Covalent modication of single glass conical nanopore channel with

6-carboxymethyl-chitosan for pH modulated ion current rectication

Li-Xiang Zhang
a
, Xiao-Hong Cao
a,b
, Yu-Bin Zheng
a
, Yao-Qun Li
a,

a
Department of Chemistry and Key Laboratory of Analytical Sciences, College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, China
b
Department of Applied Chemistry, East China Institute of Technology, Fuzhou 344000, China
a b s t r a c t a r t i c l e i n f o
Article history:
Received 6 June 2010
Received in revised form 22 June 2010
Accepted 22 June 2010
Available online 28 June 2010
Keywords:
Glass conical nanopore channel
6-carboxymethyl-chitosan
Covalent modication
Ion current rectication
In this study, a novel covalent modication method of the single glass conical nanopore channel with
amphoteric 6-carboxymethyl-chitosan (CMC) was designed to obtain a smart device responsive to a broad
range of pH stimuli. This response is highly sensitive, reversible and reproducible. The CMC modied channel
possessing carboxyl and amino groups was able to regulate ion transport selectivity and ion current
rectication properties which depend on surface charges at various pH values. Each modication step was
characterized by simply measuring the currentvoltage (IV) curves of the nanopore channel.
2010 Elsevier B.V. All rights reserved.
1. Introduction
Biological ion channels play important roles in maintaining normal
physiological conditions and serve as smart gates to ensure selective
ion transport [1]. Similar to biological voltage-gated channels,
synthetic conical nanopores possessing excess surface charge and
tip diameters comparable to the electrical double layer thickness of
electrolyte solution exhibit non-linear, rectied currentvoltage
responses [25]. Researches on synthetic nanopore with functiona-
lization have attracted broad interest due to its biomimetic features
[68]. The functional groups that can be conveniently introduced by
chemical modication on the synthetic nanopores can help tune the
ionic transport properties [9,10] or enhance and tailor their properties
for sensing [1113].
Despite the fact that many methods had been developed to
modify synthetic nanopores, previous researches concerning the
functionalization of synthetic nanopores were mainly focused on
polymer nanopores [14,15]. These modication methods utilize the
carboxyl groups that are created on the polymeric walls in the
course of the chemical etching step. In recent years, White and
coworkers reported a simple bench-top method of fabricating glass
nanopore membranes [16], which was successfully used in single
ion-channel recordings [17]. Compared with polymer nanopores,
the advantages of glass conical nanopore channel include facile and
inexpensive preparation, compactness, and mechanical robustness
[18]. Moreover, silica hydroxyl-terminated surface of the glass
conical nanopore channel is easy to form silane self-assembled
monolayers (SAMs) by silanization procedures, thus almost any
functional groups can be further introduced by covalent modica-
tion. More recently, we have reported sensing of dopamine based
on the covalent modication of the interior surface of a conical
glass micropore electrode [19].
The present work demonstrates for the rst time the integration of
the 6-carboxymethyl-chitosan (CMC) modication into single glass
conical nanopore channel to achieve a smart device responsive to pH
stimuli. CMC is a water-soluble chitosan derivative with low toxicity,
good biocompatibility and natural amphoteric properties. The
polymer backbones of CMC contains both carboxylic (COOH) and
amino (NH
2
) groups, which allow it to donate and/or accept protons
respectively in different pHenvironments [20,21]. The results showed
that the ionic transport through the CMC modied nanopore could be
well-tuned by the manipulation of the surface charges at various pH
values, which closely mimic the gating mechanisms of biological
channels.
2. Experimental section
2.1. Covalent modication of CMC
The single glass conical nanopore channels were prepared from
glass capillaries and the pore radius is determined according to the
previous literatures [16,17]. Fig. 1A shows the schematic diagram
Electrochemistry Communications 12 (2010) 12491252
Corresponding author. Tel./fax: +86 592 2185875.
E-mail address: yqlig@xmu.edu.cn (Y.-Q. Li).
1388-2481/$ see front matter 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.elecom.2010.06.031
Contents lists available at ScienceDirect
Electrochemistry Communications
j our nal homepage: www. el sevi er. com/ l ocat e/ el ecom
of the chemical modication of CMC to the channel. Firstly, the
nanopore channel was treated with piranha acid (80 C, 30 min),
followed by washing with de-ionized water to obtain clean silica
hydroxyl group on the interior surface. Then 5% 3-aminopropyl-
