Talanta 83 (2010) 42–47

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Talanta
journal homepage: www.elsevier.com/locate/talanta

A label-free electrochemical immunoassay for IgG detection based on the

electron transfer
Li-Ping Qiu a , Cun-Chang Wang b , Peng Hu a , Zai-Sheng Wu a,∗ , Guo-Li Shen a , Ru-Qin Yu a,∗
a
State Key Laboratory for Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University,
Changsha, PR China
b
Department of Chemistry, Xiangnan University, Chenzhou 423000, PR China

a r t i c l e i n f o a b s t r a c t

Article history: In this study, a highly selective, label-free electrochemical immunoassay strategy based on the charge
Received 17 June 2010 transport through the multilayer films associated with the electrocatalytic reduction of [Fe(CN)6 ]3− is
Received in revised form 19 August 2010 proposed using human immunoglobulin G (human IgG) as the model analyte. The antibody–antigen
Accepted 24 August 2010
complex formed on the sensing interface can efficiently induce change of the surface charge characteris-
Available online 29 September 2010
tics, the conductivity of multilayer film and/or electron transfer distance, resulting in an immunoreaction
signal. The current reduction is proportional to the amount of analyte. Under the optimized experimental
Keywords:
conditions, the proposed sensing strategy provides a linear dynamic range from 10 to 104 ng mL−1 and
Label-free
Electrochemistry
a detection limit of 3 ng mL−1 , indicating an improved analytical performance. This possibly makes it a
Immunoassay potential alternative in bioanalysis of proteins and other molecules.
Electron transfer © 2010 Elsevier B.V. All rights reserved.
Methylene blue
Gold nanoparticles

1. Introduction Faced with this dilemma, label-free immunoassay [13–16] has

received increasing interests, due to its intrinsic superiority. Gold
Protein, as bioorganic molecule, plays a vitally important role in nanoparticles (GNPs) [17–19] are particularly attractive for biology,
the life of human being. The determination of protein has become a due to their easy preparation, good biocompatibility, chemi-
hot topic for scientists in the era of proteomics. Thousands of analy- cal stability and great surface area-to-volume ratio. When used
ses [1–3] having been developed, among which, the antibody-based as the assembly interface, GNPs are able to increase the load-
immunoassay [4–6] attracted much attention, due to the routine ing amounts and retain the bioactivity of the biomolecules, and
tools, high selectivity and sensitivity. Enzyme linked immunosor- then improve the response performance of the biosensors. There-
bent assay (ELISA) [7,8] as the conventional immunoassay has been fore, the gold nanoparticles provide a bioactive interface for the
commonly used in clinical and environmental monitoring. How- fabrication of the third-generation biosensor without electronic
ever, it is often subject to a long assay time, high background media.
noise and complex operation. An attractive alternative is the Thionine (TH) [20–22] as a redox dye, can undergo electropoly-
immunosensor [9,10], which has many striking advantages, such merization at an appropriate potential on the gold electrode
as simple pretreatment, low cost, high sensitivity, non-essential surface. The polythionine (PTH) films not only provide a stable
separation, miniaturization, and so on. Generally, immunosensor is sensing matrix with abundant amino groups, but also improve the
divided into two categories: immunosensor with labels and label- conductivity of the interface, improving the sensitivity of the ana-
free immunosensor, most of the labeled immunosensors [11,12] lytical system.
described to date rely on the use of different enzymes attached In this study, a highly selective, label-free immunoassay based
to biomolecules that catalyze the amplification reaction. How- on the electron transfer through the multilayer films with the
ever, the activity of the prepared conjugates must be carefully unique property of gold nanoparticles was presented using human
controlled due to the intrinsic instability of enzymes, and the IgG as the model analyte. The characterization of sensing inter-
additional procedures and external reagents must be involved. face was carried out, and the optimum experimental conditions and
analytical performance were investigated in detail. The proposed
analysis scheme not only exhibited excellent response performance
∗ Corresponding author. Tel.: +86 731 88822577; fax: +86 731 88822577. but also circumvent the various problems associated with labeled
E-mail addresses: [email protected] (Z.-S. Wu), [email protected] (R.-Q. Yu). immunosensor.

