an amperometric enzyme-linked immunosensor using resveratrol as the substrates for horseradish...

CHINESE JOURNAL OF ANALYTICAL CHEMISTRY Volume 35, Issue 12, December 2007 Online English edition of the Chinese language journal

Cite this article as: Chin J Anal Chem, 2007, 35(12), 1783–1786.

Received 29 March 2007; accepted 22 July 2007 * Corresponding author. Email: [email protected]; Tel: +86 731-2618234; Fax: +86 731-2618234 This work was supported by the Natural Science Foundation of Hunan Province of China (No. 07JJ3020) and the National Natural Science Foundation of China (No. 20775010). Copyright © 2007, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences. Published by Elsevier Limited. All rights reserved.

RESEARCH PAPER

An Amperometric Enzyme-linked Immunosensor Using Resveratrol as the Substrates for Horseradish Peroxidase for Brucella Melitensis Antibody Assay Gong Fu-Chun1,*, He De-Si2, Cao Zhong1, Tan Shu-Zhen1, Tan Ya-Fei1 1School of Chemistry and Environmental Engineering, Changsha University of Science and Technology, Changsha 410076, China 2Department of Animal Science, Hunan Biological and electromechanical Polytechnic, Changsha 410128, China

Abstract: As a natural product, resveratrol was evaluated as a potential substrate for horseradish peroxidase (HRP) and was applied to the amperometric enzyme-linked immunosensing assays. The sensors based on HRP-IgG were constructed by dispersing graphite, BrAg, and paraffin wax at room temperature. Optimal measurements of resveratrol substrate for HRP were investigated. The results of the comparison studies indicate that resveratrol is more feasible for HRP and more stable in the air than o-phenylenediamine, o-benzophenone aniline, and 3,3’,5,5’-tetramethylbenzidine. In a Britton-Robinson buffer of pH 6.8, HRP-IgG could catalyze the oxidation reaction of resveratrol by H2O2, and the reductive current of the product of resveratrol at –376 mV increases in a certain concentration of HRP-IgG, binding to the Brucella melitensis antigen-modified electrode. The linear range of the Brucella melitensis antibody determination obtained with the proposed immunosensors is 3.0 × 10–4–1.65 × 10–2 g l–1 with a detection limit of 1 × 10–4 g l–1 (3σ). The immunosensor surface could be regenerated by simply polishing with an alumina paper, with an excellent reproducibility (relative standard deviation = 4.6%). The proposed method has been successfully used for the analysis of rabbit serum samples with satisfactory results. Key Words: Resveratrol; HRP substrate; Enzyme-linked immunosensor; Brucella melitensis antibody; Modified electrode

1 Introduction

Enzyme-linked immunoassay (ELISA), introduced in early seventies in 20 century and coupling the amplification effect of enzymatic reaction with the selective antigen-antibody binding, can provide sensitivity comparable with that of radioimmunoassay. Depending on the assay format, the antigen or antibody is labeled, and an enzyme activity measurement is performed as a final step of the assay. Several enzymes, such as alkaline phosphatase, β-D-galactosidase, and horseradish peroxidase (HRP), may be used as the labels. The HRP is often applied in immunoassays and nucleic acid hybridization assays, partially because of the ready availability of peroxidase-conjugated antibodies to haptens such as biotin, fluorescein isothiocyanate and digoxigenin[1,2].

Photometric[3], fluorimetric,[4] and luminometric[5] detections are widely used in the ELISA methods. The excellent sensitivity and wide linear range typical of electrochemical detection have attracted attention in recent years, with the development of electrochemical enzyme-linked immunosensor. The key point of the ELISA methods is the use of an appropriate substrate for the HRP detection.

Hydroquinone (HQ)[6], 3,3’,5,5’-tetramethylbenzidine (TMB)[7], o-phenylenediamine (OPD)[8], and o-benzophenone aniline (OAP)[9] are the most commonly amperometric substrates used for the HRP[6–9]. The enzymatic oxidation products of OD, OPD, TMB and OAP did insufficiently sensitize their electrochemical measurements in the corresponding experiment setup. The other common drawbacks of the aforementioned substrates are their

GONG Fu-Chun et al. / Chinese Journal of Analytical Chemistry, 2007, 35(12): 1783–1786

insufficient reaction rate and relatively low stability toward H2O2 in the absence of HRP and tend to cause a strong background signals. Additionally, OPD is carcinogenic effect to human being bodies[10].

