Coupled enzyme reactions, biosensors

The final method of coupling enzyme reactions to electrochemistry is to immobilize a biocatalytic material directly at the electrode surface. This biocatalytic material can be an immobilized enzyme, bacterial particles, or a tissue slice, as shown in Fig. 8. The biocatalyst converts substrate (analyte) into product, which is measured by the electrode. Electrodes of this type can be potentiometric or Faradaic, and are often referred to as biosensors. ... 

Of all types of biosensors, metabolism sensors based on the molecular analyte recognition and conversion have been most intensively studied. According to the degree of integration of the biocomponents they can be classified into monoenzyme sensors, biosensors using coupled enzyme reactions, organelle, microbial, and tissue-based sensors. The sequence of the following sections corresponds to this classification. 

Fig. 140. Internal signal processing in biosensors using coupled enzyme reactions. (Redrawn from Scheller et al., 1985b).

Fig. 141. Coupling of molecular recognition and internal signal processing, e.g. amplification and chemical filtering, by coupled enzyme reactions in biosensors.

The third chapter concentrates on metabolism sensors, which are arranged according to the degree of biocatalyst integration. The various different ways of coupling enzymes with transducers in monoenzyme sensors are exemplified by the determination of glucose and urea. The current state of the art is shown for monoenzyme sensors for some further 25 analytes and classes of analytes. Coupled enzyme reactions are shown to provide expansion of the biosensor concept to new analytes and to multiparameter assays as well as to an improvement of such analytical parameters as specificity and sensitivity. This chapter offers for the first time a complete overview of the potentials of coupled enzyme reactions in biosensors. 

The signal-concentration dependence for electrochemical biosensors is linear between one and three concentration decades. The lower limit of detection is at 0.2 mmol I" with potentiometric and 1 jUmoll with amperometric enzyme electrodes. The use of amplification reactions allows us to decrease the detection limit to the nanomolar to picomolar range. Whereas the response time of potentiometric enzyme electrodes averages 2-10 min, with amperometric ones an assay can be conducted within a few seconds up to 1 min. This permits up to several hundred determinations per hour to be performed. Increasing complexity of the biochemical reaction system, e.g. by coupled enzyme reactions, may bring about an increase in the overall measuring time. 

Types of Coupled Enzyme Reactions Used in Biosensors... 

In biosensors, interactions between the immobilized biomolecule and the sample, which is often a highly complex matrix, may cause undesired binding events or measuring effects. Particularly in biosensors that use coupled enzyme reactions the substrates of each reaction will interfere therefore, increasing complexity of biosensors results in a decreased selectivity. Interferences can also occur on the level of the transducer reaction. 

Coupled Enzyme Reactions in Electrochemical Enzyme Sensors 455 Tab. 6 Enzymatic analyte recycling for signal enhancement in biosensors... 

Coupled enzyme reactions mimic certain metabolic situations in organelles and cells that have themselves already been exploited in biosensors [367], [368], The use of enzyme sequences facilitates ... 

The simple cases where one enzyme is employed afford a limited scope of potential targets. Usually two or more enzyme reactions are coupled, as exemplified by the development of a piezoelectricaHy-transduced biocatalytic biosensor that couples two enzyme reactions to detect glucose [492-62-6] ... 

Figure 6.7 illustrates the voltammetric response of the third-generation SOD-based 02 biosensors with Cu, Zn-SOD confined onto cystein-modified Au electrode as an example. The presence of 02" in solution essentially increases both the cathodic and anodic peak currents of the SOD compared with its absence [150], Such a redox response was not observed at the bare Au or cysteine-modified Au electrodes in the presence of 02". The observed increase in the anodic and cathodic current response of the Cu, Zn-SOD/cysteine-modified Au electrode in the presence of 02 can be considered to result from the oxidation and reduction of 02, respectively, which are effectively mediated by the SOD confined on the electrode as shown in Scheme 3. Such a bi-directional electromediation (electrocatalysis) by the SOD/cysteine-modified Au electrode is essentially based on the inherent specificity of SOD for the dismutation of 02", i.e. SOD catalyzes both the reduction of 02 to H202 and the oxidation to 02 via a redox cycle of its Cu (II/I) complex moiety as well as the direct electron transfer of SOD realized at the cysteine-modified Au electrode. Thus, this coupling between the electrode and enzyme reactions of SOD could facilitate the development of the third-generation biosensor for 02". ...

The simple cases where one enzyme is employed afford a limited scope of potential targets. Usually two or more enzyme reactions are coupled, as exemplified by the development of a piezoelectrically-transduced biocatalytic biosensor that couples two enzyme reactions to detect glucose [492-62-6], C6H120 > (3) (13). In this biosensor a quartz radio crystal is functionalized with the enzyme glucose-6-phosphate dehydrogenase. As shown in Figure 3, a thin film of Prussian blue [14038 43-8], C18N18Fe7, is then coated onto the crystal. 

Table 13.2 summarises the different approaches used to construct enzyme electrochemical biosensors for application to food analysis based on the different types of enzymes available. Generally, the main problems of many of the proposed amperometric devices have been poor selectivity due to high potential values required to monitor the enzyme reaction, and poor sensitivity. Typical interferences in food samples are reducing compounds, such as ascorbic acid, uric acid, bilirubin and acetaminophen. Electrocatalysts, redox mediators or a second enzyme coupled reaction have been used to overcome these problems (see Table 13.2), in order to achieve the required specifications in terms of selectivity and sensitivity. 

Apart from the application of complex biocatalytic systems intense efforts are being made to broaden the spectrum of measurable substances and to improve the analytical parameters of biosensors by the coupling of several different enzyme reactions. If the enzymatic conversion of the analyte does not result in a readily detectable physicochemical effect, further enzyme reactions can be coupled, leading to a measurable signal by conversion of the primary reaction product. 

From the analysis of the coupling of enzyme reaction and mass transfer the following conclusions may be drawn for the design of biosensors. 

Coupled reactions with immobilized enzymes in biosensors Studio Biophys. 119 167-70... 

Figure 14-26. Basic principles of the coupling of enzyme reactions in biosensors, a) Sequential coupling ...

Up to now the practical application of biosensors has been almost entirely limited to oxidase-based amperometric monoenzyme electrodes and pH-shifting by hydrolases. However, the internal coupling of different biocatalytic reactions in biosensors will lead to a greatly extended applicability and a substantial improvement of the analytical performance characteristics. Exciting results might be achieved by applying the concepts of chemical and genetic modification of enzymes. Further, the site-to-site directed fixation of artificially coupled enzymes could improve the speed and practicability of coupled substrate conversions. 

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