0039-9140/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.talanta.2010.08.036
 L.-P. Qiu et al. / Talanta 83 (2010) 42–47 43

2. Experimental ing surface was covered with 30 ␮L of gold nanoparticles solution

and cultivated at room temperature in a water-saturated atmo-
2.1. Chemicals and apparatus sphere for 4 h. Then, 10 ␮L of antibody solution was dropped onto
the surface of the electrode and allowed to react for 1 h. To cover the
Human immunoglobulin G (human IgG), goat monoclonal anti- non-specific sites, 10 ␮L of BSA solution (10 mg mL−1 ) was dropped
human IgG antibody, and bovine serum albumin (BSA) were onto the electrode surface. After reacting for 1 h, the proposed
purchased from Dingguo Biochemical Reagents (Changsha, PR sensing interface (PTH-MB/GNP/antibody electrode) for human IgG
China). Thionine and chloroauric acid (HAuCl4 ·4H2 O) came from assay was obtained.
Sinopharm Chemical Reagents (Shanghai, China). Trisodium citrate
was obtained from Guangdong Taishan Chemicals (Guangdong, 2.3. Electrochemical measurements
China). All chemicals were of analytical-reagent grade and used
as received unless otherwise stated. The ultra-pure water (resis- The different pulse voltammograms (DPVs) were recorded from
tance > 18.2 M cm) was used throughout the experiments. 0.6 V to −0.2 V and the baseline-subtracted currents were used
Gold nanoparticles were prepared by the reduction of HAuCl4 to evaluate the response characteristics of sensing interface. The
via the help of citrate, according to the previous method [23]. The AC impedance measurements were performed in the frequency
proteins were dissolved in 10−2 mol L−1 phosphate buffered saline range from 1 Hz to 100 kHz. Cyclic voltammetry (CV) measure-
(PBS, pH 7.4) containing 0.3 mol L−1 NaCl, and stored at 4 ◦ C until ments were scanned from 0.6 V to −0.6 V at 100 mV s−1 . All the
used. electrochemical measurements were carried out in 10−2 mol L−1
Scanning electron microscopy (SEM) analysis was conducted PBS (pH 7.4) containing 0.1 mol L−1 KCl and 5 × 10−3 mol L−1
using a JSM-5600LVmicroscope (JEOL Ltd., Japan). All the K3 [Fe(CN)6 ]/K4 [Fe(CN)6 ] unless otherwise stated.
electrochemical detections were performed in a conventional
three-electrode system consisted of sensing interface, saturated 2.4. Detecting IgG
calomel reference electrode and platinum counter electrode, using
CHI 760B electrochemical workstation (Shanghai, China). The samples with different antigen concentrations were
dropped onto the as-prepared sensing interface (PTH-
2.2. Sensor fabrication MB/GNP/antibody electrode), and allowed to immunoreact
for 1 h. After rinsing thoroughly with ultra-pure water, the elec-
The gold electrode was pretreated according to the protocol trochemical measurements of the resulting electrode were carried
reported in the previous work [24]. As shown in Scheme 1, the out.
electropolymerization of the thionine monomer on the cleaned
electrode surface was carried out in two steps [20]: first, the 3. Results and discussion
gold electrode was held under a constant potential of +1.5 V for
10 min in 10−2 mol L−1 phosphate buffered saline (PBS, pH 6.0) 3.1. Sensor fabrication
containing 10−3 mol L−1 thionine monomer, 0.1 mol L−1 KCl and
4 × 10−3 mol L−1 methylene blue (MB); second, CV scanning was To fabricate the sensing interface for IgG detection, electropoly-
conducted between −0.45 V and +0.15 V at 50 mV s−1 , with 25 merization onto the gold electrode surface was conducted via
cycles in the identical solution. Subsequently, the modified sens- potential scanning in TH/MB solution. As shown in Fig. 1, a typ-
ical cyclic voltammogram during the electropolymerization is
observed. Due to the good conductivity of TH, the redox peak cur-
rent increases with the increment of scan turns, indicating the
growing process of PTH film on the gold surface, and then tends
to be stable after 25 cycles, revealing that the gold electrode is

Fig. 1. The cyclic voltammograms of electropolymerization of thionine monomer

onto a bare gold electrode in 10−2 mol L−1 PBS (pH 6.0) containing 10−3 mol L−1
thionine monomer and 0.1 mol L−1 KCl in the presence (outside) and absence (inside)
Scheme 1. Design of IgG sensing interface. of 4 × 10−3 mol L−1 MB.
44 L.-P. Qiu et al. / Talanta 83 (2010) 42–47

