Flow Injection Immunoassay with Electrochemical Detection

1 Flow Injection Immunoassay with Electrochemical Detection 

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]. 

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Five types of amperometric detection have been applied for enzyme-based ECIA. They are flow-injection analysis with electrochemical detection (FIAEC), liquid chromatography with electrochemical detection (LCEC), amperometric detection with interdigitated array electrodes (IDA), rotating disk electrode (RDE) amper-ometry, and scanning electrochemical microscopy (SECM). Of these, the two conventional types, FIAEC and LCEC, shall be discussed in this section, leaving the discussion of the other types to Section V on miniaturized immunoassays. 

Instead of immobilizing the antibody onto the transducer, it is possible to use a bare (amperometric or potentiometric) electrode for probing enzyme immunoassay reactions (42). In this case, the content of the immunoassay reaction vessel is injected to an appropriate flow system containing an electrochemical detector, or the electrode can be inserted into the reaction vessel. Remarkably low (femtomolar) detection limits have been reported in connection with the use of the alkaline phosphatase label (43,44). This enzyme catalyzes the hydrolysis of phosphate esters to liberate easily oxidizable phenolic products. 

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