Biochemical sensors enzymes

Diffusion Currents. Half-wave Potentials. Characteristics of the DME. Quantitative Analysis. Modes of Operation Used in Polarography. The Dissolved Oxygen Electrode and Biochemical Enzyme Sensors. Amperometric Titrations. Applications of Polarography and Amperometric Titrations. 

The Dissolved Oxygen Electrode and Biochemical Enzyme Sensors... 

The type of enzyme sensor described above is highly selective and can be sensitive in operation. There are obvious applications for the determination of small amounts of oxidizable organic compounds. However, it is perhaps too early to give a realistic assessment of the overall importance of enzyme sensors to analytical chemistry. This is especially so because of parallel developments in other biochemical sensors which may be based upon a quite different physical principle. 

THE DISSOLVED OXYGEN ELECTRODE AND BIOCHEMICAL ENZYME SENSORS... 

Diffusion currents. Half-wave potentials. Characteristics of the DME. Quantitative analysis. Modes of operation used in polarography. The dissolved oxygen electrode and biochemical enzyme sensors. Amperometric titrations. Applications of polarography and ampero-metric titrations. 

In a similar manner, enzyme-containing membranes have been coupled with electrochemical devices such as an oxygen electrode to form enzyme sensors for a variety of biochemical and clinical applications (Table II). Considerable effort has been concentrated on the development of these enzyme sensors (83-86). 

Pandey, P. C. 1994. Tetracyanoquinodimethane-mediated flow injection analysis electrochemical sensor for NADH coupled with dehydrogenase enzymes. Anal. Biochem. 221 392-396. 

Entrapment of biochemically reactive molecules into conductive polymer substrates is being used to develop electrochemical biosensors (212). This has proven especially useful for the incorporation of enzymes that retain their specific chemical reactivity. Electropolymerization of pyrrole in an aqueous solution containing glucose oxidase (GO) leads to a polypyrrole in which the GO enzyme is co-deposited with the polymer. These polymer-entrapped GO electrodes have been used as glucose sensors. A direct relationship is seen between the electrode response and the glucose concentration in the solution which was analyzed with a typical measurement taking between 20 to 40 s. 

Experiment 2. Cholinesterase as a sensor on the cell surface A target of the allelochemical may also be a surface sensor-cholinesterase (Fig. 10). If after the staining with Red analogue of Ellman reagent the blue colour is absent in the allelochemical treated microspore, possible target is the enzyme (Roshchina, 2001a,b) as for alkaloid berberine tested. If after the treatment by the test allelochemical, the colour is absent or light, the compound inhibits the enzyme (also see biochemical assay in Chapter 11). 

Calcium effects. The biochemical effects of Ca "" in the cytoplasm are mediated by special Ca -binding proteins calcium sensors"). These include the annexins, calmodulin, and troponin C in muscle (see p. 334). Calmodulin is a relatively small protein (17 kDa) that occurs in all animal cells. Binding of four Ca "" ions (light blue) converts it into a regulatory element. Via a dramatic conformational change (cf 2a and 2b), Ca -calmodulin enters into interaction with other proteins and modulates their properties. Using this mechanism, Ca "" ions regulate the activity of enzymes, ion pumps, and components of the cytoskeleton. 

One essential difference between flow-through sensors based on an immobilized catalyst arises from the nature of the catalyst, viz. biochemical (usually an enzyme) or chemical (normally an inorganic species). The latter are much less numerous and frequently used. 

Flow-through optical sensors bearing one or more immobilized enzymes at their sensing microzone can be classified according to the type of physical relationship between the microzone and the detection system or instrument used into those using fibre optics (photometric and luminemetric) and those integrating a biochemical reaction and detection (usually photometric). 

Any (bio)chemical reaction is accompanied by energy conversion, most often in the form of heat production, the amount of heat produced being proportional to that of substance converted. Therefore, heat is a highly nonspecific expression of a (bio)chemical reaction but can be used as indicative for a given substance when this is selectively converted (e.g. by effect of a catalyst, particularly an enzyme). This section discusses three types of sensors based on the use of as many types of devices for measurement of the heat involved in a biochemical reaction, namely fibre optics, polymer films and thermistors. 

Therefore, this sensor integrates a biochemical and a chemical reaction with a prior separation (dialysis) and chemiluminescence detection. The process involves the following steps (a) dialysis of the enzyme (6) enzymatic oxidation of the reagent (c) derivatization of hydrogen peroxide and d) detection of the chemiluminescence produced. Such an original approach offers several advantages over similar methodologies, namely ... 

Ruzicka ei al. [57,58] developed some peculiar reflectance flow-through biosensors based on a sensing microzone accommodating an enzyme and an acid-base indicator (both in immobilized form) where spacial and temporal resolution of the biochemical and chemical reaction or the reversible separation of hydrogen ions was therefore impossible. The pH sensors developed by these authors (see Section 3.5.1.1) can be regarded as precedents for these reflectance sensors. The sensing approach used relies on... 

Phase transition in gels in response to biochemical reactions [27,28]. Polymer gels were synthesized in which an enzyme (urease) or a biologically active protein (lectin) was immobilized. The volume phase transitions were observed in such gels when biochemical reactions took place. Such mechano-biochemical gels will be used in devices such as, sensors, selective absorbers, and biochemically controlled drug release. 

Correlation between the results, obtained by using the known sensors and methods and the proposed sensors, is very good. The approach demonstrates perspectives for creating enzyme-free chemical/biochem-ical sensors. It also allows the elimination of disadvantages of enzyme-containing sensors, particularly, their time and thermal instability, high cost and necessity to use substrate in the analyzed solution. 

Surface-functionalized polymers are also of interest for uses in biochemical reactors, and biomedical sensors. The immobilization of enzymes on a polymer surface is an important example. Numerous reasons exist for attempting to immobilize enzymes on the surfaces of polymers. For one thing, immobilization often enhances the length of time over which the protein maintains its catalytic activity, compared to the same... 

Just one example will be given here.195 Evaporation of water from aqueous solutions of MEEP and the enzyme urease yields films that can be cross-linked by exposure to gamma rays. The cross-linked films absorb water to form hydrogels in which the enzyme molecules are trapped within the interstices of the gel network. Some of the enzyme molecules may also be covalently grafted to the polymer side groups. The immobilized enzyme retained approximately 80% of its activity for the conversion of urea to ammonia. This system can, in principle, be used for the immobilization of a wide variety of enzymes, and for their use in biochemical flow reactors, or in sensors. 

Photoswitchable enzymes could have an important role in controlling biochemical transformations in bioreactors. Various biotechnological processes generate an inhibitor, or alter the environmental conditions (pH, for example) of the reaction medium. Photochemical activation of enzymes that adjust environmental conditions or deplete the inhibitor to a low concentration may maintain the bioreactor at optimal performance. More specifically, integration of the photoswitchable biocataly-tic matrix with a sensory electrode might yield a feedback mechanism in which the sensor element triggers the light-induced activation/deactivation of the photosensitive biocatalyst. 

One of the exciting developments associated with ion-selective electrodes has been the fabrication of microelectrodes capable of monitoring an intracellular ion concentration. The history of these developments from the mid-1950s has been reviewed.88 a symposium held in 1996 was devoted to the history of ion-selective electrodes. One paper discussed their development and commercialization,89 another described how the 1970s was the decade in which they really became established,90 a third outlined their industrial applications,91 and a fourth traced the evolution of blood chemistry analyses using them.92 The first attempts to construct biochemical sensors by immobilizing enzymes on electrodes date from the 1960s.93... 

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