triethoxylsilane (APTES, 99%, Acros Organics) in absolute ethanol
was used to react with the interior pore surface for 30 min,
followed by rinsing with absolute ethanol and drying at 110 C for
30 min. Afterwards, the resulting nanopore channel was then
treated with 2.5% glutaraldehyde (25% solution in water, Acros
Organics) in phosphate buffer (10 mM, pH=7.4) for 2 h, followed
by rinsing with phosphate buffer. Finally, the aldehyde groups
terminated surface was treated with 2 mg/mL CMC (Nantong
Xingsheng Biological product Co. Ltd., China) in phosphate buffer
(10 mM, pH=7.4) solution overnight, followed by rinsing with
phosphate buffer and de-ionized water, respectively.
2.2. Apparatus and method
Glass nanopore channels were lled with electrolyte using a
100 L microsyringe. After removal of air bubbles remaining inside
the channel by a brief and mild sonication, an Ag/AgCl electrode
(0.5 mm in diameter) was inserted into the capillary (near the
channel bottom), and another similar Ag/AgCl electrode was placed
in bulk solution (near the tip). Linear sweep voltammetry
experiments were carried out on a CHI 660C electrochemical
workstation (Shanghai CHI Instrument Co. Ltd., China). The
measurements of the ion current owing through the nanopore
channel were performed by scanning the voltage from 1 V
to +1 V at a scanning rate 100 mV/s. Laser scanning confocal
uorescence experiment was performed on an inverted confocal
microscope (Leica TCS SP5) to obtain the uorescence image of a
glass conical nanopore channel (inset of Fig. 1A). The excitation
source was an argon ion laser with wavelength 488 nm. The
channel lled with 100 M Rhodamine 6G (R6G) was placed
directly over a 10 (NA 0.4) objective.
3. Results and discussion
3.1. Monitoring of the modication procedure
Each modication step was conrmed by recording the IV curves
of the pores in 1 mMKCl at pH=3 and 7.4. The chemical modication
has a profound effect on the ionic transport properties of the pores.
The rectication ratio is dened as a ratio (I (on state)/I (off state))
of the absolute value of currents recorded for voltages of the same
value but opposite polarity (1 V).
Unmodied single glass conical nanopore channel shows a
typical rectied IV curve in KCl solution (Fig. 2A). This happens
because unmodied glass surface is negatively charged due to the
dissociation of the hydroxyl groups. The IV curve is non-linear,
and the absolute value of the current recorded at negative voltage
is higher than that recorded at positive voltage, with rectication
ratio ~2.6 at pH=7.4.
After silanization by APTES, silica hydroxyl groups on the glass
surface were converted into terminal amino groups, which were
conrmed by the reversal of the rectication direction and
increase of the rectication extent (rectication ratio ~6.3 at
pH=3), as shown in Fig. 2B. The nanopore channel shows a high
conducting (on) state at positive potential while a low conduct-
ing (off) state at opposite potential polarity. The amino groups
Fig. 1. (A) Schematic diagramof the covalent modication of single glass conical nanopore channel with CMC. The inset is the confocal uorescence image of the channel. (B) Scheme
of the pH dependent surface charge transformations in the CMC modied channel.
1250 L.-X. Zhang et al. / Electrochemistry Communications 12 (2010) 12491252
on the surface are protonated, making the pore positively charged.
Afterwards, glutaraldehyde was further used to react with the
amino modied pore surface. Almost linear IV curve was recorded
in pH=7.4 electrolyte, with the rectication ratio ~1.0 (Fig. 2C).
The loss of rectication reects the presence of electrically neutral,
reactive aldehyde end groups on the surface.
3.2. CMC modied glass nanopore channel for pH modulated current
rectication
Single glass conical nanopore channel with reactive aldehyde
end group was nally cross-linked with CMC, which was aimed to
achieve a ne-tuning of the surface charges to manipulate the ion
transport through the pore. A schematic illustration of pH
dependent surface charge transformations in such CMC modied
channel is presented in Fig. 1B. Fig. 3 demonstrates the pH
dependence of ion current rectication properties of a single glass
conical nanopore channel modied with CMC. At pH=2, the
ionized amino groups (NH
3
+
) are positively charged due to
protonation, while the protonated carboxylic groups (COOH)
are neutral, rendering the pore positively charged and selective for
anionic species (Fig. 3A). The selectivity of the pore is based on the
interactions between translocating ions and the pore wall. Excess
surface charges on the pore wall lead to the preferential direction
for ion ow. On the contrary, at pH=12, reversed rectication
curve is observed and the channel shows a preferential ion ow for
cations. The deprotonated amino groups (NH
2
) are in neutral
form, while the carboxylic groups (COO

