Flow injection electrochemical enzyme immunoassay based on the use of an immunoelectrode strip integrate immunosorbent layer and a screen-printed carbon electrode

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Abstract

A flow injection amperometric immunoassay system based on the use of screen-printed carbon electrode for the detection of mouse IgG was developed. An immunoelectrode strip, on which an immunosorbent layer and screen-printed carbon electrode were integrated, and a proposed flow cell have been fabricated. The characterization of the flow immunoassay system and parameters affecting the performance of the immunoassay system were studied and optimized. Amperometric detection at 0.0 V (versus Ag/AgCl) resulted in a linear detection range of 30–700 ng ml−1, with a detection limit of 3 ng ml−1. The signal variation among electrode strips prepared from variant batch did not exceed 8.5% (n=7) by measuring 0.5 μg ml−1 antigen standard solution.

Introduction

Enzyme immunoassay (EIA) has been widely applied in bioanalytical chemistry owing to its high sensitivity and specificity, availability of many enzyme markers, long-term stability of the labeled reagents and operational safety [1], [2]. With the extension of the applications of EIA, including electrochemical enzyme immunoassay, there has been growing interests in the development of flow injection EIA system due to a few important advantages of flow injection analysis (FIA) [3], [4], [5]. Enzyme immunoassay coupling with a flow injection system and an amperometric detection has become a powerful analytical tool for the determination of low levels of analytes such as atrazine [6], bacteria [7], cortisol [8], cephalexin [9], gentamicin [10], human chorionic gonadotrophin [11] and progesterone [12] in different biological fluids.

As some special designs, Ghindilid et al. presented a kind of flow-through immunosensor based on a high-surface-area carbon immunoelectrode. Dispersed carbon material serves as a carrier for immobilized antibodies and at same time as an electrode material [13]. The same group have developed a flow injection amperometric immunofiltration assay system. The system was based on the use of disposable porous nylon membranes, which acted as a support for the immobilization of antibody, and an amperometric detector [7]. Liu et al. [14] proposed a flow injection solid-phase chemiluminescent immunoassay using a membrane-based reactor. The membrane to which antigen was attached was mounted in a flow cell and could be replaced after each measurement. In their system, a disposable immunosensor containing an immobilized antibody was used on-line with an FIA system. The system was capable of continuously carrying out each of the steps involved in solid-phase sandwich immunoassays, including the immune reaction, the washing, the sandwich reaction, and the enzymatic reaction.

One of the advantages of screen-printing as a means of sensor production is its ability to produce sensor sufficiently cheaply and reproducibly to allow them to be used for a few or single determination in batch analysis [15]. There have been a lot of reports about enzyme biosensor or immunosensor using screen-printed electrode [16], [17], [18], [19], some of these works involved a flow injection system. A study involving flow injection analysis of organophosphate pesticides incorporated a cobalt phthalocyanine modified screen-printed carbon electrode into a thin-layer cell, demonstrated that an on-line approach using screen-printed electrodes would be feasible [20]. Collier et al. [21] presented a special design of screen-printed biosensor and corresponding wall-jet flow cell. The work reported by Killard et al. [22] has indicated the possibility of using a screen-printed carbon electrode-based immunosensor in an amperometric flow cell. And then, the reports about planar working electrodes employed in flow immunoassay systems have been published, including gold film electrodes [23] and the screen-printed electrodes [12], [24].

In the present work, an electrochemical flow immunoassay system based on the screen-printed electrode-based immunoelectrode strip was presented. A proposed immunoelectrode strip and flow cell with a three-electrode configuration were used for the immunoreaction steps and the electrochemical detection. The optimization of determination condition using mouse IgG as a model analyte was presented and analytical characteristics of the immunoassay system were evaluated.

Section snippets

Chemicals and reagents

Goat anti-mouse IgG (whole molecular, M8642), mouse IgG (MIgG, I5381), goat anti-mouse IgG-HRP conjugates (anti-MIgG-HRP, A4416) were purchased from Sigma. NaI, bovine serum albumin (BSA), poly-oxyethylene-sorbitan monolaurate (Tween 20) and glutaraldehyde (25% solution) were obtained from Sigma. Carbon ink (Electrodag 423SS) was obtained from Acheson Colloids (Japan). Hydrogen peroxide (30% (v/v) aqueous solution) was purchased from Beijing Chemical Reagent Factory (Beijing, China).

Unless

Construction of the immunoelectrode strips and electrochemical flow-through cell

The immunosorbent reactor with separated electrochemical detector (downstream of immunoreactor) coupled to flow injection system was powerfully promoted the development of FIA-EIA [26], [27], [28]. According to the similar principle, a deviation from the traditional “stacked layers” of the biosensor geometry was adopted in this study. The construction of the immunoelectrode strip was shown in Fig. 2A. The immunoelectrode strip included immunosorbent layer (1mm×17 mm) with immobilized antibody

Conclusion

In this work, a flow injection enzyme-linking immunoassay system with electrochemical detection was developed using the immunoelectrode strips based on the screen-printed carbon electrode. The proposed electrochemical flow cell with the three-electrode configuration showed the preferable characterization and practicability. The feasibility of the idea about integrating biorecognition layer and electrochemical detector on the same electrode stripe but placing respectively at upstream and

Acknowledgements

This work was supported by the National Key Basic Research Development Project “Research on Human Major Disease Proteomics” (2001CB5102) and the National Natural Science Foundation of China (No. 20299030).

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