Biosensors enzyme-catalyzed reactions

The use of additional membranes, which selectively convert nonionic analytes into ionic species that can be determined via ISEs is another common approach. An abundance of ingenious designs make use of biocatalysts for the development of potentiometric biosensors. Much of the earlier designs have made use of enzymes as the molecular recognition element. The products that are associated with such enzyme-catalyzed reactions can be readily monitored with the potentiometric transducer by coating the traditional electrodes with the enzyme. 

During glucose addition to oxygen saturated solution the reduction current response of the biosensor decreased, which resulted from the electrocatalytic reaction restrained to the enzyme catalyzed reaction between the oxidized form of GOD and glucose. With increasing glucose concentration the catalytic reduction current of oxygen decreased. 

As mentioned in the previous section, the response, the stability and the enzyme activity found greatly enhanced at the MWCNT platform. Other than CNTs, AuNPs also possess some unique properties and recent years it has been widely employed in the biosensors to immobilize biomolecules. Thus in this section we discuss about the application of AuNP matrix for the immobilization of AChE for pesticide sensor development. With the use of AuNPs, the efficiency and the stability of the pesticide sensor gets greatly amplified. Moreover, the nanoparticles matrix offers much friendly environment for the immobilized enzyme and thus the catalytic activity of the enzyme got greatly amplified. Interestingly, Shulga et al. applied AChE immobilized colloidal AuNPs sensor for the nM determination of carbofuran, a CA pesticide [16], The enzyme-modified electrode sensor was also utilized for the sensitive electrochemical detection of thiocholine from the enzyme catalyzed hydrolysis of acetylthiocholine chloride (ATCl). The fabrication and the enzyme catalyzed reaction at the AuNPs coated electrode surface is shown in Fig. 6. 

The first applications of enzymes in bioanalytical chemistry can be dated back to the middle of nineteenth century, and they were also used for design of first biosensors. These enzymes, which have proved particularly useful in development of biosensors, are able to stabilize the transition state between substrate and its products at the active sites. Enzymes are classified regarding their functions, and the classes of enzymes are relevant to different types of biosensors. The increase in reaction rate that occurs in enzyme-catalyzed reactions may range from several up to e.g. 13 orders of magnitude observed for hydrolysis of urea in the presence of urease. Kinetic properties of enzymes are most commonly expressed by Michaelis constant Ku that corresponds to concentration of substrate required to achieve half of the maximum rate of enzyme-catalyzed reaction. When enzyme is saturated, the reaction rate depends only on the turnover number, i.e., number of substrate molecules reacting per second. 

CNTs present good electrical communication, which renders feasible the electron transfer from protein to the electrode. For this reason many laboratories have turned their scientific interests in the fabrication of CNT-modified electrodes onto which enzymes or nucleic acids are immobilized. As it can be seen from Table 2.3, most of the works in the field of CNT-protein conjugates are about the development of new biosensors. CNT-biosensors have shown efficient electrical communications and promising sensitivities required for applications as antigen recognition, enzyme-catalyzed reactions and deoxyribonucleic acid (DNA) hybridizations [124]. The presence of CNTs facilitates the transportation of the signal from the enzyme to the electrode. The use of CNT-modified electrodes permits... 

The kinetics of enzyme-catalyzed reactions can be very complex, and the mathematical representations for the effect of the concentrations of substrate, product, cofactors, and inhibitors are presented in a variety of textbooks in this field [1]. The exact form of this dependence of enzyme activity on these factors might have a profound effect on the behavior of an enzyme biosensor. However, one can delineate general rules of thumb concerning the properties of enzymes for the preliminary design of enzyme-based sensors. 

Of comparable general importance is the bacterial luciferase system [226-228], which opens up the opportunity to combine any NAD(P) " -dependent enzyme-catalyzed reaction with a luminometric measurement. Even the chemiluminescent luminol reaction can be used for biosensing, because it can be coupled to any oxidase reaction that produces HjOj [225, 229]. The logical further development of these systems towards real optical biosensors has recently been reported by Blum et al. [230], who immobilized the light-producing systems onto the tip of optical fibers and thus obtained fiber-optic luminescence probes. 

Biosensors based on membrane electrodes are attractive from several perspectives. Firsl. complex organic molecules can be determined with the convenience, speed, and ease that characterize ion-selective measurements of inorganic species. Second, enzyme-catalyzed reactions occur under mild conditions of temperature and pH and at relatively low suhstrale concentrations. Third, combining the selectivity of the enzymatic reaction and the electrode response provides results that are free from most interferences. 

In the simplest format, amperometric biosensors are constructed in such a way that in the absence of other electroactive sample components, the faradaic current produced at the electrode surface is produced solely due to presence of the product of an enzyme-catalyzed reaction as illustrated in Figure 1. Three possible configurations available for an amperometric biosensing electrode system [4] are illustrated in Figure 2 ... 

In potentiometric biosensors the biological recognition reaction causes a modulation of a redox potential, a transmembrane potential, or the activity of an ion. So the potentiometric biosensors utilize the measurement of a potential at an electrode in reference to another electrode (Bard and Faulkner, 1980). Mostly, it is comprised of a permselective outer layer and membrane or sensitive surface to a desired species (a bioactive material), usually an enzyme. The enzyme-catalyzed reaction generates or consumes a species, which is detected by an ion-selective electrode. Usually a high-impedance voltmeter is used to measure the electrical potential difference or electromotive force (EMF) between two electrodes at near-zero current. The basis of this type of biosensor is the Nemst equation, which relates the electrode potential (E) to the concentration of the oxidized and reduced species. For the reaction aA + ne bB, the Nemst equation can be described as the following. 

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 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 °. 

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