In this study, resveratrol (3’,4’,5-trihydroxystlbene, Fig.1), a natural product[11], was for the first time evaluated as an amperometric substrate for HRP-catalyzed reaction and applied to an enzyme-linked immunosensing system using Brucella melitensis antibody (BrAb) as a model analyte. The properties of resveratrol used as a potential amperometric substrate for HRP were investigated. Immunosensors were prepared by dispersion of Brucella melitensis antigens (BrAg), graphite and paraffin at low temperature. The surface of immunosensors could be renewed by polishing the used biocomposites layer, and the resulting surface serves as a platform for the immunoreaction and HRP-enzymatic reaction. In the enzymatic reaction step, HRP catalyzes the conversion of resveratrol into quinone compounds. The content of HRP-IgG attached to the aforementioned immunosensor surface is proportional to the reductive current of enzymatic products. In the use of the novel substrate resveratrol, the proposed approach allows to detect lower level of enzyme labels for BrAb assays. The proposed BrAg-graphite-paraffin based immunosensors have been applied to the determination of BrAb in rabbit serum samples with the aid of HRP-IgG.

Fig.1 Chemical structure of resveratrol

2 Experimental 2.1 Apparatus

Amperometric measurements and cyclic voltammetric experiments were performed on a CHI-660A Electrochemical Analyzer (Shanghai Chenhua Electronic Co., Shanghai, China). A three-electrode cell, using a Brucella melitensis antigen modified-immunosensors as the working electrode, a saturated calomel reference electrode (SCE) and a platinum plate auxiliary electrode, was used. A Model CSS501 thermostat (Chongqing, China) was used to control the incubation temperature. 2.2 Reagents

Horseradish peroxidase (HRP), resveratrol (3’,4’,5-

trihydroxysilbene), HQ, TMB, o-phenylenediamine (OPD), and o-benzophenone aniline (OAP) were obtained from Sigma,

USA. H2O2, bovine serum albumin (BSA), graphite, and paraffin wax were purchased from Shanghai Reagents (Shanghai, China). Brucella melitensis antibody (BrAb), Brucella melitensis antigen (BrAg), and HRP-IgG (goat anti-rabbit IgG) were obtained from Hunan Biological and electromechanical Polytechnic, China. All other reagents were of analytical reagent grade, and triply distilled water was used throughout.

Resveratrol solution: In all, 2.28 mg resveratrol were dissolved in a small volume of ethanol. To this solution, ethanol was added to bring the final volume 100 ml. This solution is diluted with a B-R buffer solution (pH 5.8). 2.3 Preparation of amperometric immunosensors

The amperometric immunosensors were prepared according

to literature [12] with slight modifications. The BrAg-BSA- modified graphite was prepared as follows: 5 mg of BrAg and an appropriate amount of BSA (16 mg) were dissolved in 1 ml of a cold B-R buffer solution (4 ºC), and the solution was mixed with 2.5 g of graphite powder. The mixture was left to dry in a desiccator at 4 ºC. The BrAg-BSA-modified graphite and the paraffin wax dissolved in THF were thoroughly mixed for a paraffin wax to carbon-weight ratio of 2:3. The resulting paste squeezed into the Polyvinyl Chloride (pvc) tube of 6 mm i.d. to a depth of 1 cm. Inside the tube, the mass was in contact with a conducting graphite rod, which was in turn connected with an electric wire to accomplish the measurement circuit. When not in use, the immunosensors were stored in a dry state at 4 ºC. The configuration of the BrAg-modified immunosensor is shown in Fig.2.

Fig.2 Configuration of BrAb-immunosensor 1, electric wire connected with a graphite rob; 2, head of PVC bolt; 3, PVC tube; 4, PVC bar with graphite rod entrapped; 5, BrAg-paraffix- graphite matrix; 6, BrAg

2.4 Renewal of the immunosensor surface

The surface of the immunosensor could be renewed by

turning the nut to extrude 0.1-mm thick outer paste layer and by polishing with an alumina paper (0.05 μm) to produce a


smooth, shiny surface. The electrode surface was finally cleaned with doubly distilled water. 2.5 Measurement procedure

The BrAg-modified immunosensors were immersed in a

2-ml B-R buffer (pH 7.2) containing analyte BrAb and were incubated for 45 min at 37 ºC. The BrAg-BrAb–modified immunosensors were rinsed thoroughly with the B-R buffer, followed by an additional incubation of 45 min in the same B-R buffer solution containing 1.0 × 10–5 M HRP-IgG. The resulting sensors with HRP-IgG (goat anti-rabbit IgG) were stored in the blocking buffer prior to the amperometric measurement.

