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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
A Sensitive Potentiometric Immunosensor Based on
Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
1
Liu Yuanyuan, 2 Feng Chunliang
School of Chemistry and Chemical Engineering, Inner Mongolia University of Science and
Technology, Baotou, China, [email protected]
2
Faculty of Chemistry and Chemical Engineering, Institute of Chemistry for Functionalized
Materials, Liaoning Normal University, Dalian, China, [email protected]
1
Abstract
A potentiometric immunosensor with the immobilization procedure of antibody (mouse anti-IgG) on
nano-Au/Cys/nafion-modified platinum disk electrode has been investigated through nanotechnology,
nafion membrane and self-assembled technology. The immunosensor obtained by the immobilization
procedure showed lower detection limit of response to antigen (mouse IgG) compared to the current
methods, the detection limit is as low as 2×10-4 ng/mL with a wider linear range from 2×10-4 to 1
ng/mL. Otherwise, the cyclic voltammetry the optimal conditions for immunosensor’s measurement
were also discussed in this article.
Keywords: Potentiometric Immunosensor, Nafion, Colloidal Au
1. Introduction
Potentiometric immunosensors have attracted growing attention with high sensitivity, label-free,
small size, and ease in use. The immobilization of biomolecules or the design technology of sensing
interface is one of the key factors for immosensors [1]. Generally, direct immobilization of proteins
onto bare metal surfaces (such as platinum, gold and silver) can frequently result in their denaturation
and bioactivity loss of antibody or antigen. Many new immobilized means [2-8] for proteins (such as
enzyme, antibody and antigen) have reported in recent years, and the most popular method for
immobilization built up through nano-technology [9-12]. Colloidal particles have a number of
attractive properties for the purpose of surface nano-fabrication. Their ability of self-organizing can be
utilized to form ordered structures, and the parallel nature of the assembly process makes it possible to
produce large numbers of nano-sized features and/or cover large surface areas. High biological
compatibility of nanoparticles was another advantage for protein immobilization.
Nafion, highly stable membrane, has found many fields of application including dialysis, osmosis,
and electrochemical reactors due to its chemical, thermal and mechanical properties [13-16]. Recently,
some papers have reported on application of nafion in amperometric sensors for analytes [17-19] to
enhance the stability of protein film. Otherwise, the self-assembled technology [20] was a usual
method to immobilize immunoaffinity on the metal surface of transducers and the self-assembled
monolayer of thiolate compounds has been widely used.
In this paper, a potentiometric immunosensor was constructed by immobilizing antibody (mouse
anti-IgG) on a nano-Au/cysteine/nafion modified platinum disk electrode through nafion membrane,
L-cysteine and Au colloids by electrostatic interactions. The successful immobilization procedure was
proved by Cyclic voltammograms. The results of immunosensor showed lower detection limit of
response to antigen (mouse IgG) compared with the current methods, the detection limit is as low as
2×10-4 ng/mL with a wider linear range from 2×10-4 to 1 ng/mL. The optimal work environments of
immunosensor were also discussed in this paper.
2. Experimental
2.1 Apparatus and Reagent
Goat-anti-mouse IgG (anti-IgG, 14 mg/ml) and mouse IgG (IgG, 27 mg/ml), 4-mercapto benzoic acid
(4-MBA) was purchased were purchased from Yingjun Bioengineering Company (shanghai, China).
International Journal of Digital Content Technology and its Applications(JDCTA)
Volume7,Number7,April 2013
doi:10.4156/jdcta.vol7.issue7.67
569
A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
Gold chloride was purchased from Shenyang Reagent Factory (China). All other reagents were
prepared with chemicals of analytical grade.
