Glucose Thermistor

In order to provide a steady-state signal, heat must be continuously evolved. An ideal reaction for this is the biocatalytic enzymatic reaction, which combines high substrate specificity with a high amplification factor. Thus, an enzyme-containing layer is deposited over the thermal probe, and the substrate is allowed to diffuse in. 

As it diffuses in, it reacts according to a general Michaelis-Menten equation (2.21), and the molar heat equal to the enthalpy of that reaction is evolved. For an enzymatic reaction 

This is a typical diffusion-reaction scheme, as discussed in Chapter 2 (2.13). The major difference is the unconfined heat flow, which makes the solution of the response equation, even by digital simulation, difficult. 

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Fig. 3.3 Response of glucose oxidase thermistor to glucose in the presence of different concentrations of hydrogen peroxide (adapted from Tran-Minh and Vallin, 1978, p. 1874)...

To illustrate this principle, we have chosen a lectin-glycoprotein system (130). The glycoenzymes glucose oxidase and peroxidase are bound to immobilized Concanavalin A or lentil lectin coupled to Sepharose. The immobilized lectin is packed in a small column inside a simple flow calorimeter. A continuous buffer stream (flowrate 0.75 mL/min) is pumped through a small column, at the outlet of which is placed a thermistor. This unit is well insulated from the surroundings. 

Mattiasson and Borreback [128] employed glucose oxidase and peroxidase in a thermistor devices containing immobilized Con A for the measurement of... 

Figure 20.6 Calibration curve for glucose obtained with a miniaturized enzyme thermistor containing glucose oxidase/catalase on superporous agarose using 20 n samples injected in a flow of 100 /zl min. ...

Bowers L D and Carr P W 1976 Immobilized-enzyme flow-enthalpimetric analyzer application to glucose determination by direct phosphorylation catalyzed by catalase Clin. Chem. 22 1427-33 Danielsson B and Mosbach K 1988 Enzyme thermistors Methods Enzymol. 137 181-97... 

Kiba N, Tomiyasu T and Furusawa M 1984 Flow enthalpimetric determination of glucose based on oxidation of 1,4-benzoquinone with use of immobilized glucose oxidase column Talanta 31 131-2 Scheller F, Siegbahn N, Danielsson B and Mosbach K 1985 High-sensitivity enzyme thermistor assay of L-lactate by substrate recycling Anal. Chem. 57 1740-3... 

Enzyme thermistors have so far been described for the determination of urea, penicillin, and other amides [179], for glucose, sucrose, cholesterol, uric acid, and lactate, and they have been used preferentially for medical analyses [180]. However, process control [181], environmental monitoring, and detection of enzyme activities in eluates from chromatographic columns [177] are further applications of these sometimes sensible , but sensitive and universal biosensors. 

Test strips, which are available for the determination of about ten low-molecular mass substances (metabolites, drugs, and electrolytes) and eight enzymes [356], can be considered as precursors of optoelectronic biosensors. Efficient optoelectronic sensors based on immobilized dyes have been devised for the determination of glucose, urea, penicillin, and human serum albumin [357]. Other approaches use immobilized luciferase or horseradish peroxidase to assay ATP or NADH or, when coupled with oxidases, to measure uric acid or cholesterol. These principles have not yet been generally accepted for use in routine analysis. Thermistor devices involving immobilized enzymes or antibodies for a number of clinically relevant substances have also been described. Thermometric enzyme linked immunosorbent assays are being routinely employed for monitoring the production of monoclonal antibodies. 

These examples indicate the in situ applicability of enzyme electrodes however, numerous problems have still to be solved. At present, coupling of enzyme sensors for fermentation control in a bypass arrangement appears to be more favorable [412]. Following this concept, an invertase thermistor incorporating a sterilizable filter unit has been developed [413] for the monitoring of alcoholic fermentation by immobilized yeast cells. Another thermistor has been successfully used for on-line glucose measurement under real cultivation conditions of Cephalosporium acremonium [414]. Similar calorimetric devices are suitable for other fermentation processes and in environmental analysis. 

The problems of adequate sampling are obvious upon examination of Figures 22-11 and 22-12. In both cases, an enzyme thermistor was used for quasi on-line glucose determination of a fed-batch cultivation of Cephalosporium acremonium [86]. Sampling was performed by micro- or ultraflltration. Only the analysis of ultraflltered samples shows good agreement between enzyme thermistor and off-line data. 

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