Voltammetric immunosensors

Neves, M.M.P.S., Gonzdlez-Garcia, M.B., Santos-Silva, A. and Costa-Garcfa, A. (2012) Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies. Sens. Actuators, B, 163, 253-259. 

In contrast to direct immunosensors, indirect immunosensors, using as a label an enzyme [307-309] or an ionophore [310], have been more developed. This technique is derived from well established and widely utilized enzyme immunoassays evaluated by photometric methods. Enzyme immunoassay with electrochemical detection (EEIA) represents an important innovation of EIA by the replacement of optical detection with voltammetric or potentiometric measurements. It may also be adapted to flow-through systems [311, 312]. 

This immunosensor-type assay uses ferrocene methanol as co-substrate (redox mediator) of glucose oxidase (GOD). The various steps of the assay, shown in Fig. 8.20, are as follows (1) Covalent immobilization of protein A on graphite-polystyrene screen-printed electrodes (SPEs) (2) Addition of the rabbit IgG to be quantified which is captured specifically by protein A (3) Addition of a biotinylated goat anti-rabbit antibody (4) Addition of avidin-GOD conjugate (5) Addition of glucose and ferrocene methanol (6) Measurement of catalytic current by flow injection immunoassay. The voltammetric current corresponds to the one-electron oxidation of the ferrocenyl group to ferricinium. The electrode can subsequently be regenerated up to 30 times. The feasibility of the assay has been demonstrated for the case of monoclonal mouse anti-human prolactin (PL) with a detection limit of 0.02 pg mL [90]. 

Amperometric or voltammetric biosensors typically rely on an enzyme system that catalyt-ically converts electrochemically non-active analytes into products that can be oxidized or reduced at a working electrode. Although these devices are the most commonly reported class of biosensors, they tend to have a small dynamic range due to saturation kinetics of the enzyme, and a large overpotential is required for oxidation of the analyte this may lead to oxidation of interfering compounds as well (e.g., ascorbate in the detection of hydrogen peroxide). In addition to the use in enzyme-based biosensors, amperometric transducers have also been used to measure enzyme-labelled tracers for affinity-based biosensor (mainly immunosensors and genosensors). Enzymes which are commonly used for this purpose include horseradish peroxidase (HRP) [17] and alkaline phosphatase (AP) [18,19,21]. 

The electrochemical determination of cyt c was performed by utilizing its direct electroactivity of heme of cyt c (Fe(III)/Fe(II)) specifically bound to anti-cyt c. Under controlled experimental conditions, the developed immunosensors were exposed to solutions of cyt c to form immunocomplex. After the immunological binding of cyt c to anti-cyt c, a cyclic voltammetric curve was recorded. Cyt c solutions with different concentrations were prepared and applied onto the anti-cyt c/SAM/GNP/PPy/SPE immunosensor. The redox peak current due to cyt c increased with increase in cyt c concentration owing to the direct electron transfer of cyt c (Figure 4.16). 

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