IMPROVED BIOSENSOR AND THE METHOD OF ITS USE

Abstract: The concentration of glucose in a fluid is measured by detecting the initial increase in heat when the glucose in the fluid is contacted with glucose oxidase, resulting in a heat-generating enzymatic reaction. The rate at which the heat is generated by the enzymatic reaction in the case of glucose has been discovered to have a linear relationship with the concentration of glucose in the fluid over the range of medically interesting glucose concentrations.

Description: DESCRIPTION IMPROVED BIOSENSOR AND THE METHOD OF ITS USE Field of the Invention This invention relates generally to the field of determining the presence or concentration of analytes in fluid samples and more specifically to an improved apparatus and method for making such determinations by detecting the initial increase in the heat of enzymatic reactions. Background of the Invention Biosensors for detecting the concentration of analytes in fluid samples are not new. Thermistors which detect the change in temperature of a fluid sample due to the heat of an enzymatic reaction are disclosed by Mosbach in US 4,021,307 and in Anal. Chem. , 1981, 53/1, 83A-94A. The use of thermistors for this purpose is also disclosed by Weaver, et al, in Biochim. Biophys. Acta, 1976, 452, 285-291. In each case the change in temperature is correlated with a concentration of analyte in the fluid sample. The use of thermopiles for such a purpose is disclosed by Guilbeau, et al in ASAIO, 1987, 10/3, 329- 335. Guilbault et al in Anal. Chem, 1983, 55 1582-1585 disclose the use of two methods for calculating the heat of an enzymatic reaction of alcohol and alcohol oxidase in a fluid sample. One method is described as the "total heat change" method and the other is described as the "rate of change in the temperature with time method". Guilbault et al show that both methods result in linear correlations between the heat detected and the concentration of alcohol in the sample over a certain low range of concentration. At higher concentrations both methods produce curves which are non-linear. There is no showing or suggestion by Guilbault et al that the "rate of change in the temperature with time method" would show a linear correlation over a large range of concentrations of glucose in fluid following an enzymatic reaction with glucose oxidase. On the contrary the teachings of Guilbault et al would suggest that other enzymes and substrates would give results similar to those given by the alcohol system; that is, that both methods of calculation would give linear curves at low concentrations and non-linear curves at higher concentrations. Linear curves are important if a test is to be commercially useful in testing fluids, such as body fluids, for constituents such as glucose. It is desirable for the curves to be linear over not only the normal range of glucose concentrations in blood (from about 73 mg/dl to about 100 mg/dl) but also over a wider range of glucose concentrations, some of which indicate the need for medical intervention (from about 20 mg/dl to about 350 mg/dl) . Curves for glucose in blood which are non-linear at higher concentrations, such as the curves shown for alcohol by Guilbault et al, would not be useful in the treatment of conditions such as diabetes because they would show about the same response for all higher concentrations of glucose in blood. Such non-linearity of curves at higher glucose concentrations is a disadvantage which must be overcome before biosensors which detect heat of enzymatic reactions are to be medically or commercially useful in sensors to test for glucose concentrations in blood. SUMMARY OF THE INVENTION This and other disadvantages of the prior art are overcome by the present invention which, in one aspect, relates to a device for determining the concentration of glucose in a fluid by detecting the heat produced by an enzymatic reaction between the glucose and glucose oxidase, the device comprising: a) detecting means for detecting the initial increase in heat, and b) correlating means, responsive to the detecting means, for correlating the initial increase in heat with concentration of glucose. In another aspect the invention relates to a method for determining the concentration of glucose in a fluid comprising: a) contacting the glucose in the fluid with the enzyme glucose oxidase to cause a heat-producing enzymatic reaction; b) detecting the initial increase in heat and; c) correlating the initial increase in heat with the concentration of the glucose in the fluid. This invention is based on the surprising and unexpected discovery that although the "total heat change" method as taught by Guilbeaut et al gives a curve which is non-linear at higher concentrations in the case of glucose and glucose oxidase reactions in a fluid, the "rate of change in the temperature with time method" provides a linear curve for such reactions across the medically interesting range of glucose concentrations in blood. For convenience, the "rate of change in the temperature with time method" is hereinafter referred to generally as measuring or correlating the initial increase in heat of the enzymatic reaction. Stated another way, the initial increase in heat refers to the increase in heat over time until the point at which a steady temperature of the fluid in the vicinity of the enzymatic reaction is attained. BRIEF DESCRIPTION OF THE DRAWING FIG. 1 is a chart showing correlations between glucose concentration and the heat of a glucose/glucose oxidase enzymatic reaction measured by both the total heat change method and the initial increase in heat method. A more detailed description of the drawing is found in connection with the Examples. DETAILED DESCRIPTION Devices for the practice of the present invention may include any useful detecting means for the initial increase in heat of the enzymatic reaction. Examples of such useful detecting means are thermistors and thermopiles. Detectors on which glucose oxidase has been immobilized by the methods taught in the prior art are practical commercial embodiments of useful detectors. In use, detectors on which glucose oxidase has been immobilized are contacted with a drop of blood containing an amount of glucose to be determined. The glucose oxidase on the detector causes an enzymatic reaction which produces heat. The change in heat over time in the vicinity of the reaction is measured until there is no further increase in temperature. In the case of thermistors and thermopiles this temperature change is seen as a voltage change in, for example, micro volts/sec. The correlating means may be any useful means for translating the output of the detector means. One example of a useful correlating means is a voltmeter which displays the voltage output of the detector means with a needle reading or digitally in volts or some other useful inditia. Another example is software embedded in a computer chip which translates the voltage output of the detector means into a digital mg/dl glucose-in-blood reading. EXAMPLES Example 1 in this comparative example a thermopile having Glucose Oxidase immobilized on its surface was placed in a 23 degree C. solution of water and an amount of glucose was added sufficient to result in a specific mg/ml concentration of glucose. The total change in voltage between the fluid without glucose and the fluid with glucose, which corresponds to the total heat produced by the enzymatic reaction, was noted. The procedure was repeated using different amounts of glucose to result in different concentrations of glucose in fluid. The data obtained is shown in the chart below and in FIG. 1 as the curve marked by solid circles. Concentration Voltage (mg/ml) (micro volts) 20 0.3 58 1.1 96 1.8 134 2.5 171 3.1 208 3.4 245 3.4 282 3.4 318 3.4 One can see by this comparative example and by the line marked by the solid circles in FIG. 1 that the curve which correlates voltage with concentration is non-linear at higher concentrations. A patient or physician using a device which calculates mg/ml glucose in blood by the "total heat change" method would not be able to clearly differentiate between 171 mg/ml and 318 mg/ml, although the difference is medically significant. Example 2 In this example the procedure t>f Example 1 was followed except that the rate of the initial increase of the heat generated by the enzymatic reaction was measured in microvolts/second. This is also known as measuring the initial slope of the temperature curve as the temperature builds toward the total temperature change. The following data was recorded: In contrast to Example 1, this data shows linearity in the correlation between glucose concentration and initial increase across substantially the entire range of medically interesting glucose concentrations. This relationship can be graphically observed and compared with the curve of Example 1 by referring to FIG. 1 wherein the curve of Example 2 is identified by solid squares. The present invention has been disclosed in the above teachings and drawing with sufficient clarity and conciseness to enable one skilled in the art to make and use the invention, to know the best mode for carrying out the invention and to distinguish it from other inventions and from what is old. Many variations and obvious adaptations of the invention will readily come to mind, and these are intended to be contained within the scope of the invention as claimed below.