Electrochemical workstation (CHI660D) was used to measure the potential change of the
immunosensor before and after the antigen-antibody immunoreaction and test cyclic voltammetric
characteristics in ferricyanide phosphate buffer solution. A three-compartment electrochemical cell
contained a platinum auxiliary electrode, a saturated calomel reference electrode (SCE) and a modified
platinum disk electrode as working electrode. Ultrapure water (18.3MΩ) produced by a Human LIP900
system (Human, Korea) was used throughout the experiment. The size of gold nanoparticles was
estimated from transmission electron microscopy (H600, Hitachi Instrument Co., Japan).
2.2 Preparation of colloidal Au solution
The 20 nm Au colloid was prepared according to the literature [18]. Solution A: 1mL of 1% HAuCl4
solution was added to 99mL Ultrapure water. Solution B: 4mL of 1% trisodium citrate solution.
Solution A was heated up to boiling as stirring, then solution B was added to solution A immediately.
The mixture was heated for 30 min sequentially. The solution color was claret. Gold nanoparticle was
investigated by UV-visible spectroscopy (Fig. 1) and the size of prepared gold nanoparticles was
confirmed by transmission electron microscopy (see Fig. 2).
100nm
Figure 1. UV-visible spectroscopy of
Figure 2. The TEM images of Au
gold nanoparticles.
colloid particles. The size of the Au
colloid particle is from 15 to 20 nm.
2.3 Immunosensor modification
Scheme.1 showed the assembled steps of the immunosensor. The platinum disk working electrode
was first immersed in piranha solution (H2O2: H2SO4 1:3) for 15 min, then ultrasonicated in nitric acid
(1:1), ethanol and water for 10 min, respectively. The cleaned platinum disk electrode was immerged in
the nafion methanol solution (v/v, 2%) for 5 min at room temperature and then removed to parch with
an infrared light for 20 min. The resulting electrode was thoroughly rinsed with water. Following that,
the nafion-modified platinum disk electrode was saturated in L-cysteine (Cys, 20 mM in ultrapure
water) and Au colloids solution for 24 h, respectively. Finally, the Au colloid-modified electrode was
incubated in anti-IgG solution (20 ng/mL in 0.01M acetate buffer solution, pH 5.5) at 30 ℃ for 2 h.
The immusosensor was prepared for use.
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
Scheme.1 Immunosensor modification
2.4 Potentiometric response of immunosensor
Antibody and antigen have ability for the selective molecular recognition of a target analyte from a
complex mixture, and the antigen-antibody interaction has been widely used as the molecular
recognition part of immunosensor due to its highly specific interaction.
The detection is based on the change in the potentiometric response before and after
antigen-antibody reaction. The potentiometric response of the immunosensor towards antigen-antibody
is evaluated as the equation:
E  E 2  E1
Where E1 is the value of steady-state potentiometric response (versus Ag/AgCl electrode) in a buffer
solution before the antigen-antibody reaction, and E2 represents the value of the steady-state
potentiometric response (versus Ag/AgCl electrode) after antigen-antibody reaction under the same
conditions.
In this study, the modified immunosensor was tested as above statements.
3. Results and discussion
3.1 Cyclic voltammetry characteristics of immunosensor
The cyclic voltammetry of ferricyanide is a valuable and convenient tool to monitor the barrier of the
modified electrode. Therefore, it was chosen as a marker to investigate the changes of the electrode
behavior after each assembly step.
Figure 3 shows cyclic voltammograms (CVs) of differently modified electrodes in a 10mM
ferricyanide phosphate buffer solution. Well-defined CVs, characteristic of a diffusion-limited redox
process, are observed at the bare platinum disk electrode (Fig. 3a.). After dipping the electrode in the
nafion solution, the redox peak disappeared (Fig. 3e.). The reason is that the nafion can act as an inert
electron and mass transfer blocking layer, and it hinders the diffusion of ferricyanide toward the
electrode surface. When cysteine was chemisorbed on the electrode, a remarkable current increase was
observed (Fig. 3c.) due to the fact that cysteine acts as a so-called promoter in the electron transfer
procedure. After gold nanoparticles were self-assembled on the cysteine-modified electrode, the current
remarkably increased (Fig. 3b.), for the nanometer-sized Au colloid immobilized on the surface acts
like an electron-conducting tunnel, making it easier for electron transfer to take place. When anti-IgG
molecules were adsorbed on the electrode surface, a remarkable current decrease was observed (Fig.
