A Status Report on Electroanalytical Techniques for Immunological

Chapter 12

A Status Report on Electroanalytical Techniques for Immunological Detection

Downloaded by NORTH CAROLINA STATE UNIV on October 11, 2012 | http://pubs.acs.org Publication Date: October 23, 1996 | doi: 10.1021/bk-1996-0646.ch012

Omowunmi A. Sadik and Jeanette M. Van Emon Characterization Research Division, National Exposure Research Laboratory, U.S. Environmental Protection Agency, P.O. Box 93478, Las Vegas, NV 89193-3478

Immunoassays are commonly used for the detection of low levels of a specific target analyte or group of analytes. The search for new nonradioactive detection methods has resulted in a plethora of immunoassay techniques utilizing different types of labels, the most common being enzymes. Electrochemical immunoassays are based on modifications of enzyme immunoassays with the enzyme activities being determined potentiometrically or amperometrically. Other nonradioactive assays have been designed which are not adapted from spectrophotometric detection methods. Each of these assays, including electrochemical enzyme immunoassays, is discussed briefly in this chapter. In addition, various electrochemical immunosensors reported recently and focuses on the use of electropolymerized conducting polymers (CPs) in amperometric immunochemical sensors are discussed. An electrochemical immunosensor is described for the analysis of polychlorinated biphenyls using a CP-based immunosensor. The sensor produced adequate linear response characteristics and sensitivities that are comparable with results obtained using the enzyme-linked immunosorbent assay (ELISA) technique.

Immunochemical techniques are based on the interaction of antibodies (Ab) with antigens (Ag). An antibody is a protein that recognizes a target analyte (i.e., antigen) or group of analytes and then reacts specifically with it to form a complex. An immunogen is a compound that induces the formation of specific antibodies. Various labels can be used to produce a range of assays, including radioimmunoassays (RIA), fluorescence immunoassays (FLIA), enzyme immunoassays (EIA) and chemiluminescence immunoassays (CLIA). The wide dynamic range and low detection limits of electroanalytical techniques are helpful in the development of electrochemical immunoassay as an alternative approach to spectrophotometric and radiometric detection procedures.

0097-6156/96/0646-0127$15.25/0 © 1996 American Chemical Society In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.


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The use of electrochemical methods for immunoassay procedures follows a tradition of technical expansion. Breyer and Radcliff first demonstrated the polarographic detection of azo-labeled antigen in a homogeneous immunoassay (1). In the 1950s, Berson and Yalow developed the area of radioimmunoassays (2). Since the first electrochemical immunoassay experiments, significant progress has been made in modern electrochemical instrumentation, microelectronics, and the development of new classes of electrode materials. These developments have further stimulated interest in combining immunoassay procedures with electrochemical measurements. The principles have been used to achieve very low detection limits when coupled to chemical amplification systems, such as enzymes. Electrochemical detection techniques are generally inexpensive, fast, and amenable to automation. Electrochemical monitoring of immunological reactions holds great promise as a practical alternative to assays involving radioactive labels. Several electrochemical immunoassay approaches have been proposed. These include: multianalyte immunoassays involving anodic stripping voltammetric detection of different metal labels (3), capillary electrochemical enzyme immunoassay coupled to flow injection analysis systems for digoxin and atrazine (4,5), the use of interdigitated array electrodes for small-volume voltammetric enzyme immunoassay (6), and homogeneous amperometric immunoassay for theophylline (7,8). The successful implementation of electrochemiluminescence labels for immunometric assays of hormones, cancer markers and nucleic-acid hybridization assays was reported by Yang et al., (9). A separation-free enzyme immunoassay utilizing microporous gold electrodes with self-assembled monolayer antibody electrodes was also reported (10). A new and highly promising approach for the detection and amplification of Ab-Ag interactions involves the incorporation of immobilized antibodies into conducting polymer films or membranes (11,12). Conducting polymer membranes (CPMs) are particularly attractive for biosensor applications where a range of different immobilized analytes can easily be assessed. The interaction with the analytes can also be controlled electrochemically through the application of electrical potentials. Such sensors have been used successfully with pulsed amperometric detection in a flow injection system (11- 14). CPM-based biosensors have been proposed for direct and continuous detection of low concentrations of biological and organic analytes in process streams, environmental samples and biological fluids (1517). The use of bilayer lipid membranes for electrochemical transduction of immunological reactions has also been reported (18). A review that discusses the principles of electrochemical immunoassay protocols based on the measurement of a faradaic current was recently reported (19). Other recent work discusses the concept of light-addressable potentiometric immunosensors (20). Our aim in this chapter is to give a status report on electroanalytical techniques employed for immunological detection, and also to present some results obtained in the successful application of conducting polymer-based immunosensors for environmental applications.

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

12. SADIK & VAN EMON

Electroanalytical Techniques

129


Electrochemical Immunoassay Immunoassays are commonly categorized as "heterogeneous", (in which Ab-bound Ag is separated from free Ag), or "homogeneous", (in which no separation steps are involved). Features of electrochemical immunoassays are shown in Table I. Direct electrochemical immunoassays monitor changes in electrical properties of the Ab-Ag binding events. In this case, the sensitivity is directly proportional to the amount of Ab present. However, in some competitive assays, labeled Ags compete with unlabeled Ags for a limited number of Ab binding sites. The Ab-bound Ags (labeled and unlabeled) are separated from the free Ags and the signal produced by the Abbound labeled antigens is then measured. The signal intensity of the bound phase is inversely proportional to the concentration of the unlabeled Ag. Competitive sandwich assays involve the incubation of excess primary Ab with an Ag. The AbAg complex is then incubated with a labeled secondary Ab which binds to the first Ab. The unbound labeled Ab is rinsed away and the bound labeled Ab is measured. The signal intensity is related to the amount of primary Ab present, which is inversely related to the amount of Ag (analyte) in the sample. A class of heterogeneous enzyme immunoassays with electrochemical detection has emerged and is known as electrochemical enzyme immunoassay (ECIA). ECIA is the result of the modifications of enzyme immunoassays with the enzyme activity being determined electrochemically (21,22). ECIA is based on the labeling of specific Ab or target analyte with an enzyme that catalyzes the production of an electroactive product. Different formats of ECIA have been reported that depend on the label type, assay format, and the electrochemical techniques employed. They are discussed below. Competitive ECIA: Microwell plates are first prepared by attaching specific Ab to the inside walls through passive adsorption or covalent bonding. An equilibrium is established between the bound Ab, the analyte, and enzyme-labeled Ag. This may take several minutes or hours depending on the analyte and the configuration of the orientating reagents. After the incubation step, the unbound reagents are washed away and the substrate is added. At a fixed time, the sample is withdrawn and analyzed for electroactive products. The general procedure in ECIA for the determination of analyte is represented in Figure 1. Alkaline phosphatase is used in voltammetric immunoassays with phenyl phosphate as the substrate and it catalyzes the hydrolysis of the /?-nitrophenyl phosphate ester to yield phenol and phosphoric acid. The enzyme-generated phenol is easily detectable by either a flow injection analysis with electrochemical detection (FIA-EC), or by liquid chromatography with electrochemical detection (LC-EC). A typical quantitation curve shows current vs. concentration of analyte standard. The electrochemical signal decreases with increasing analyte concentration. ECIA of this type has been demonstrated for IgG, α-feroprotein, digoxin and morphine using unmodified electrodes (21-23 ).

In Environmental Immunochemical Methods; Van Emon, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1996.

130

ENVIRONMENTAL IMMUNOCHEMICAL METHODS

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A Status Report on Electroanalytical Techniques for Immunological

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