Electrochemical immunoassay immobilization

Electrochemical immunoassays include a wide variety of devices based on the coupling of immunological reactions with electrochemical transduction. All of them involve the immobilization of an immunoreagent component on the surface of the electrode transducer. Electrochemical detection is based on the direct intrinsic redox behavior either of an analyte species or of some reporter molecule. For the detection no expensive equipment is needed, with the measurement of either a simple current or a voltage charge. Different electrochemical detection strategies are used, but ampero-metric detection is most widely used. Potentiometric and conductometric detection are applied in different assays as well. 

The modification of electrodes with enzymes and other biological macromolecules was well underway before 1978, and a detailed history of this field is beyond the scope of the present paper. A brief discussion of biological systems is given, however, to place them in context with other modification layers. A recent review by Frew and Hill (121) discusses past and future strategies for design of electrochemical biosensors. Topics discussed were enzyme electrodes, electron transfer mediators, conducting salts, electrochemical immunoassay, enzyme labels, and cell-based biosensors. In general, the bioactive molecule or cell is immobilized in proximity to an electrochemical transducer and exposed to the analyte solution for real-time analysis. 

Fu, Y., Li, P, Bu, L. et al (2010) Chemical/biochemical preparation of new polymeric bionanocomposites with enzyme labels immobilized at high load and activity for high-performance electrochemical immunoassay. J. Phys. Chem. C, 114,1472-1480. 

Chatrathi, M.P, Wang, J., and Collins, G.E. (2007) Sandwich electrochemical immunoassay for the detection of Staphylococcal enterotoxin B based on immobilized thiolated antibodies. Biosens. Bioelectron., 22 (12), 2932. 

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. 

Competitive immunoassays may also be used to determine small chemical substances [10, 11]. An electrochemical immunosensor based on a competitive immunoassay for the small molecule estradiol has recently been reported [11]. A schematic diagram of this immunoassay is depicted in Fig. 5.3. In this system, anti-mouse IgG was physisorbed onto the surface of an SPCE. This was used to bind monoclonal mouse anti-estradiol antibody. The antibody coated SPCE was then exposed to a standard solution of estradiol (E2), followed by a solution of AP-labeled estradiol (AP-E2). The E2 and AP-E2 competed for a limited number of antigen binding sites of the immobilized anti-estradiol antibody. Quantitative analysis was based on differential pulse voltammetry of 1-naphthol, which is produced from the enzymatic hydrolysis of the enzyme substrate 1-naphthyl phosphate by AP-E2. The analytical range of this sensor was between 25 and 500pg ml. 1 of E2. 

FIGURE 5.6 Schematic representation of the immunosensor based on a Protein A-GEB biocomposite as a transducer, (a) Immobilization of RlgG on the surface via interaction with Protein A, (b) competitive immunoassay using anti-RIgG and biotinylated anti-RIgG, (c) enzyme labeling using HRP-streptavidin and (d) electrochemical enzyme activity determination. (Reprinted from [31] with permission from Elsevier.)... 

Immunosensors have been designed which use both direct and indirect immunoassay technology to detect specific analytes within a minute or less in a variety of matrices (see Fig. 9). Indirect immunosensors may employ ELA, FLA, or CLIA principles whereby enzyme-, fluorophore- or chemiluminescent-labeled analyte competes with the target (nonlabeled) analyte for binding sites on the immobilized antibody. Unbound (free) labeled analyte is then quantitated using an electrochemical, optical, or electromechanical transducer and compared to the amount of target analyte in the sample. 

Heterogeneous immunoassay has also been conducted with the antibody immobilized on beads. For instance, mouse IgG (50-100 ng/mL) was detected by ELISA in a glass chip. First, mouse IgG (antigen) was captured by magnetic beads coated with sheep anti-mouse antibody (1.02 x 107 beads/mL). Then the secondary antibody, which was rat anti-mouse conjugated with alkaline phosphatase (0.7 pg/mL), was delivered. Thereafter the substrate, PAPP, was added. It was enzymatically converted to p-aminophenol (PAP), which was electrochem-ically detected by the on-chip interdigital microelectrodes [1016]. 

Several heterogeneous electrochemical enzyme immunoassays have been demonstrated. These are based on the enzyme-linked immunosorbent assay (ELISA) technique in which antibody is immobilized on the walls of a small volume plastic vessel. The ELISA technique can follow either a competitive equilibrium or a sandwich format. Both formats have been used with electrochemical detection. The general protocol for these two formats is shown in Fig. 9. 

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