Fig. 2. The cyclic voltammograms of PTH film-modified electrode (a) and PTH-MB
film-modified electrode (b) in 10−2 mol L−1 PBS (pH 7.4) containing 0.1 mol L−1 KCl.

almost saturated by the PTH film and further electropolymerization

is hindered.
When adding MB to the deposition solution, the redox peak
current relatively increases. We speculate that MB as an excellent
electroactive substance improves the conductivity of the sens-
ing interfaces. As shown in Fig. 2, the redox peak current of the
PTH-MB electrode (b) is 9.967 × 10−7 A at voltage potential of
−0.334 V, which is 113.8% more sensitive than that of the PTH elec-
trode (a) (8.761 × 10−7 A) by potential scanning in 10−2 mol L−1
PBS containing 0.1 mol L−1 KCl. The resultant sensing interface is
expected to improve the response performance of our analysis
system.
The AC impedance characteristics of the modified interfaces
were investigated. As shown in Fig. 3, no detectable change in the
charge-transfer resistance (AC impedance) is obtained even after Fig. 4. The SEM images for the PTH-MB/GNP/antibody electrode before (A) and after
the formation of the electroactive polymer film (b), compared with (B) the addition of the antigen.
the bare gold electrode (a). The conductivity also almost does not
change when gold nanoparticles adsorb onto the surface of the PTH-
MB film-modified electrode via the strong covalent bond between working interface (d). This demonstrates the accomplishment of
the amino groups and gold (c). The insulated protein layer formed antibody-based biorecognition interface.
on the modified gold electrode impedes the interfacial electron To confirm that the biorecognition reaction was successfully
transfer, leading to an obvious increase in the resistance of the conducted, the morphology of the PTH-MB/GNP/antibody elec-
trode before and after the addition of antigen was characterized
using SEM. As shown in Fig. 4, the antibody molecules look like
bright particles with the general size of 1–2 ␮m (A). After reacting
with antigen, the particles grow bigger (2–3 ␮m) and look smoother
(B) due to the lower conductivity. The difference of the two inter-
faces indicates the attachment of antigens, indicating the feasibility
of our scheme.
Despite being immobilized successfully, the concentration of
antibody directly affects the signal intensity corresponding to tar-
get molecule. Thus, the effect of antibody concentration on the
current signal was investigated, and the results are shown in
Fig. 5. The original antibody solution (10 mg mL−1 ) was diluted with
10−2 mol L−1 PBS (pH 7.4) containing 0.3 mol L−1 NaCl to prepare
a series of antibody solutions. The redox peak current increases
with the increment of antibody concentration, and then reaches
the highest at 3 mg mL−1 . The current signal gradually decreases
at higher concentration. Due to the limited amount of loading
sites on the sensing surface, the higher antibody concentration
might increase the disorder in alignment of adsorbed antibody,
hampering the following immunoreaction. Therefore, the antibody
Fig. 3. The AC impedances of the sensing interfaces at different preparation stages:
bare gold electrode (a); PTH-MB film-modified electrode (b); gold nanoparticle- concentration of 3 mg mL−1 is chosen as the optimal in the present
modified electrode (c); antibody-immobilized electrode (d). experiment.
 L.-P. Qiu et al. / Talanta 83 (2010) 42–47 45

Fig. 5. Effect of the dilution ratio of antibody immobilized onto the gold
nanoparticle-modified electrode on the signal intensity. The original antibody con-
centration was 10 mg mL−1 .

