Page 199 - Analytical Electrochemistry 2d Ed - Jospeh Wang

184 ELECTROCHEMICAL SENSORS

The antibody is a globular protein produced by an organism to bind to foreign
molecules (i.e., antigens) and mark them for elimination from the organism. The
remarkable selectivity of antibodies is based on the stereospeci®city of the binding
5
site for the antigen, and is re¯ected by large binding constants (ranging from 10 to
9
1
10 L mol †. Antibody preparations may be monoclonal or polyclonal. The former
are produced by a single clone of antibody-producing cells, and thus have the same
af®nity. Polyclonal antibodies, in contrast, are cheaper but possess varying af®nities.
Electrochemical immunosensors, combining speci®c immunoreactions with elec-
trochemical transduction, have gained considerable attention in recent years (37±41).
Such sensors are based on labeling of the antibody (or antigen) with an enzyme that
acts on a substrate and generates an electroactive product that can be detected
amperometrically. Enzyme immunosensors can employ competitive or sandwich
modes of operation (Figure 6-13). In competitive-type sensors, the sample antigen
(analyte) competes with an enzyme-labeled antigen for antibody-binding sites on a
membrane held on an amperometric or potentiometric sensing probe. After the
reaction is complete, the sensor is washed to remove unreacted components. The
probe is then placed in a solution containing the substrate for the enzyme, and the
product or reactant of the biocatalytic reaction is measured. Due to the competitive
nature of the assay, the measurement signal is inversely proportional to the
concentration of the analyte in the sample. Several enzymes, such as alkaline
phosphatase, peroxidase, or catalase, have been particularly useful for this task.
Sandwich-type sensors are applicable for measuring large antigens that are
capable of binding two different antibodies. Such sensors utilize an antibody that
binds the analyte-antigen, which then binds an enzyme-labeled second antibody.
After removal of the nonspeci®cally adsorbed label, the probe is placed into the
substrate-containing solution, and the extent of the enzymatic reaction is monitored

FIGURE 6-13 Enzyme immunosensors based on the competitive or sandwich modes of
operation. (Reproduced with permission from reference 40.)