Enzyme biosensors oxidation reactions

Hydrogen peroxide (H2O2) is one of the most important products or substrate of enzyme catalyzed oxidation reactions. Most common enzymes used in biosensors are oxidases, which catalyze the model oxidation reactions ... 

Several biosensors are commercially available. One of the most useful is the glucose sensor. The standard sensor determines glucose concentration based on the glucose oxidase enzyme. The chemical reaction for oxidation of glucose is ... 

More than 90% of commercially available enzyme-based biosensors and analytical kits contain oxidases as terminal enzymes responsible for generation of analytical signal. These enzymes catalyze oxidation of specific analyte with molecular oxygen producing hydrogen peroxide according to the reaction ... 

The first enzyme biosensor was a glucose sensor reported by Clark in 1962 [194], This biosensor measured the product of glucose oxidation by GOD using an electrode which was a remarkable achievement even though the enzyme was not immobilized on the electrode. Updark and Hicks have developed an improved enzyme sensor using enzyme immobilization [194], The sensor combined the membrane-immobilized GOD with an oxygen electrode, and oxygen measurements were carried out before and after the enzyme reaction. Their report showed the importance of biomaterial immobilization to enhance the stability of a biosensor. 

Bioelectrocatalysis involves the coupling of redox enzymes with electrochemical reactions [44]. Thus, oxidizing enzymes can be incorporated into redox systems applied in bioreactors, biosensors and biofuel cells. While biosensors and enzyme electrodes are not synthetic systems, they are, essentially, biocatalytic in nature (Scheme 3.5) and are therefore worthy of mention here. Oxidases are frequently used as the biological agent in biosensors, in combinations designed to detect specific target molecules. Enzyme electrodes are possibly one of the more common applications of oxidase biocatalysts. Enzymes such as glucose oxidase or cholesterol oxidase can be combined with a peroxidase such as horseradish peroxidase. 

The results obtained with the biosensor for five different honey samples were compared with those obtained with a commercial enzyme kit from R-biopharm using spectrophotometric detection (Table 16.1). This method is based on the phosphorylation of gluconate by gluconate kinase in the presence of ATP, then the 6-phospho-D-gluconate produced was oxidized with NADP+ by 6-phosphogluconate dehydrogenase. The reduced form of NADP, NADPH, formed in this oxidation reaction is measured spectrophotometrically at 340 nm. 

This enzyme-based biosensor uses glucose oxidase (GO) as a chemical recognition element, and an amperometric graphite foil electrode as the transducer. It differs from the first reported glucose biosensor discussed in the introduction to this chapter in that a mediator, 1,1 -dimethylferricinium, replaces molecular oxygen as the oxidant that regenerates active enzyme. The enzymatic reaction is given in Eq. 7.15, and the electrochemical reaction that provides the measured current is shown in Eq. 7.16. 

Most of bacterial biosensors are based on the operon luxCDABE that codes for the bacterial luciferase founded in the marine bacteria V. fischeri and V. harveyi, and for an essential aldehyde substrate that would otherwise have to be supplied exogenously. The cluster luxAB cassette codes for the luciferase whereas luxCDE encodes a fatty acid reductase complex. The latter enzymes are responsible for the synthesis of the long-chain aldehyde that is required as substrate in the bioluminescence reaction (Meighen and Dunlap, 1993 Hakkila et al., 2002). Luciferase catalyses the oxidation reaction of flavin mononucleotide (FMNH2). A long-chain (7 to 16 carbons) aldehyde is reduced in presence of oxygen by the aldehyde reductase. The outcome of the bioluminescent reaction can be expressed as follows ... 

The process depicted for phenol in equations 5 consists of an enzyme-catalyzed oxidation to a quinone, and a reduction process taking place at the electrode these reactions may serve for electrode calibration. The development of AMD biosensors for detection of phenols in environmental waters has been described for phenoloxidases such as tyrosinases and laccases and less specific oxidases such as peroxidases. Such biosensors may be part of a FIA system for direct determination of phenols or may serve as detectors for LC °. 

Enzymes were historically the first molecular recognition elements [2-6] included in biosensors and continue to be the basis for a significant number of pubUcations in this field. Amperometric biosensors based on glucose oxidase (GOx) are the most famous example of biosensors applied to medical diagnostic. The three modes of oxidation reactions that occur in redox enzyme-based biosensors, like GOx, are referred to as first, second, and third generation as follows [7] ... 

The electrochemical potential of a redox mediator (EJJ,) must be such that it provides a thermodynamic driving force to facilitate electron transfer with the enzyme to be used. Because of this requirement, the redox potential of the mediator determines the operational potential of the biocatalytic electrode. Thus for an enzymatic oxidation reaction, must be higher than the redox potential of the enzyme ( en) whereas the reverse is true for an enzymatic reduction reaction. The difference between and is defined as the mediator-induced overpotential (A et) and is the potential required for electron transfer to occur between the enzyme and mediator. In the context of a biosensor, a large overpotential can lead to an artificially inflated signal due the oxidation of biological interferants such as ascorbate. Additionally a large overpotential limits the open circuit potential in the context of a biofuel cell. It is therefore desirable to minimize the electrochemical overpotential however, there exists a limit to the minimum overpotential required to facilitate rapid electron exchange between the enzyme and mediator. 

Oxidation of P-nicotinamide adenine dinucleotide (NADH) to NAD+ has attracted much interest from the viewpoint of its role in biosensors reactions. It has been reported that several quinone derivatives and polymerized redox dyes, such as phenoxazine and phenothiazine derivatives, possess catalytic activities for the oxidation of NADH and have been used for dehydrogenase biosensors development [1, 2]. Flavins (contain in chemical structure isoalloxazine ring) are the prosthetic groups responsible for NAD+/NADH conversion in the active sites of some dehydrogenase enzymes. Upon the electropolymerization of flavin derivatives, the effective catalysts of NAD+/NADH regeneration, which mimic the NADH-dehydrogenase activity, would be synthesized [3]. 

In AChE-based biosensors acetylthiocholine is commonly used as a substrate. The thiocholine produced during the catalytic reaction can be monitored using spectromet-ric, amperometric [44] (Fig. 2.2) or potentiometric methods. The enzyme activity is indirectly proportional to the pesticide concentration. La Rosa et al. [45] used 4-ami-nophenyl acetate as the enzyme substrate for a cholinesterase sensor for pesticide determination. This system allowed the determination of esterase activities via oxidation of the enzymatic product 4-aminophenol rather than the typical thiocholine. Sulfonylureas are reversible inhibitors of acetolactate synthase (ALS). By taking advantage of this inhibition mechanism ALS has been entrapped in photo cured polymer of polyvinyl alcohol bearing styrylpyridinium groups (PVA-SbQ) to prepare an amperometric biosensor for... 

Although the inhibition-based biosensors are sensitive, they are poor in selectivity and are rather slow and tedious since the analysis involves multiple steps of reaction such as measuring initial enzyme activity, incubation with inhibitor, measurement of residual activity, and regeneration and washing. Biosensors based on direct pesticide hydrolysis are more straightforward. The OPH hydrolyzes ester in a number of organophospho-rus pesticides (OPPs) and insecticides (e.g. paraoxon, parathion, coumaphos, diazinon) and chemical warfare agents (e.g. sarin) [53], For example, OP parathion hydrolyzes by the OPH to form p-nitrophenol, which can be measured by anodic oxidation. Rainina... 

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