3d.) because the anti-IgG biomacromolecules hinders the diffusion of ferricyanide toward the electrode
surface again. CV results confirmed that nafion, Au colloids and anti-IgG were successfully assembled
on the platinum disk surface.
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
Figure 3.
Cyclic voltammograms of (a) a bare platinum disk electrode, (b)
nano-Au/Cys/nafion-modified platinum disk electrode, (c) Cys/nafion-modified platinum disk
electrode, (d) anti-IgG/nano-Au/Cys/nafion-modified platinum disk electrode, (e) nafion-modified
platinum disk electrode in 10mM PBS (pH=7.4)+ 0.4g NaCl+0.01g KCl+10mM Fe(CN)64-/3solution at 50 mVs−1.
3.2 Detection for Antigen
The potentiometric immunosensor has been studied for the detection of antigen-antibody interaction.
By injection of antigen (mouse IgG), the response with antibody (mouse anti-IgG)can test by potential
signal.
Fig. 4 indicates the potentiometric response of anti-IgG/nano-Au/Cys/nafion-modified platinum disk
electrode. The electrode exhibits the linear dependence on the logarithm of IgG and potential changes.
The linear regression equation was ΔE= 4.5331 [IgG] + 17.085 with a correlation coefficient of 0.9947
and the concentration of IgG is from 2×10-4 to 1 ng/mL with a detection limit of 2×10-4 ng/mL. The
detection limit of this method is lower at least one quantitative grade than the other current
immunosensors.
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
Figure 4.
Calibration curves for the anti-IgG/nano-Au/Cys/nafion-modified
immunosensor in phosphate buffer solution (0.01 M, pH=7.4) in the presence of
different concentrations of antigen.
3.3 Optimization of experimental conditions and characteristics of immunosensor
3.3.1 pH for immunoreaction
The effect of pH is important to the immunosensor behavior, for antigen and antibody are proteins
that have their own isoelectric point and colloid Au also has its own pH restriction. pH was studied
from 5.0 to 8.5 in PBS (Fig.5). The immunosensor potentiomtric response increases with increasing pH
value from 5.0 to 7.0 and decreases as the pH increases further. The experimental results showed that
the maximum response occurs at pH 7.0 where most immunoreactions exhibit optimal binding.
Therefore, a pH 7.0 of PBS was used as the medium for the immunoreaction.
Figure 5. The pH effect to immunoreaction
Figure 6. The effect of the nano-Au
size to antibody immobilization
3.3.2 The size of Au colloids for immobilization
The size of the Au nanoparticles plays an important role in the immunosensor behavior. Doron et al.
[21] confirmed that the larger sized Au colloids can discontinuously assemble and smaller sized Au
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
colloids may generate continuous arrays of particles on the base monolayer. However, the very smaller
gold nanoparticles, the stronger coulomb repulsion effect among charges. The reason is that the gold
nanoparticles are negatively charged species as a result of the adsorption of citrate ions in the
immobilization process [22]. Fig.6 Showed the potentiometric responses of the immunosensor with
different size of colloidal gold among 10, 15, 20, 30 and 40 nm for original immobilization on panar
gold electrode, the result showed the immunosensor fabricated with about 20 nm gold nanoparticles
exhibits a larger response than other sizes, therefore, 20 nm gold nanoparticles was chosen as the
immobilized matrix.
