Enzyme Competition Electrodes

Renneberg R., Pfaffer D., Scheller F. and Janchen M. (1982) Enzyme sequence and competition electrodes based on immobilized glucose oxidase, peroxidase and catalase. Anal. Chim. Acta, 134, 359-364. 

Catalase has also been used as an enzyme label in competitive heterogeneous enzyme immunoassays. Catalase generates oxygen from hydrogen peroxide with the oxygen determined amperometrically with an oxygen electrode. This approach has been demonstrated for a-fetoprotein theophylline and human serum albumin... 

Many problems involving competitive reaction kinetics may be treated by invoking the steady-state assumption within the digital simulation this has been done in at least two instances [29-34]. The first of these involves the development of a model for enzyme catalysis in the amperometric enzyme electrode [29-31]. In this model, the enzyme E is considered to be immobilized in a diffusion medium covering an electrode that is operated at a fixed potential such that the product (P) of enzyme catalysis is electroactive under diffusion-controlled conditions. (This model has also served as the basis for the simulation of the voltammetric response of the enzyme electrode [35].) The substrate (S) diffuses through the medium that contains the immobilized enzyme and is catalyzed to form P by straightforward enzyme kinetics ... 

This line has been initiated in the late 70s by Rechnitz group (1), who introduced enzyme sequences into enzyme electrodes. The field has been considerably widened by introducing the competitive (parallel), recycling and accumulation sensors. The following main merits of such sensors shall be pointed out ... 

Aizawa et al. reported the development of a competitive enzyme immunoassay using electrodes (A2). In this method, antibody immobilized on the membrane is attached to the electrodes for oxygen. When catalase-antigen conjugate binds to the immobilized antibody, production of oxygen can be... 

Haga et al. developed another type of immunosensor by combining an enzyme membrane immunoassay and an enzyme sensor using oxygen electrodes (HI). In this assay antigen molecules (theophylline) are attached on the surface of the liposomes and an enzyme (horseradish peroxidase) is encapsulated in the sensitized liposome. When antibody (antitheophylline antibody) and complement are added, the enzyme is released by the liposome lysis. The enzyme activity with the NADH-NAD reaction can be determined by the oxygen electrode. When antigen is added, it competitively binds to antibodies, then liposome lysis and enzyme activity are decreased. The sensitivity of this method for theophylline determination was reported as 0.7 ng/ml. 

Such interference falls into two classes competitive substrates and substances that either aaivate or inhibit the enzyme. With some enzymes, such as urease, the only substrate that reacts at reasonable rate is urease hence, the urease-coated electrode is specific for use (59, 165). Likewise, uricase acts almost specifically on uric acid (167), and aspartase on aspartic acid (8, 168). Others, such as penicillinase and amino oxidase, are less specific (63,169,170). Alcohol oxidase responds to methanol, ethanol, and allyl alcohol (171, 172). Hence, in using electrodes of these enzymes, the analyte must be separated if two or more are present (172). Assaying L-amino acids by using either the decarboxylative or the deaminating enzymes, each of which acts specifically on a different amino... 

An enzyme immunoelectrode suitable for the assay of human serum albumin and insulin uses an oxygen electrode covered with an antibody-containing nylon net kept in place with an O-ring. From 1 to 25 ng/L of albumin and 5 to 100 ng/L of insulin can be assayed (306). A specific sensor for the tumor antigen a-fetoprotein (AFP) is prepared by immobilizing anti-AFP antibody covalendy on a membrane prepared from cellulose triacetate, 1,8-diamino-4-aminomethyl octane, and GA (307). The sensor is applied to an EIA based on competitive Ab/... 

Bovine serum albumin (BSA) and cyclic AMP (cAMP) are determined by a competitive binding enzyme immunoassay (315). With urease as label, an ammonia gas-sensing electrode is used to measure the amount of urease-labeled antigen bound to a double-antibody solid phase by continuously measuring the rate of ammonia produced from urea as substrate. The method yields accurate and sensitive assays for proteins (BSA less than 10 ng/mL) and antigens (cAMP less than 10 nM), with fairly good selectivity over cGMP, AMP, and GMP. 

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. 

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