Homogeneous electrochemical enzyme

Homogeneous electrochemical enzyme immunoassays for both phenytoin and digoxin have been developed. In both cases the label was glucose-6-phosphate dehydrogenase, which catalyzes the reduction of NAD to NADH. The NADH produced was detected by LCEC at a carbon paste electrode. 

A homogeneous electrochemical enzyme immunoassay for 2,4-dinitrophenol-aminocaproic acid (DNP-ACA), has been developed based on antibody inhibition of enzyme conversion from the apo- to the holo- form Apoglucose oxidase was used as the enzyme label. This enzyme is inactive until binding of flavin adenine dinucleotide (FAD) to form the holoenzyme which is active. Hydrogen peroxide is the enzymatic product which is detected electrochemically. Because antibody bound apoenzyme cannot bind FAD, the production of HjOj is a measure of the concentration of free DNP-ACA in the sample. 

FIGURE 5.7. Effect of changing the cosubstrate and the pH on the kinetics of an homogeneous redox enzyme reaction as exemplified by the electrochemical oxidation of glucose by glucose oxidase mediated by one-electron redox cosubstrates, ferricinium methanol ( ), + ferricinium carboxylate ( ), and (dimethylammonio)ferricinium ( ). Variation of the rate constant, k3, with pH. Ionic strength, 0.1 M temperature 25°C. Adapted from Figure 3 in reference 11, with permission from the American Chemical Society. 

Fig. 3 Cyclic voltammetric analysis of the kinetics of an homogeneous redox enzyme reaction using a reversible one-electron mediator as the cosubstrate, taking as example the catalysis of the electrochemical oxidation of j8-D-glucose by glucose oxidase (6.5 pM) with ferrocene methanol as the cosubstrate at pH = 7 (ionic strength 0.1 M, temperature ...

Quantitative examination of the dynamics of these systems by analysis of their time-resolved electrochemical responses is also an important objective. This is true even in the case of catalysis by a homogeneously dispersed enzyme for which a rigorous treatment has allowed the... 

In another study, a microchip-based electrochemical enzyme immunoassay was developed by Chatrathi and colleagues [65] and its performance is demonstrated for the determination of monoclonal mouse IgG as a model analyte. The direct homogeneous immunoassay requires the integration of electrokinetic mixing of ALP-labeled anti-mouse IgG antibody (Ab-E) with the mouse IgG antigen (Ag) analyte in a precolumn reaction chamber, injection of immunochemical products into the separation channel, followed by rapid electrophoretic separation of enzyme-labeled free antibody and enzyme-labeled antibody-antigen complex. The separation is followed by a postcolumn reaction of enzyme tracer with p-aminophenyl... 

Mainly, three approaches have been used to immobilize the enzyme on transducer or electrode surface, single layer, bilayer, and sandwich configurations [69, 98], In some studies enzymes are covalently linked with sol-gel thin films [99], Sol-gel thin films are highly convenient for fast, large, and homogeneous electron transfer [17]. With an increase in gel thickness the signal decays and diffusion of analytes to biomolecule active site becomes difficult eventually these factors lead to poor response. By employing thin films various biosensors such as optical and electrochemical biosensors have been reported. 

Chlorobenzonitrile and adrenaline, our second example, both give electrode products that are unstable with respect to subsequent chemical reaction. Because the products of these homogeneous chemical reactions are also electroactive in the potential range of interest, the overall electrode reaction is referred to as an ECE process that is, a chemical reaction is interposed between electron transfer reactions. Adrenaline differs from/ -chlorobenzonitrile in that (1) the product of the chemical reactions, leucoadrenochrome, is more readily oxidized than the parent species, and (2) the overall rate of the chemical reactions is sufficiently slow so as to permit kinetic studies by electrochemical methods. As a final note before the experimental results are presented, the enzymic oxidation of adrenaline was known to give adrenochrome. Accordingly, the emphasis in the work described by Adams and co-workers [2] was on the preparation and study of the intermediates. 

This chapter presents an approach to perform enzyme linked immunosorbent assays (ELISA) in a microfluidic format with electrochemical detection. This field of analytical chemistry has shown a strong activity in recent years, and many reports have presented the use of capillary-sized reactors for running immunoassays either in homogeneous format (where the antigen-antibody complex and the labelled revelation reagents are separated prior to detection, as for instance by capillary electrophoresis [1-3]) or in heterogeneous format (where the antibody is immobilised on the inner surface of the microsensor device [4] or on microbeads [5,6]). 

Mayer determined acetylcholine and choline by enzyme-mediated liquid chromatography with electrochemical detection [195]. The two compounds were separated by passing the eluted fractions through a post-column reactor containing immobilized Acetylcholineesterase and choline oxidase. In the presence of either compound, the dissolved oxygen was converted into hydrogen peroxide, which was detected amperometrically at a platinum electrode. This method was used to determine choline in rat brain homogenates. 

The mechanism and theory of bioelectrocatalysis is still under development. Electron transfer and variation of potential in the electrodeenzyme-electrolyte system has therefore to be investigated. Whether the enzyme is soluble and the electron transfer process occurs through a mediator, or whether there is direct enzyme immobilization on the electrode surface, the homogeneous process in the enzyme active centre has to be described by the laws of enzyme catalysis, and the heterogeneous processes on the electrode surface by the laws of electrochemical kinetics. Besides this there are other aspects outside electrochemistry or... 

Ideally, PFV requires a film of active molecules (monolayer or submonolayer) that behave independently of each other and homogeneously with regard to their electrochemical and catalytic properties. Interactions between centres in neighbouring molecules are naturally minimised by the surrounding polypeptide and ensure that PFV is relatively free from complications induced by intermolecular interactions of the type that frequently distort the voltammetry of surface-confined small molecules. Usually, however, peak widths are larger than expected due to inhomogeneity (dispersion). The activity of enzyme molecules with dispersed and poor interfacial electron-transfer kinetics (low will distort the voltammogram and complicate analysis. 

In summary, it can be stated that methods of electrochemical detection are very applicable to enzyme immunoassays. Broadly speaking, the above examples demonstrate that homogeneous EIA are faster and simpler but often less sensitive and more subject to interference than heterogeneous EIA. The latter are less sensitive to interference and electrode fouling because the measuring chamber in front of the electrode is rinsed before determination of the marker activity. However, none of the methods described is suitable for continuous measurement. 

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