Enzymes, detection immobilized

Immobilized Enzymes. The immobilized enzyme electrode is the most common immobilized biopolymer sensor, consisting of a thin layer of enzyme immobilized on the surface of an electrochemical sensor as shown in Figure 6. The enzyme catalyzes a reaction that converts the target substrate into a product that is detected electrochemicaHy. The advantages of immobilized enzyme electrodes include minimal pretreatment of the sample matrix, small sample volume, and the recovery of the enzyme for repeated use (49). Several reviews and books have been pubHshed on immobilized enzyme electrodes (50—52). 

In previous papers it was shown that the enzymatic pool of Pectolyase Y23 possesses high catalytic efficiency either as free or immobilized form in solution of pectins [28, 29] and of fresh vegetable tissues [30]. According to Baldwin and Pressor [31] the following enzymatic activities were detected in the preparation PL, PG and PE. The amount of the different enzyme detected per mg of Pectolyase Y23 and the main enzyme characteristic are quoted in Table 1... 

The enzyme is immobilized on a nylon mesh, which also acts as a diffuse reflector for the light. The dynamic range of this sensor is between 1(T5 and 10 3M. Although the primary process that determines the steady-state concentration of the p-nitrophenoxide ion is the diffusion-reaction mechanism (which is governed by concentrations of all participating species), the detection of its concentration is again subject to the limitations of optical sensing of ionic species (Section 9.4.1). There are many similar optical enzyme biosensor schemes that utilize detection of... 

When the feedback mode is possible (i.e. the enzymes is immobilized on an insulating support, Fig. 37.5a), it provides a much better lateral resolution but has only a very limited sensitivity. Therefore, it can only be applied for very active enzymes or if enzymes are bound in high surface concentrations. Bard et al. [15] gave a quantitative detection limit for this situation ... 

In one report, the activity of the HRP enzyme was studied by CL detection using the xanthine/xanthine oxidase (XOD)/luminol system. The enzymes were immobilized on glass beads trapped in a reaction chamber by weirs. It was found that when HRP was immobilized, a greater CL signal was generated than with free-solution HRP. However, when XOD (not HRP) was immobilized, enzymatic reaction products were not detectable. This was probably because the immobilized XOD enzyme had a low specific activity [721]. 

Acetylcholineesterase and choline oxidase Au foil was treated with cystamine to produce a base layer of ami-nothiolate units, was derivatized by reaction of the amino group and disodium-4,4 -diisothiocyanato-trans-stilbene-2,2 -disulfonate. Enzymes were immobilized at the isothiocyanate group via thiourea link. The bifunctional sensor for ACh was prepared by stepwise immobilization of four layers of the enzyme ChO and three layers of AChE. Choline generated was detected amperometircally with the use of 2,6-dichloro-phenolindophenol as a mediator in solution. Electrical communication between the enzyme and the electrode is achieved either by the use of ferrocenecar-boxylic acid as mediator in the assay buffer or by immobilization of [(ferrocenyl methyl)amino] hexa-noic acid on the enzyme layer. [92]... 

Both enzymes were immobilized on beads (125-315 tm) and serially used in enzyme reactors either as packed beds or as expanded beds. Again detection was based on amperometric H2O2 detection. Co-reagents, such as NADH and NH3, were added to the carrier. The expanded bed reactors showed a lower sensitivity and longer response times compared to the packed bed system. However, the risk of clogging due to sample constituents... 

The sensitive part of an electrode is covered with a membrane on which the enzyme is immobilized in immunocomplexes. The enzyme-catalyzed reaction takes place near the sensor (Mattiasson and Nilsson, 1977). The method is as fast as the thermometric assay but less sensitive. Electrode-based EIA using urease conjugates have been reviewed by Meyerhoff and Rechnitz (1980). This method has reasonably low detection limits. These promising potentiometric EIA are discussed by Boiteux et al. (1981) and Gabauer and Rechnitz (1982). 

Target analyte Immobilized enzyme Detected species... 

In many cases it is not possible or desirable to register the reaction catalyzed by an enzyme. It may be that the product is difficult to detect or that the sensitivity in the analysis one needs to apply is not high enough. Then the use of one or more additional enzymes is quite common. The strategy has been worked out for soluble enzymes, and, in the flow systems, the enzymes are either immobilized separately, or coimmobilized. The latter approach has certain advantages in the sense that a better kinetic performance can be observed in a coimmobilized enzyme sequence as compared to when the enzymes are immobilized separately [52]. However, since most assays are based on the use of an excess of immobilized enzymes, no dramatic differences are observed. 

The natural specificity of enzyme-catalyzed reactions can be used as the basis for selective detection of analytes (49, 64-68). One fruitful approach has featured potentiometric sensors with a structure similar to that of Figure 2.4.5, with the difference that the gap between the ion-selective electrode and the polymer diaphragm is filled with a matrix in which an enzyme is immobilized. 

As a first step in this direction, a surface consisting of electric eel AChE and horse serum BChE was designed. Both of these enzymes are inhibited by TPPSi and the characteristic absorbance peaks for the porphyrin-enzyme complexes are different (421 vs. 446 nm). This allows for co-immobilization of the two enzymes from a simple mixture of equal concentrations onto the entire slide surface [25]. Exposure to those compounds inhibiting BChE competitively results in a loss in absorbance at 421 nm while compounds inhibiting AChE competitively result in a loss at 446 nm. Compounds inhibiting both enzymes result in a loss at both 421 nm and 446 nm. This combination of two enzymes allows for class discrimination of those compounds, which are inhibitors of BChE, inhibitors of AChE, inhibitors of both enzymes, and inhibitors of neither enzyme. Detection limits are approximately the same for the dual enzyme system as those observed for the single enzyme systems. 

Fig.4. (top) Reaction scheme for the enzymatic determination of starch as executed in the FIA manifold shown below, where AMG refers to amyloglucosidase and GDH to glucose dehdrogenase, both of which enzymes are immobilized on porous glass in separate packed-bed reactors. The detection of the generated NADH is facilitated amperometrically by means of a modified electrode at 0 mV vs. Ag/AgCl, 0.1 M KCl. Flow rates are given in ml/min injected volume 30... 

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