Enzyme biosensors product concentration

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. 

The response of an enzyme sensor in the steady state depends largely on the ratio of the substrate concentration [5] to the enzyme Michaelis constant K. When [S K is large, the reaction rate reaches a maximal value V,, which is proportional to the number of active sites of the immobilized enzyme. The reaction rate is independent of the substrate concentration, and the product concentration at the contact with the electrode is the same for all high substrate concentration. The quantify of enzyme in the layer determines the linear zone in the response to the substrate concentration. This zone corresponds to first-order kinetics with respect to substrate concentration, whereas the region with a plateau has zeroth-order kinetic. When the substrate concentration is very high([5] K ), the biosensor is no longer capable of determining the substrate but may determine inhibitors which affect the minimal rate of the enzymatic reaction... 

Mizutani et al. (2003) determined the concentration of acetic acid in a manner similar to that of Mieliauskiene et al. (2006), the detection being performed using a combination of FIA with amperometric tri-enzyme biosensor detection. The biosensor was prepared by immobilizing AK, PK, and PyOx on a poly(dimethylsiloxane) (PDMS)-coated electrode. The oxygen consumption was monitored using the PDMS-coated electrode without interference from the PyOx reaction product (hydrogen peroxide). Thus, the biosensor-based system could be used for the determination of acetic acid from 0.05 to 20 mM with a sampling rate of 20 h and it remained stable for one month. This system was applied in the analysis of wine samples (Mizutani et al., 2003). 

When an amperometric electrode is used as the transducer of a biosensor, there is a consumption of reaction products this is the major difference from a potentiometric electrode. The diffusion-reaction equations ((3) and (4)) still apply, assuming that the product concentration at the transducer-active layer interface is zero ([P] = 0). This hypothesis corresponds to the maximal sensitivity of the biosensor. The flow of product towards the transducer can be limited by mass transfer, either in the semi-permeable membrane, which separates the enzyme from the sample medium [139], or in the active enzymatic layer [140]. When the semi-permeable membrane is the limiting factor of a diffusion process, all of the product formed is... 

Potcntiomctric Biosensors Potentiometric electrodes for the analysis of molecules of biochemical importance can be constructed in a fashion similar to that used for gas-sensing electrodes. The most common class of potentiometric biosensors are the so-called enzyme electrodes, in which an enzyme is trapped or immobilized at the surface of an ion-selective electrode. Reaction of the analyte with the enzyme produces a product whose concentration is monitored by the ion-selective electrode. Potentiometric biosensors have also been designed around other biologically active species, including antibodies, bacterial particles, tissue, and hormone receptors. 

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... 

Tissue electrodes [2, 3, 4, 5, 45,57], In these biosensors, a thin layer of tissue is attached to the internal sensor. The enzymic reactions taking place in the tissue liberate products sensed by the internal sensor. In the glutamine electrode [5, 45], a thick layer (about 0.05 mm) of porcine liver is used and in the adenosine-5 -monophosphate electrode [4], a layer of rabbit muscle tissue. In both cases, the ammonia gas probe is the indicator electrode. Various types of enzyme, bacterial and tissue electrodes were compared [2]. In an adenosine electrode a mixture of cells obtained from the outer (mucosal) side of a mouse small intestine was used [3j. The stability of all these electrodes increases in the presence of sodium azide in the solution that prevents bacterial decomposition of the tissue. In an electrode specific for the antidiuretic hormone [57], toad bladder is placed over the membrane of a sodium-sensitive glass electrode. In the presence of the antidiuretic hormone, sodium ions are transported through the bladder and the sodium electrode response depends on the hormone concentration. 

Electrodes based on enzyme activity. These are selective and sensitive devices that may be used to measure substrate concentrations. A biosensor based on glucose oxidase is used to measure the concentration of glucose by detecting the production of H202. 

Enzymatic reactions coupled to optical detection of the product of the enzymatic reaction have been developed and successfully used as reversible optical biosensors. By definition, these are again steady-state sensors in which the information about the concentration of the analyte is derived from the measurement of the steady-state value of a product or a substrate involved in highly selective enzymatic reaction. Unlike the amperometric counterpart, the sensor itself does not consume or produce any of the species involved in the enzymatic reaction it is a zero-flux boundary sensor. In other words, it operates as, and suffers from, the same problems as the potentiometric enzyme sensor (Section 6.2.1) or the enzyme thermistor (Section 3.1). It is governed by the same diffusion-reaction mechanism (Chapter 2) and suffers from similar limitations. 

OPH-based biosensors have been fully discussed in previous reviews [2,165]. AChE-based biosensors are based on the principle that OP pesticides have an inhibitory effect on the activity of AChE that may be permanent or partially reversible. The extent of the inhibition is directly related to the concentration of the pesticide and therefore enzyme activity may be used as a measure of the inhibition [166]. The amperometric measurement of AChE activity can be based on the measurement of any of the following three mechanisms [167] (1) production of hydrogen peroxide from choline, (2) oxygen consumption during the enzyme reaction or (3) production of electroactive compounds directly from the oxidation of acetylthiocholine chloride such as thiocholine. The measurement of hydrogen peroxide and oxygen consumption has been described in more details in other reviews [167]. 

A biosensor utilizes a biological component to translate the concentration of a specific analyte of interest into a signal detectable by some chemical or physical means (7). Successful operation of a biosensor requires that the biological component and the signal it transduces be localized to and concentrated within a region in close proximity to the detection system. Immobilization of enzymes within a polymeric matrix ensures concentration and localization of the enzymic reaction, and creates a convective barrier, thus preventing dilution and convective removal of the product species before it is detected. Enzymes immobilized on or near the detection system are frequently used as the... 

Amperometric sensors monitor current flow, at a selected, fixed potential, between the working electrode and the reference electrode. In amperometric biosensors, the two-electrode configuration is often employed. However, when operating in media of poor conductivity (hydroalcoholic solutions, organic solvents), a three-electrode system is best (29). The amperometric sensor exhibits a linear response versus the concentration of the substrate. In these enzyme electrodes, either the reactant or the product of the enzymatic reaction must be electroactive (oxidizable or reducible) at the electrode surface. Optimization of amperometric sensors, with regard to stability, low background currents, and fast electron-transfer kinetics, constitutes a complete task. 

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