) are ionized, resulting

in the net pore charge negative. The inversion of the rectied
direction and pore selectivity measured under acidic and basic
conditions further conrm the successful modication of the
amphoteric CMC.
Compared with pH=2, at pH=3 and 4, the numbers of
protonated amino groups (NH
3
+
) decrease, leading to the less
positive surface charge and decrease of the rectication extent
(Fig. 3B). At pH=5, close to the isoelectric point of CMC
(pI ~4.9 as reported [22]), the net pore charge is almost zero,
and nearly linear IV curve is recorded (Inset of Fig. 3A).
Increasing the pH above pI promotes the formation of nega-
tively charged carboxylate groups COO

. When gradually
adjusting pH from 6 to 11 (Fig. 3B) the surface charge becomes
more and more negative on account of the growing amount of
ionized carboxylate groups (COO

), thus resulting in the

increase of rectication degree.
Fig. 3C shows the effect of pH on the absolute values of currents
recorded under voltages of the same amplitude but opposite
polarities. Currents recorded at positive voltage shows a decreasing
trend as the pH values increase, while at negative voltage, an opposite
trend is observed. At pHb5, currents for Vb0 are lower than that for
VN0 and the positively charged pore is selective to anions. While at
pHN5, currents for Vb0 are higher than that for VN0, the rectication
direction is reversed and the negatively charged pore is cation
selective. The turning point of pH is near 5, close to the pI value where
the currents recorded at two opposite voltage polarites are similar.
The variation of the current upon switching pH between 2 and 12
shown in Fig. 3D reects that the manipulating of the ion current
owing through the CMC modied nanopore channel is highly
reversible and reproducible.
4. Conclusions
We integrated the chemical modication of CMC into the glass
conical nanopore channel to control ionic transport through nano-
meter-scale opening in response to a broad range of pH stimuli,
simulating the process of ion transport in living organisms. The
response of such amphoteric CMC modied nanopore channel is
highly sensitive, reversible and reproducibly, and may suggest its
future applications in biosensors and actuators.
Acknowledgments
We are grateful for the nancial support from the National
Natural Science Foundation of China (20975084, 20575055), the
National Basic Research Program of China (973Program,
2007CB935600) and the Science and technology program of Fujian
Province (2009Y0046). We thank Professor Richard N. Zare for the
illumination in the applications of nanopores, Professor Henry S.
White and his coworkers for sharing the experience of glass
nanopore fabrication, and for their kindness in the revisions of the
manuscript. We are also grateful to Dr Jia-Hai Wang for the helpful
discussions.
Fig. 2. Monitoring of the modication procedure on a single glass conical nanopore
channel (6 nm in orice radius, with half-cone angle ~7 and pore depth ~80 m) by
recording IV curves in 1 mM KCl. (A) unmodied (B) APTES modied (C) further
reacted with glutaraldehyde.
1251 L.-X. Zhang et al. / Electrochemistry Communications 12 (2010) 12491252
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Fig. 3. pHdependence of the current recticationof a single glass conical nanopore channel (6 nminorice radius) modiedwithCMCin10 mMKCl. (A) IVcurves recordedat pH=2, 12
and 5 (the inset). (B) IV curves recorded at various pH values. (C) The absolute values of currents measured at negative (red columns) and positive voltage polarity (blue columns),
respectively. The inset describes the changes in the rectication ratio upon variation of pH. (D) Reversible variation of the current upon switching pH between 2 and 12 at +1 V.
1252 L.-X. Zhang et al. / Electrochemistry Communications 12 (2010) 12491252