Amperometric measurements were performed in 20 ml of B-R buffer of pH 5.8 containing 1.0 × 10–4 M H2O2. A three-electrode system was used with an applied potential of –376 mV (vs. SCE). After stabilizing the background current, the response was subsequently recorded after the addition of a 2-ml resveratrol solution.

3 Results and discussion 3.1 Properties of resveratrol

The comparison studies on the stability of resveratrol, (OPD),

(OAP), and TMB were carried out. Fig.3A shows the results obtained with the substrate solution stored at room temperature for different period followed by amperometric determination. It can be observed that the reductive current decrease rate of resveratrol was 6% after a period of 7 day-store at room temperature (curve a), similar to that of TMB (curve b). The OPD (curve c) and OAP (curve d) exhibit insufficient stability, and they are assigned are used.

The sensitivity to temperature was examined with the substrates exposing toward different temperature ranging from 25 ºC to 65 ºC (Fig.3B). The results indicate that the reductive current decrease rate of resveratrol was 8% at 65 ºC (curve e), whereas the current decrease values for OPD (curve f), OAP (curve h), and TMB (curve g) were 16%, 27% and 20%, respectively. The result implies that resveratrol is sufficiently stable to the temperature.

Fig.3 Effect of stored time and temperature on the response current of resveratrol, OPD, OAP and TMB The comparison studies on the current response

characteristics of resveratrol, TMB, OPD and OAP reacting with H2O2 and H2O2-HRP were carried out with the results shown in Table 1. It can be observed that the response of resveratrol reacting with H2O2 is more obtuse than that of TMB, OPD and OAP. The current response of resveratrol in the presence of H2O2-HRP is more significant than that of the same substrates examined. This implies that a more sensitive detection of HRP using resveratrol as a substrate can be realized due to the relatively low background signals derived from H2O2.

Table 1 Analytical results of resveratrol, OAP, OPD and TMB

reacting with H2O2 and HRP-BrAb /H2O2 Sample ∆i (H2O2) (μA) ∆i (HRP-BrAb /H2O2) (μA) Resveratrol OAP OPD TMB

1.3 11.0 15.0 9.3

45.0 31.0 35.0 40.0

3.2 Optimization of substrate concentration and Km determination

The effect of resveratrol concentration on the reductive current performance was investigated. 3.0 × 10–4 M H2O2-1.0 × 10–5 M HRP-IgG and different concentration of resveratrol were added into 2 ml B-R buffer solutions. After 2 min, the resulting solutions were subjected to amperometric measurement. The results indicate that the current response increases with the increase of the amount of resveratrol up to 5.0 × 10–4 M and then tends to saturate.

Only when the determination of enzymatic velocity is the initial velocity, the Michaelis equation can be established and a genuine Km can be detected. The Km for resveratrol-HRP-H2O2 system was measured with the initial velocity method. According to the countdown law, the fitting linear regression equation for resveratrol can be represented as: 1/v = 6.8 × 10–13

× 1/C + 4 × 10–3 (r = 0.992). Vmax and Km can be given from the regression equation with a value of 2.5 × 102 s–1 and 1.7 × 10–10 M, respectively. According to the same procedure, the Vmax for TMB, OPD and OAP is 1.8 × 102 s–1, 12.8 s–1 and 1.2 × 102 s–1, respectively. The results indicate that the reaction rate of resveratrol-HRP-H2O2 system is greater than that of the other substrates.


3.3 Optimization of experimental parameters The selection of buffer solution such as B-R, HCl-Tris,

Na2CO3-NaHCO3, NaOH-H3BO3-KCl by measuring the reductive current of the HRP-enzymatic product of resveratrol was carried out. The results indicate that a sensitive response can be obtained with B-R buffer solution, and it was used as the reaction medium in most experiments.

Both the current response of enzymatic product of resveratrol and the enzymatic reaction rate are influenced by the pH value. The effect of pH on the resveratrol-based enzymatic equilibrium solution was carried out. The results indicate that the current of products increases with the increase of pH and reaches a peak at pH 5.8 and then begins to decrease, which may be associated with the fact that pH can affect the protonation of the products.

