Immobilized enzyme reaction detection

Thermal Titration and Immobilized Enzyme Reaction Detection. . 319... 

Nevertheless, both immobilized enzyme reactors and biosensors are used as detection units in flow injection analysis and Hquid chromatography. Some of these flow systems are illustrated in Figure 3. The choice of flow system will be determined both by the number of analytes to be measured and by the complexity of the sample. Flow injection in combination with immobilized enzymes is used for single solute determinations, as in the systems shown in Figure 3A-C. Multiple solute determination requires a separation step whereby the chromatography column is introduced (Figure 3D). There is also an additional need for separation power when interfering matrix components in complex samples need to be eliminated in order to permit accurate quantitation of the analyte. The use of multiple flow lines with immobilized enzyme reactions in flow injection systems has been demonstrated, whereby each flow line measures a... 

Yao, T., M. Satomura, and T. Nakahara, 1994. Simultaneous determination of sulfite and phosphate in wine by means of immobilized enzyme reactions and amperometric detection in a flow-injection system. Talanta 41 2113-2119. 

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). 

Multienzyme Electrodes. Coupling the reactions of two or more immobilized enzymes increases the number of analytes that can be measured. An electro-inactive component can be converted by an enzyme to a substrate that is subsequentiy converted by a second enzyme to form a detectable end product (57). For example, a maltose [69-79-4] sensor uses the enzymes glucoamylase and glucose oxidase, which convert... 

Impractical and Theoretical Considerations The operation of an enzyme electrode is illustrated in Figure 6-1. The immobilized enzyme layer is chosen to catalyze a reaction, which generates or consumes a detectable species ... 

Other solutions to dealing with interferences in the detection of H O have included the use of a copperfll) diethyldithiocarbamate precolumn to oxidize the sample before it reaches the immobilized enzyme, as well as the use of a palladium/gold sputtered electrode which catalyzes the oxidation of hydrogen peroxide In addition, peroxidase has been used to catalyze the reaction between hydrogen peroxide and iodide ferrocyanide and organo-fluorine compounds Am-... 

The same group reported in 1986 a sensitive and selective HPLC method employing CL detection utilizing immobilized enzymes for simultaneous determination of acetylcholine and choline [187], Both compounds were separated on a reversed-phase column, passed through an immobilized enzyme column (acetylcholine esterase and choline oxidase), and converted to hydrogen peroxide, which was subsequently detected by the PO-CL reaction. In this period, other advances in this area were carried out such as the combination of solid-state PO CL detection and postcolumn chemical reaction systems in LC [188] or the development of a new low-dispersion system for narrow-bore LC [189],... 

Figure 3 CL detection systems in combination with HPLC. P, pump I, injector C, column M, mixing tee D, detector RC, reaction coil MC, mixing coil RE, recorder E, eluent R, reagent W, waste IMER, immobilized enzyme reactor.

System C is used when an immobilized enzyme reactor (IMER) is introduced into system B. The analyte(s) separated by HPLC is converted to a suitable species for CL detection with an IMER, and then mixed with the CL reagent. In this system, a buffer solution as a mobile phase and an ion-exchange-type column are preferable for an enzyme reaction. 

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). 

We also examined the preservation stability of the photoimmobilized antibodies. Although the reactivity of the antibodies dropped away over a period of 10 days when they were stored at room temperature, it was maintained for about 2 months when stored at 4°C. This result is acceptable in terms of commercial viability, though further increase in stability would be preferable. We next examined the sensitivity for the immunochips. An immu-nochip usually has a two-dimensional surface, so the detection limit for antigens can be estimated from the amount of immobilized antibodies that are present. It was difficult to increase the sensitivity of a detection system which uses a photoluminescence probe. However, we succeeded in obtaining higher sensitivity for an immunochip in which we adopted a chemiluminescence detection system using an enzyme reaction. 

Figure 27.18 Common configuration for postcolumn reactors with electrochemical analysis. (A) LC-chemical reaction-EC. Postcolumn addition of a chemical reagent (for example, Cu2+ or an enzyme). (B) LC-enzyme-LC. Electrochemical detection following postcolumn reaction with an immobilized enzyme or other catalyst (for example, dehydrogenase or choline esterase). (C) LC-EC-EC. Electrochemical generation of a derivatizing reagent. The response at the second electrode is proportional to analyte concentration (for example, production of Br2 for detection of thioethers). (D) LC-EC-EC. Electrochemical derivatization of an analyte. In this case a compound of a more favorable redox potential is produced and detected at the second electrode (for example, detection of reduced disulfides by the catalytic oxidation of Hg). (E) LC-hv-EC. Photochemical reaction of an analyte to produce a species that is electrochemically active (for example, detection of nitro compounds and phenylalanine). Various combinations of these five arrangements have also been used. [Reprinted with permission from Bioanalytical Systems, Inc.]...

Another approach to dealing with the nonelectrochemically active nature of most amino acids is to generate, in situ, chemical reactions at the electrode surfaces to produce electrochem-ically active products for detection. Related to this concept, is the online use of immobilized enzymes (142) to react with amino acids. A by-product of this reaction is hydrogen peroxide, which is then quantified by amperometric detection. 

Several techniques have been developed for the determination of purine and pyrimidine derivatives in food sample and in particular for hypoxanthine quantification biosensors (220-223) and electrochemical methods making use of immobilized enzyme electrode (224 -227), electrochemical enzymatic-based HA methods (228,229), enzyme reaction with fluorimetric detection (230), radioimmunoassay (231), colorimetric methods (232), capillary electrophoresis (233), and TLC (234). Many HPLC methods have also been developed and are reported in Table 4 (235-247) the most recent ones are described next. 

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