Electrochemically coupled enzymatic

Figure 1. (a) Scheme for the reaction sequence of the electrochemically coupled enzymatic oxidation of glucose, (b) The effect of the enzymatic reaction on the electrochemistry of ferrocene using a gold disc of 25lim diameter at 5mV s... 

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is carried out in a test tube under controlled conditions with aliquots being taken at timed intervals. These aliquots are then analyzed for the electroactive product and the enzyme activity in the sample calculated from the generated kinetic information. 

Fig. 10 Schematic representation of the two approaches mainly used in EzILAs. (a) The enzyme conjugate and the analyte compete for the selective binding sites of the polymer finally, a substrate is converted into a product that generates a chemical signal (e.g., fluorescence, absorbance, electrochemical) at a rate which is proportional to the amount of bound enzyme and hence to the concentration of analyte in the sample, (b) Direct assay where the analyte is the enzyme which is quantified by a coupled enzymatic reaction...

The simplest method of coupling enzymatic reactions to electrochemical detection is to monitor an off-line reaction using FIAEC or LCEC. The enzymatic reaction is... 

Hoogvliet, J.C., L.C. Lievense, C.V. Kijk, and C. Veeger (1998). Redox mediators coupling enzymatic and electrochemical reactions for coenzyme regeneration. Eur. /. Biochem., 174,273. 

The demand for monitoring common metabolites of diagnostic utility such as glucose, urea and creatinine continue to provide the impetus for a staggering research effort towards more perfect enzyme electrodes. The inherent specificity of an enzyme for a given substrate, coupled with the ability to electrochemically detect many of the products of enzymatic reactions initiated the search for molecule-selective electrodes. 

An additional path to protonation of coordinated NO is the reduction of the M-NO unit electrochemically (73). Such coupled reduction/proto-nation schemes have been argued to be relevant to enzymatic nitrogen oxide reductases (41). Farmer and coworkers (74) accomplished such reductions by using graphite electrodes modified by depositing... 

In MET, a low-molecular-weight, redox-active species, referred to as a mediator, is introduced to shuttle electrons between the enzyme active site and the electrode.In this case, the enzyme catalyzes the oxidation or reduction of the redox mediator. The reverse transformation (regeneration) of the mediator occurs on the electrode surface. The major characteristics of mediator-assisted electron transfer are that (i) the mediator acts as a cosubstrate for the enzymatic reaction and (ii) the electrochemical transformation of the mediator on the electrode has to be reversible. In these systems, the catalytic process involves enzymatic transformations of both the first substrate (fuel or oxidant) and the second substrate (mediator). The mediator is regenerated at the electrode surface, preferably at low overvoltage. The enzymatic reaction and the electrode reaction can be considered as separate yet coupled. 

The preparation and application of SAM systems patterned by STM and their use in catalysis was demonstrated by Wittstock and Schuhmann [123]. The patterning (local desorption) of SAMs from alkane thiols on gold was performed by scanning electrochemical microscopy (SECM), followed by the assembly of an amino-deriva-tized disulfide and coupling of glucose oxidase to form a catalytically active pattern of the enzyme. The enzymatic activity could be monitored/imaged by SECM. 

Catalytic Oxidation of NAD(P)H A Continuously Improved Selection of Suitable ROMs This research is triggered hy at least two reasons (1) the importance of NAD(P)H/NAD(P)- - redox couples in biological systems is known, as is known the dependence of oxidation mechanisms on the oxidants [14, 82, 172-174] (2) the possibility of developing amperometric biosensors for NAD(P)+-dependent dehydrogenases. As a consequence, much attention is devoted to the regeneration of these coenzymes in their reduced or oxidized forms for their application in biosensors or in enzymatic synthesis [180]. Here, we are concerned with electrochemical regeneration [181]. 

Electro-generated and regenerated bis(bipyridine)rhodium(I) complexes were able to catalyze the selective non-enzymatically coupled electrochemical generation of NADH from NAD . The direct cathodic reduction even at very negative working potentials leads to the formation of large amounts of enzymatically inactive NAD dimers, while the indirect electrochemical reduction via the rhodium complex acting as... 

Further research for useful mediators led to the anthracycline antibiotic adria-mycin which serves as a novel mediator for the FNR or diaphorase (DP) catalyzed electrochemical reduction of NAD(P)+ [107]. This regeneration system has been satisfactory combined with NAD(P)H-dependent enzymatic reactions. The NADP+-dependent FNR/adriamycin system was coupled with GluDH. GluDH was entrapped together with FNR on the electrode surface. NADPH was efficiently... 

Association between enzymatic and electrochemical reactions has provided efficient tools not only for analytical but also for synthetic purposes. In the latter field, the possibilities of enzymatic electrocatalysis, e.g., the coupling of glucose oxidation (catalyzed either by glucose oxidase or glucose dehydrogenase) to the electrochemical regeneration of a co-substrate (benzoquinone or NAD+) have been demonstrated [171, 172]. An electroenzymatic reactor has also been developed ]172] to demonstrate how the enzyme-electrode association can be used to produce biochemicals on a laboratory scale. 

Electrochemical detection is inherently a chemical rather than a physical technique (such as ultraviolet, infrared, fluorescence, or refractive index). It is, therefore, not surprising to hnd that many imaginative postcolumn reactions have been coupled to LC-EC. These include photochemical reactions, enzymatic reactions, halogenation reactions, and Biuret reactions. In each case, the purpose is to enhance selectivity and therefore improve limits of detection. While simplicity is sacrihced with such schemes, there are many published methods that have been quite successful. 

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