
become less negative and affects the mechanism of operation for the current fluctuations.
15
KCl @ pH 8 (no cobalt)
Co @ pH 8
Co @ pH 7
Figure 9: A Comparison of a Nanopore Behavior at Cobalt Solutions Buffered to Different pH Values
(Pore Opening Diameters d≈3nm, D≈270nm)
Cobalt causes fluctuations in the ionic current through the pore at certain voltage ranges when buffered
to a pH of 8. At lower pH levels, this behavior disappears, and the current through the pore becomes
essentially identical to that without the cobalt ions in solution.
Conclusion
We introduce a system with single conically shaped nanopores in a PET film which
produces voltage-gating behavior in the presence of sub-milimolar concentrations of
cobalt ions in solution. These current instabilities are seen only in certain voltage ranges,
as well as only in certain concentrations of cobalt. Additionally, buffering the pH of the
electrolyte to any pH less than 8 eliminates the current instabilities seen at higher
acidities. Future efforts will focus on the modeling and quantitative description of ion
current fluctuations in nanopores induced by the presence of cobalt ions.
Acknowledgments
Irradiation
of
polymer
foils
was
performed
at
the
Gesellschaft
fuer
Schwerionenforschung, Darmstadt, Germany. The support of the University of Irvine,
California (UCI), the UCI Undergraduate Research Opportunities Program (UROP) and
the National Science Foundation (NSF) is greatly acknowledged. The direction of Dr.
Zuzanna S. Siwy was fundamental for the entire experience, and cannot be overstated. In
16
addition, MS is very grateful to Matthew Powell and Eric Kalman for many discussions
and guidance.
References
1. H. Bayley, C.R. Martin, Chem. Rev. 2000, 100, 2575.
2. Z. Siwy, Adv. Funct. Mat. 2006, 16¸735.
3. B. Hille, Ionic Channels of Excitable Membranes, 2nd ed., Sinauer Association,
Sunderland, MA 1992.
4. J. J. Kasianowicz, E. Brandin, D. Branton, D. W. Deamer, Proc. Natl. Acad. Sci. USA.
1996, 93, 13770.
5.J. Li, D. Stein, C. McMullan, D. Branton, M. J. Aziz, J. A. Golovchenko, Nature 2001,
412, 166.
6. Z. Siwy, A. Fulinski, Phys. Rev. Lett. 2002, 89, 198103.
7. Z. Siwy, A. Fulinski, Am. J. Phys. 2004, 72, 567.
8. Z. Siwy, M.R. Powell, E. Kalman, R.D. Astumian, R.S. Eisenberg, Nano Lett. 2006, 6,
473.
9. Z. Siwy, M.R. Powell, Al Petrov, E. Kalman, C. Trautmann, R.S. Eisenberg, Nano
Lett. 2006, 6, 1729.
10. R. Spohr, German Patent DE 2 951 376 C2, 1983.
11. P. Apel, Y. E. Korchev, Z. Siwy, R. Spohr, M. Yoshida, Nucl. Instrum. Methods
Phys. Res., Sect. B 2001, 184, 337.
12. Z. Siwy, P. Apel, D. Baur, D. Dobrev, Y. E. Korchev, R. Neumann, R. Spohr, C.
Trautmann, K. Voss, Surf. Sci. 2003, 532-535, 1061.
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