Thermal enzyme sensors

Thermal enzymes sensors, or enthalpimetric enzyme sensors, measure the concentration of a substrate using the variation in the enthalpy of an enzymatic reaction. According to whether the reaction is exothermic or endothermic, the increase or decrease in temperature of the reaction medium can be monitored using a temperature sensor. Thermistors or thermopiles are usually employed because of their large sensitivity to small variations in temperature. Two thermistors are placed in a thermally stable environment to allow a differential measurement of small changes in temperature. The method used to determine the variation in enthalpy depends on whether the enzyme is immobilized at the reactor or directly on the thermosensitive transducer. 

When the enzyme is immobilized inside a reactor, usually an enzymatic column [219], two thermistors are placed on the outside of the column. One of the thermistors serves as a reference, and the other is the working thermistor they can be placed in series or in parallel. When they are placed in series, only one enzymatic column is used, the reference thermistor is upstream from the column and the working thermistor is downstream from it. When they are mounted in parallel, the incoming flow of substrate is divided between two columns, one of which contains the immobilized enzyme. The working thermistor is placed downstream from the enzymatic column and the reference thermistor is placed downstream from the reference column. Although this second method is more complex and requires two pumps, it offers a better analytical performance because of the symmetry of the system. 

The use of an enzymatic reactor associated with one or two thermistors on the outside of the column to measure the increase in temperature arising from the reaction, cannot be considered as a real biosensor. This is really microcalorimetry and requires cumbersome equipment (reactors, pumps, etc.), a large amount of immobilized enzyme and hence entails a large consumption of substrate. 

A real thermal enzyme sensor must involve, as its name suggests, a thermal transducer (thermistors or thermopiles) with a layer of immolnlized enzyme. The enzyme transforms die substrate and ensures the liberadon or consumption of calories that are detectable in situ by the transducer. 

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Development of simple, low-cost calorimeters for routine analysis, called thermal enzyme probes (TEP), has been attempted by several groups. These are fabricated by attaching the enzyme directly to a thermistor [4, 5]. However, in this configuration, most of the heat evolved in the enzymic reaction is lost to the surrounding, resulting in lower sensitivity. The concept of TEP was essentially designed for batch operation, in which the enzyme is attached to a thin aluminum foil placed on the surface of the Peltier element that acts as a temperature sensor [6]. 

Next we have to define the boundary and the initial conditions. For so called zero flux sensors there is no transport of any of the participating species across the sensor/enzyme layer boundary. Such condition would apply to, e.g., optical, thermal or potentiometric enzyme sensors. In that case the first space derivatives of all variables at point x are zero. On the other hand amperometric sensors would fall into the category of non-zero-flux sensors by this definition and the flux of at least one of the species (product or substrate) would be given by the current through the electrode. 

This reaction produces a low variation in enthalpy and so a glass sheath must be positioned around the thermistor to limit thermal diffusion. This device improves the sensitivity and the detection limit of the biosensor. The same pH effect is found for this sensor (Rgure 4.38) as was found for other enzyme sensors, but the response time is much shorter (less than 10 seconds). 

Possibilities for Enzymes in Implantable Fuel Cells There is significant and increasing demand for power supplies for implantable medical devices, including continuous glucose monitors for diabetic patients, thermal sensors for... 

In this paper we have immobilized an enzyme within a thermally reversible hydrogel. Immobilized enzymes have been used in a variety of applications, ranging from treatment of diseases to sensors, assays, and industrial processes (15-20). When an enzyme is immobilized within a gel which exhibits reversible shrinking and swelling as the ten rature is raised and lowered through the LCST of the gel matrix polymer, the enzyme may be switched off and on as the substrate diffusion rate is regulated by the gel pore size (5). In adcfition to enzymes, a variety... 

This is the reaction taking place at the surface of the thermal sensor, the pellistor, discussed in Chapter 3. An example of a biocatalyst is the enzyme glucose oxidase (GOD) which highly selectively promotes oxidation of D-glucose to gluconic acid. 

The geometry shown here corresponds to a semi-infinite planar diffusion. Other geometries (e.g., radial geometries) typical for microsensors can be used. The enzyme-containing layer is usually a hydrogel, whose optimum thickness depends on the enzymatic reaction, on the operating pH, and on the activity of the enzyme (i.e., on the Km). Enzymes can be used with nearly any transduction principle, that is, thermal, electrochemical, or optical sensors. They are not, however, generally suitable for mass sensors, for several reasons. The most fundamental one is the fact... 

In spite of all their advantages, sensitivity and selectivity, bio-sensors, however, do possess disadvantages connected with thermal and timely instability, high cost of bio-receptors and the need to add substrates in the solution under analysis as signal-generating substances. Some attempts to synthesize and use as receptors chemical organic catalytic systems, which will ensure the required selectivity and response rate, have become the basis for developing enzyme-free sensors [11], or biomimetic sensors. 

For monitoring catalytic (enzymatic) products, various techniques, such as spectrophotometry [32], potentiometry [33,34], coulometry [35,36] and amperometry [37,38], have been proposed. An advantage of these sensors is their high selectivity. However, time and thermal instability of the enzyme, the need of a substrate use and indirect determination of urea (logarithmic dependence of a signal upon concentration while measuring pH) cause difficulties in the use and storage of sensors. 

Correlation between the results, obtained by using the known sensors and methods and the proposed sensors, is very good. The approach demonstrates perspectives for creating enzyme-free chemical/biochem-ical sensors. It also allows the elimination of disadvantages of enzyme-containing sensors, particularly, their time and thermal instability, high cost and necessity to use substrate in the analyzed solution. 

Why is the operational stability of an enzyme-based thermal sensor for oxalic acid expected to be poorer than an amperometric oxalate sensor prepared using the same enzyme, oxalate oxidase ... 

The integrated system, including transducer and enzyme reactor, provides improved reliability and stability in multianalyte determinations, as compared with discrete thermal sensor systems. In addition, application of micromachining and IC technologies is of benefit for the manufacture of uniform, cheap thermal transducers with flexible shape, size, and resistance, as well as delicate microstructure on the chips. The good thermal insulation of the transducers from the flow stream eliminates interference from the reactants on the transducers, and the intrinsic stability of the transducers obviates the need for frequent recalibration of the sensors. 

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