Microchip-based amperometric immunoassays using redox tracers

Abstract

A new chip-based electrochemical immunoassay protocol, based on the use of a ferrocene redox label, is described. Two reaction formats, based on direct (noncompetitive) and competitive modes of operation, were employed for illustrating the use of redox tracers in chip-based electrochemical immunoassays. The direct assay consisted of mixing the ferrocene-tagged antibody and the antigen analyte, a rapid electrophoretic separation of labeled free antibody and the labeled antigen/antibody complex, and a downstream anodic detection of the ferrocene tracer at gold-plated carbon screen-printed electrode detector. The competitive assay integrates precolumn reactions of the labeled antigen and the target antigen with the antibody with electrophoretic separation of the free and bound labeled antigens, along with amperometric detection of the redox tag. An internal standard has been used to normalize the peak area for the construction of calibration plots. Fundamental operating variables are examined and optimized. The use of a redox tracer offers the advantages of simplified protocol, wider linear range, higher stability, and higher separation efficiency compared to an analogous use of enzyme tags. The direct mouse-immunoglobulin G (IgG) assay and the competitive 3,3',5-triiodo-L-thyronine (T(3)) one were accomplished within less than 150 and 130 s (with field strengths of 256 and 192 V/cm), and offer minimum detectable concentrations of 2.5 x 10(-12) and 1 x1 0(-6) g/mL, respectively. Such use of redox labels for chip-based amperometric immunoassay protocols offers considerable promise for decentralized clinical or environmental testing.

Why the move to microfluidics for protein analysis?

“…Another field where the time-to-result is crucial is that of clinical immunoassay: several approaches have been presented, such as homogeneous immunoassays with antibody-antigen complex detection and quantification by capillary electrophoresis (CE) and laser-induced fluorescence or electrochemical means [32,33]. By contrast, heterogeneous immunoassays replace CE with a washing step and have been demonstrated on microchip platforms [34 ,35-40].…”

Recent developments in amperometric detection for microchip capillary electrophoresis

“…In one application, anti-mouse-immunoglobulin G (IgG) was employed as the antibody and mouse IgG antigens were evaluated [10,11]. First, a direct (or noncompetitive) immunoassay was developed.…”

“…The three compounds were allowed to react and then separated electrophoretically. Following separation, the labeled complexes (T 3 -antibody) and unbound competing labeledantigen (T 3 ) were detected electrochemically and the amount of T 3 was then calculated [11].…”

“…The feasibility of integrating several laboratory procedures onto a chip, including sample preparation, separation, and detection, has been demonstrated by several groups [8][9][10][11][12]. The numerous advantages of incorporating several previously distinct processes into a miniaturized format include faster analysis times, decreased cost and waste production, portability, disposability, and the potential for point-of-care use [2,3,7].…”

An end-channel amperometric detector for microchip capillary electrophoresis

“…García and Henry [14] adopted analogous alignment method except two PDMS layers were irreversibly bonded. Wang et al [15][16][17][18] performed the alignment by a plastic screw and the distance between working electrode and separation channel was about 50 m. Zeng et al [19] firstly designed and fabricated a home-made guide tube in order to precisely alignment and quickly replacement the working electrode, the result was fairly good but the fabrication process of the guide tube, the working electrode as well as the detector was rather laborious and complicated.…”