Enzyme sensors principles

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. 

In principle, there are two possible ways to measure this effect. First, there is the end-point measurement (steady-state mode), where the difference is calculated between the initial current of the endogenous respiration and the resulting current of the altered respiration, which is influenced by the tested substances. Second, by kinetic measurement the decrease or the acceleration, respectively, of the respiration with time is calculated from the first derivative of the currenttime curve. The first procedure has been most frequently used in microbial sensors. These biosensors with a relatively high concentration of biomass have a longer response time than that of enzyme sensors. Response times of comparable magnitude to those of enzyme sensors are reached only with kinetically controlled sensors. 

The interplay of mass transfer, partition and enzymatic substrate conversion determines the dynamic measuring range, response time, and accessibility towards interferences of enzyme sensors. New principles for designing the analytical performance by coupled enzyme reactions are presented in this paper ... 

Aizawa presented an overview on the principles and applications of the electrochemical and optical biosensors [61]. The current development in the biocatalytic and bioaffinity bensensor and the applications of these sensors were given. The optical enzyme sensor for acetylcholine was based on use of an optical pH fiber with thin polyaniline film. 

Amperometric Sensors Principle and Evolution of Enzyme Electrodes... 

A new development in the field of potentiometric enzyme sensors came in the 1980s from the work of Caras and Janata (72). They describe a penicillin-responsive device which consists of a pH-sensitive, ion-selective field effect transistor (ISFET) and an enzyme-immobilized ISFET (ENFET). Determining urea with ISFETs covered with immobilized urease is also possible (73). Current research is focused on the construction and characterization of ENFETs (27,73). Although ISFETs have several interesting features, the need to compensate for variations in the pH and buffering capacity of the sample is a serious hurdle for the rapid development of ENFETs. For detailed information on the principles and applications of ENFETs, the reader is referred to several recent reviews (27, 74) and Chapter 8. 

At the beginning of the 70s the first enzyme sensors with calorimetric indication (Mosbach, 1977) and later on those using optical indication, e.g., the light-emitting diode/photoreceptor system (Lowe et al., 1983), were developed. The extent of practical application of these measuring principles is far lower than that of enzyme electrodes. 

The choice of the indicator electrode is largely determined by the species involved in the sensing reaction. Oxygen and H2O2, which are the cosubstrate and product of oxidases, as well as NAD(P)H—the cosubstrates of about 300 pyridine nucleotide-dependent dehydrogenases—can be determined amperometrically. Based on this principle, many enzyme sensors of the first generation have been developed and commercialized (see section 17.4 and table 17.2). 

Abstract. The biosensors described in this work, for the monitoring of pesticides, are based on acetylcholinesterase immobilized on the surface of screen-printed electrodes. The principle of the biosensor is that the degree of inhibition of an enzyme sensor by a pesticide is dependent on the concentration of that pesticide. The DPV technique was used as a detection method and methyl-paraoxon as a reference pesticide for sensor calibration. 

Figure 77. Operating principle of a mediator-modified am-perometric biosensor, involving an oxidase or a dehydrogenase (in the case of a reductase, electron transfer would proceed from the electrode to the analyte substrate) a) Amperometric enzyme sensor b) Mediator-modified electrode c) Redox electrode...

The original electrode was developed for detecting urea in blood and urine samples. An enzyme sensor for measuring urea in milk has been reported by an Indian group [31]. The working principle is identical to that of Guilbault s sensor. The sensor prepared for milk analysis, however, is a flat cell fabricated with screen-printed technology. 

Table 1 lists the main sensors used for diagnosis or cure. Moreover, classifications, measurement principles, and applications for diagnosis or cure also will be described. If commercial availability can be confirmed, it is so indicated. For enzyme sensors, the detection method (electrode) for enzyme reaction products is stated in the supplemental column. 

D-glucose and the three-enzyme system GOx, mutarotase and invertase for sucrose estimation. A common format was adopted to facihtate design and operation, in this case immobilization method, the fact that all enzymes used were oxidases and that a common detection principle, reoxidation of H2O2 generated product, was chosen (except for ascorbic acid which was estimated directly). Pectin, a natural polysaccharide present in plant cells, was used as a novel matrix to enhance enzyme entrapment and stabilization in the sensors. Interferences related to electrochemi-caUy active compounds present in fruits under study were significantly reduced by inclusion of a suitable cellulose acetate membrane diffusional barrier or by enzymatic inactivation with ascorbate oxidase. Enzyme sensors demonstrated expected response with respect to their substrates, on analyte average concentration of 5 mM. 

An enzyme sensor can be considered as the combination of a transducer and a thin enzymatic layer, which normally measures the concentration of a substrate. The enzymatic reaction transforms the substrate into a reaction product that is detectable by the transducer. An extension of this definition is that the concentration of any substance can be measured provided that its presence affects the rate of an enzymatic reaction this is especially true for enzyme inhibitors. We will first examine the principle of enzyme sensors and the theoretical and practical aspects of their operation. We will then describe the various sensors, classified according to their detection mode. 

Once the enzyme sensor has been in contact with the inhibitor, it is rinsed with a solution containing a reactivating agent. In principle, the injection of substrate should give the reference peak again. 

Kakehi N, Yamazaki T, Tsugawa W, Sode K. 2007. A novel wireless glucose sensor employing direct electron transfer principle based enzyme fuel cell. Biosens Bioelectron 22 2250-2255. 

Another possibility is to immobilise enzymes either on the sensor element itself or in the vicinity of the sensing element. The operation principle is in most cases a semi-continuous spectral difference measurement in combination with a kinetic data evaluation. A sample containing the analyte of interest is recorded by the sensor immediately after contact with the sample and again after a certain time. Provided that no other changes in the composition of the sample occur over time, the spectral differences between the two measurements are characteristic for the analyte (and the metabolic products of the enzymatic reaction) and can quantitatively evaluated. Provided that suitable enzymes are available that can be immobilised, this may be a viable option to build a sensor, in particular when the enzymatic reaction can not (easily) be monitored otherwise, e.g. by production or consumption of oxygen or a change of pH. In any case, the specific properties and stumbling blocks related to enzymatic systems must be observed (see chapter 16). 

The detection modes commonly applied in enzyme-based optical fiber sensors are based on one of the following principles ... 

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