The activity of enzyme labels attached to the immunosensor surface is affected by the pH of enzymatic reaction medium. A higher or lower pH of medium would inhibit the activity of enzyme. Additionally, the pH of the reaction medium has a strong effect on the reduction of the catalytic product on the immunoelectrode surface. The current response obtained increases with the increase in pH up to 5.8 and then decreases with a maximum response at pH 6.5.

Generally, the optimal temperature of immunoreaction and enzymatic reaction would be 37 ºC. However, at this temperature, a long incubation time would decrease the activity of the antigen, antibody, and enzyme, leading to the deterioration of the response signals and a shorter lifetime of the sensors. The results indicate that the signal increases with the increase of temperature up to 37 ºC. Therefore, an incubation and enzymatic reaction temperature of 37 ºC were used in most experiments. 3.4 Measurements with the proposed amperometric immunosensor

Cyclic voltammetric studies of substrate resveratrol and its

HRP-catalytic products were carried out at a blank paraffin–graphite electrode and BrAg-modified electrode (Fig.4). One irreversible oxidation peak in cyclic voltammograms of resveratrol appears at the potential of 155 mV (Fig.4a). The addition of H2O2 to the above solutions results in the decrease of the aforementioned oxidation peak (Fig.4b), which attributes to the oxidation of resveratrol in the presence of H2O2. A distinct reduction peak at –376 mV appears with the addition of HRP-IgG (Fig.4c), which was used as the working

potential for the BrAb determination. The immunosensors prepared were used to determine BrAb

in serum samples. A linear dependence occurred between the reductive current response and the concentration of BrAb in the initial incubation solution. The detective range for the assay extended between 3.0 × 10–4 g l–1 and 1.65 × 10–2 g l–1 with a detection limit of 1.0 × 10–4 g l–1 (S/N = 3).

Fig.4 Cyclic voltammograms obtained with a BrAg-modified

electrode In the same procedure, the immunosensing determination of

BrAb using TMB as the substrate for HRP was carried out. The pseudolinear detection range for the BrAb assay extended between 7.2 × 10–4 g l–1 and 6.5 × 10–2 g l–1 with a detection limit of 3.5 × 10–4 g l–1. This result demonstrates that the detection limit for the BrAb determination can give a marked improvement compared with those achieved with substrate TMB using the same immunoreagent.

The reproducibility of the immunosensors was tested with four regenerated biocomposite surfaces of the same immunoelectrode. A series of four measurements resulted in a relative standard deviation of 4.6%, which means that a good reproducibility can be obtained using different surfaces of a one-signal immunosensing system.

The sample-recovery experiments were carried out. The results are shown in Table 2. It indicates that the results obtained with the proposed HRP-linked immunosensing method, and a resveratrol substrate is satisfactory for the determination of BrAb. The applicability of the sensors to the determination of BrAb has been tested with serum samples of rabbit. The results obtained were compared with those obtained by a conventional ELISA method in the clinical laboratory. It indicates that the detectable concentration of BrAb using proposed immunosensing system meets the requirements of clinical analysis and can be used for the direct determination of BrAb concentration in rabbit serum.

Table 2 Results of sample recovery

Sample BrAb initially present (mg l–1) BrAb added (mg l–1) Recovered (mg l–1) Recovery (%, n = 4)

Standard BrAb 10.00

Plasma 0

1.50 3.00 1.00 5.00

1.47 2.95 0.99 5.04

98.0 98.3 99.0 100.1


3.5 HRP-catalyzed reaction mechanism

It is well known that HRP could catalyze the oxidative reaction of phenol or aminophenol compound by H2O2 to form quonine analogues, and the enzymatic methods relying on this reaction are in widely used. In the HRP-resveratrol system, resveratrol is a polyphenol compound and can be oxidized by H2O2 in the presence of HRP forming a quonine compound similar to the mechanism reported in the literatures [13,14].

4 Conclusions

Resveratrol provides an alternative electrochemical substrate

for HRP for the enzyme-linked immunoassays by directly monitoring the concentration of BrAb in serum samples. This novel substrate exhibits satisfactory physical and chemical stability, low background signal, and a relatively sensitive response to HRP. After a short incubation time, the HRP-based amperometric immunosensor using resveratrol as a substrate offers a relatively sensitive and stable response with an HRP-IgG tracer. The proposed electrochemical immunosensor could provide a simple and sensitive detection for BrAb. References [1] Li Z L, Wang S, Alice L, Robin D, Allan I R. Anal. Chim.

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an amperometric enzyme-linked immunosensor using resveratrol as the substrates for horseradish...

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