Claims: What is claimed is: 1. A device for determining the concentration of glucose in a fluid by detecting the heat produced by an enzymatic reaction between the glucose and glucose oxidase, the device comprising: a) detecting means for detecting the initial increase in heat over time, and b) correlating means, responsive to the detecting means, for correlating the initial increase in heat with the concentration of glucose. 2. The device of Claim 1 wherein the detecting means is a thermister. 3. The device of Claim 2 wherein the enzyme glucose oxidase is immobilized on the thermister. 4. The device of claim 1 wherein the detecting means is a thermopile. 5. The device of Claim 4 wherein the enzyme glucose oxidase is immobilized on the thermopile. 6. The device of Claim 1 wherein the means for correlating comprises a software program driving a visible indicia. 7. A method for determining the concentration of glucose in a fluid comprising: a) contacting the glucose in the fluid with the enzyme glucose oxidase to cause a heat-producing enzymatic reaction; b) detecting the initial increase in heat; and c) correlating the initial increase in heat with the concentration of the glucose in the fluid.

Inventors: WULFF KARL, WULFF, KARL
Applicants: BOEHRINGER MANNHEIM CORP, BOEHRINGER MANNHEIM CORPORATION
Application year: 1989
Main classification: C12Q1
IPC codes: C12Q1, G01N25