3.2. Amplification of electrochemical signal

To confirm the feasibility of our present proposal, two

control sensing interfaces were prepared. The preparation of
PTH/GNP/antibody electrode (electrode B) was the same as that
of PTH-MB/GNP/antibody electrode (electrode A) described in Sec-
tion 2.2 except without MB in the electrodeposition solution;
PPD-MB/GA/antibody electrode (electrode C) chose glutaraldehyde
(GA) as cross-linker instead of gold nanoparticles in the fabri-
cation. As shown in Fig. 6, before the addition of IgG, electrode
C presents only 29% of the current response provided by elec-
trode A. After immunoadsorption of IgG on the sensing interface,
the current change of electrode C is only 9% of that provided by
electrode A. Compared with glutaraldehyde (GA), gold nanopar-
ticles show better conductivity, more excellent biocompatibility
and higher surface area-to-volume ratio. Therefore, electrode A
provides higher sensitivity than electrode C. Electrode B provides
only 57% (Fig. 6B line a) of the peak current (Fig. 6A line a) of
electrode A before the addition of IgG. On the other hand, the
current change of electrode B upon the immunoadsorption of IgG
is only 44% of that of electrode A. We speculate that MB as an
excellent electroactive substance is able to promote the interfa-
cial electron transfer and improve the electrocatalytic reduction
of ferricyanide [25], resulting in an enhanced current. In short, an
improved response performance offered by the proposed sensing
interface comes from the combination of MB and gold nanoparti-
cles.
Fig. 6. Baseline-corrected DPVs of Fe (CN)6 3− /Fe (CN)6 4− at sensing interfaces
prepared by different methods before (line a) and after (line b) the addition of
3.3. Response characteristics of electrochemical immunosensor 1 ␮g mL−1 IgG. The working electrode used was PTH-MB/GNP/antibody electrode
(A); PTH/GNP/antibody electrode (B); PTH-MB/GA/antibody electrode (C).
To assess the analytical performance of the proposed biosen-
sor, a series of samples with different antigen concentrations
were prepared and the DPVs were performed under the opti- sion equation is Ipeak = 1.252 × 10−5 log C − 4.358 × 10−6 with a
mized experimental conditions. All the DPVs in the present section correlation coefficient of 0.996 (as shown in Fig. 7B). The detec-
were the baseline-subtracted currents. As shown in Fig. 7, the tion limit (defined as three times the standard deviation of the
current change (Ipeak ) increases with the increment of IgG con- blank solution) is about 3 ng mL−1 . Such a good performance
centration within a certain range. The reason is that the redox should be attributed to the striking change of the peak current
reaction between Fe(II) and Fe(III) occurring on the electrode sur- originating from the immunoreaction between IgG and IgG anti-
face is closely related to the surface-adsorbed elements, and the body.
increase in the amount of antigen immunoadsorpted on the sens- To test the applicability and reliability of the presented strategy,
ing surface causes the decrement of the redox peak current. When the recovery experiment of samples with different concentrations
the peak current change is plotted versus the logarithm of the was carried out and the results were listed in Table 1. Recoveries
target concentration, a linear relationship in the concentration in the range of 96.4–109.0% with the relative standard deviation of
ranging from 10 to 104 ng mL−1 is achieved, and the regres- 7.4–9.1% are achieved (n = 3).
46 L.-P. Qiu et al. / Talanta 83 (2010) 42–47

Table 1
Recovery of IgG assay.

Sample Added IgG Found IgG Recovery (%) R.S.D. (%)

(ng/mL) (ng/mL)

1 10.0 10.9 109.0 9.1

2 100.0 98.5 98.5 8.2
3 1000.0 963.6 96.4 7.4

3.4. Specificity

Selectivity is important for a presented sensor, in order

to confirm that the current response was generated from
antibody–antigen specific immunoreaction rather than non-
specific adsorption, the affect of HSA (1 ␮g mL−1 ), fibronectin
(1 ␮g mL−1 ), BSA (10 mg mL−1 ) and IgE (36 ␮g mL−1 ) was investi-
gated under the same conditions. As shown in Fig. 8, compared with
IgG (1 ␮g mL−1 ), the current change induced by introduction of the
four proteins could be negligible. That is to say, the present system
reveals excellent selectivity.

4. Conclusion

A highly selective, label-free immunoassay based on the combi-

nation of the charge transport through the multilayer films and the
unique properties of MB and gold nanoparticles has been presented.
This approach can avoid various difficulties related with synthesiz-
ing the additional materials (e.g. reporters and signal amplifiers)
or controlling and preserving the biological activity of enzymes.
Given attractive performances, the proposed technique appears to
provide a promising platform for the direct immunoassay of IgG or
other biomolecules.

Acknowledgement

The work was financially supported by the National Natural Sci-

ence Foundation (Grant 20775023) of China and Scientific Research
Fund of Hunan Provincial Education Department (08A065).
Fig. 7. The current response of the sensing system for detecting IgG at different
concentrations (A); the linear relationship between the change of current response
and log of IgG concentration (B). References

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