Electrode-Based Enzyme Immunoassays

Electrode-Based Enzyme Immunoassays Using Urease Conjugates... 

Meyerhoff, M.E. Rechnitz, G.A. Electrode-based enzyme immunoassays using urease conjugates. Anal. Biochem. 1979, 95, 483-493. 

Fig. 118. Homogeneous electrode-based enzyme immunoassay. Fecp = ferrocene.

H. Dong, C.M. Li, Q. Zhou, J.B. Sun, and J.M. Miao, Sensitive electrochemical enzyme immunoassay microdevice based on architecture of dual ring electrodes with a sensing cavity chamber. Biosen. Bioelectron. 22, 621-626 (2006). 

Biosensors Biosensors are used in the final measurement stage of immunoassays such as an electrochemistry-based enzyme-linked immunoassay (ELISA) or the measurement of catalyse-labelled antigens at an antibody-coated oxygen electrode. 

Q. Gao, Y. Ma, Z.L. Cheng, W.D. Wang and M.R. Yang, Flow injection electrochemical enzyme immunoassay based on the use of an immuno-electrode strip integrate immunosorbent layer and a screen-printed carbon electrode, Anal. Chim. Acta, 488 (2003) 61-70. 

A sandwich assay for HCG using GOD as label is also described (309). The procedure is based on an amperometric enzyme immunoassay with an electrode-immobilized antibody. The antibody electrode (activated glassy carbon) is used both to separate the assay and to monitor the activity of the bound enzyme label. Two monoclonal antibodies directed against different antigenic sites are used ... 

An enzyme immunoassay using adenosine deaminase as the enzyme label has been described (318). Potentiometric rate measurements were made with an ammonia gas-sensing electrode. The immunoassay system employed is based on competition between a model haptenic group, dinitrophenyl (DNP) covalently coupled to adenosine deaminase, and free DNP hapten for the available binding sites on the anti-DNP antibody molecules. The detection limit is 50 ng antibody. 

Ho, W. O., Athey, D., and McNeil, C. J. Amperometric detection of alkaline phosphatase activity at a horseradish peroxidase enzyme electrode based on activated carbon - Potential application to electrochemical immunoassay. Biosens. Bioelectron. 1995,10, 683-691. 

A collection of redox enzymes for which efficient DET with electrodes has been observed is given in Table 2.3. Most of them are metaUoenzymes containing iron or copper. Many of these enzymes are part of electron transfer chains, i.e., have macromolecular redox partners, or react on large substrates. The evidence for DET has not always been presented by direct electrochemical measurements. In many cases the DET has been proved indirectly by measurement of a substrate dependent catalytic current. Various metabolites ranging from sugars such as fructose, cellobiose and gluconate [6], amines like methylamine and histamine [123], lactate [91],p-cresol [93] and drugs such as benzphetamine [74] can be measured with enzymes in direct contact to an electrode. The bioelectrocatalytic reaction of peroxide is one of the most important reactions not only for the determination of peroxide(s) in various media but also substrates of coupled oxidase [8] and enzyme inhibitors [130, 252]. Furthermore, enzyme immunoassays have been developed based on DET of peroxidase and laccase and electrodes [7,131,132]. 

See also DNA Sequencing. Enzymes Enzyme-Based Electrodes. Forensic Sciences Blood Analysis. Immunoassays, Techniques Enzyme Immunoassays. Microelectrodes. Polarography Techniques Organic Applications. Purines, Pyrimidines, and Nucleotides. Sensors Chemically Modified Electrodes. Voltammetry Organic Compounds. 

See also Blood and Plasma. Clinical Analysis Glucose. Enzymes Immobilized Enzymes Enzyme-Based Electrodes Enzymes in Physiological Samples Industrial Products and Processes. Ethanol. Flow Injection Analysis Principles Detection Techniques. Food and Nutritional Analysis Alcoholic Beverages. Forensic Sciences Alcohol in Body Fluids. Immunoassays, Techniques Enzyme Immunoassays. Lipids Determination in Biological Fluids. Pesticides. Process Analysis Bioprocess Analysis. 

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. 

Electrochemical immunosensors based on screen-printed electrodes have recently been applied to the detection of environmental pollutants such as PCBs, PAHs, pesticides [17-20] and of important molecules in clinical and food field [21-23]. In this case, the screen-printed electrodes are both the solid-phase for the immunoassay and the electrochemical transducers antibody or antigen molecules are directly immobilised at the sensor surface (transducer) and one of these species is enzyme-labelled in order to generate an electroactive product which can be detected at the screen-printed electrode surface. 

The use of capillaries to run immunoassay reactions was presented in the 1980s and 1990s by Halsall group [8] for the detection of atrazine. The system was based on a flow injection analysis protocol where different solutions (sample, conjugate) were injected and reacted in the capillary itself the affinity-captured enzyme conjugate then reacted with a substrate solution injected in the capillary prior to detection. The reacted substrate solution was then pushed towards an electrode for amperometric detection. The authors showed that the incubation time reached equilibrium after about 20 min, but they worked with a capillary of 360 pm diameter. In order to really reduce the incubation time below lmin, we developed disposable polymer chips with an internal diameter of 40 pm (almost one order of magnitude smaller than... 

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