3.3.3 Temperature for immunoreaction
Temperature is vital to the immobilization of antibody because antibody will lose its bioactivity on
high temperature value and inhibited its bioactivity on low temperature. The effect of temperature were
studied from 15 ℃ to 40 ℃ . Fig.7 showed the potentiometric response characteristic of the
immunosensor at different immobilization temperature, results showed at 30℃ the immobilization
exhibit optimal binding. Then , 30℃ was chosen for antibody immobilization through the whole
experiment.
Figure 7.
The temperature effect
on antibody immobilization
Figure 8.
Response time of antigen at the concentration
of 6ng/mL
3.3.4 Response time of immunosensor
Time is a standard for discerning the capability of immunosensor. One of the original aims of a good
immunosensor is detecting samples fleetly. Fig.8 is the potentiometric response time of a certain
concentration of antigen. After 10 minutes, the potentiometric response has almost finished. This result
can prove the immunoreaction on the novel immunosensor can be detected in a short time because of
high electric characteristics of gold nanoparticles.
4. The preliminary detection of actual samples
Blood of mouse (3months old, Dalian, China) was mixed with trisodium citrate solution (0.1mg/mL)
according to the ratio 1:0.9 after the mouse blood was gained, the obtained blood samples were then
placed in refrigerator for 24h at 4℃. The solution was finally divided into two layers automatically and
the upper layer, blood serum, was needed in detecting IgG during the experiments.
The samples were detected by the resulting immunosensor for five times. The parallel operating
steps were shown as followed: 2μL blood serum was added into the buffer solution after the freshly
fabricated immunosensor which was steady in the solution. And the potential will steady again
following the immunoreaction. The potentiometric response (the potential change ΔE) was then
obtained. The results which were calculated in the same equation: ΔE= 4.5331 [IgG] + 17.085 were
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
listed in Table 1. It was proved that the resulting immunosensor can detect IgG in mouse blood serum
and the results were centralized in certain range. The primary results showed the resulting
immunosensor could supply a feasible method for detection.
Table1. The results of IgG in actual mouse
bl d
Added volume Potential changΔE
Results
(mg/mL)
(μL)
(mV)
2
17.264
0.0394
2
17.226
0.0310
2
17.233
0.0326
2
17.219
0.0295
2
17.251
0.0366
RSD (%)
12.09
5. Method Validation
In this section, horseradish peroxidase were immobilized through the same method and satisfied
results which were shown in Fig.9. Fig. 9 indicated the potentiometric response of horseradish
peroxidase/nano-Au/Cys/nafion-modified platinum disk electrode. The electrode exhibited the linear
dependence on the logarithm of H2O2 and potential changes. The linear regression equation was
ΔE=23.46 [H2O2] + 11.182 with a correlation coefficient of 0.9921 and the concentration of H2O2 was
from 2×10-1 to 10 ng/mL with a detection limit of 2×10-1 ng/mL.
Figure 4.
Calibration curves for the peroxidase/nano-Au/Cys/nafion-modified
immunosensor in phosphate buffer solution (0.01 M, pH=7.4) in the presence of
different concentrations of antigen.
There was an interesting result that the potentiometric response of horseradish peroxidase was higher
than anti-IgG, the mainly reason was that the horseradish peroxidase had better conductive ability than
anti-IgG though these two moleculars were both large proteins. However, the sensitivity and the
detection limit of anti-IgG were higher than horseradish peroxidase.
6. Conclusion
A potentiometric immunosensor based on nano-Au/Cys/nafion-modified platinum disk electrode is
confirmed. The simple manipulations of construction of modified electrode, label-free, stable and
improved detection limit are main features of proposed immunosensor. And the results of optimal
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A Sensitive Potentiometric Immunosensor Based on Nano-gold/cysteine/nafion-modified Platinum Disk Electrode
Liu Yuanyuan, Feng Chunliang
measurements showed the immunosensor can work under mild conditions. The further studies are in
progress. The method is a good means to immobilize the large proteins in trace analysis and can
hopefully contribute to significance on practical application on medical testing and signal processing in
future.
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