This invention relates to a method and apparatus for measuring the concentration of a substrate the reaction of which is catalyzed with a microorganism or an enzyme to form a vaporous product or for measuring the concentration of a microoganism or an enzyme which catalyzes the reaction of a substrate to form a vaporous product or for measuring the concentration of an inhibitor of a reaction catalyzed by a microorganism or enzyme.
Enzymes and certain microorganisms such as bacteria or yeasts are known to be selective in catalyzing a reaction involving a specific substrate. Based upon this property, these materials have been employed in a wide variety of detection tecniques to determine the presence of and the concentration of the substrate. For example, enzymes and microorganisms have been employed in colormetric reactions wherein the reaction product has a different color than the starting material and the degree of color change is measured by light absorbence. This measurement then can be related to the concentration of the reactant based upon a prior-obtained calibration curve. It is emphasized that such colorimetric determinations are primarily measurements of the accumulation of reaction product and thereby measure the time integral of the reaction rate rather than the rate directly. In addition, biochemical sensors have been employed for determining the concentration of the molecules involving the use of a reference electrode and a biochemical electrode whereby change in potential is measured and this change is correlated to the concentration of the molecule. The biochemical electrode is intimately contacted with an enzyme or bacteria which enzyme or bacteria reacts selectively with the molecule being surveyed to cause a change in the potential between the biochemical electrode and the reference electrode. The use of these biochemical sensors is limited since they require the use of electrodes adapted to measure the presence of a specific ion generated during reaction are subject to interference from the same ions already present in a sample structure. Furthermore, they must be used in conjunction with an electrolyte, which electrolyte will differ depending upon the type of electrode and type of reaction being employed. Thus, these biochemicals sensing systems are undesirably limited in that only an undesirably limited number of reactants can be monitored therewith. Furthermore, because of their bulk and because of the need for employing an electrolyte, a relatively large volume of reactant is necessary in order to obtain accurate results.
Another common method for measuring the concentration or quantity of various compounds is by means of mass spectroscopy wherein the sample to be analyzed is vaporized, ionized and subjected to an electrical or magnetic field to separate the ions on the basis of mass. While this method has wide application and is considered to be a sensitive and accurate technique, it has limitations particularly as applied to the measurement of relatively high mass molecules. This is because there is some degradation and fragmentation of relatively high mass materials during ionization resulting in a reduced detection of the material present. Also, a significant limitation in the measurement of large mass molecules is due to the complexity of their mass spectra that arises from fragmentation. Thus direct mass spectroscopy of a mixture of large mass molecules usually yields an unuseable superposition of complicated mass spectra, so that identification and measurement of the large mass molecules in mixtures is difficult and often impossible. For this reason it is usual to precede mass spectroscopy of mixtures by one or more separation techniques, particularly gas chromatography. Even then, however, due to overlapping gas chromatography peaks and fragmentation problems, identification and measurement can be ambiguous. In some prior art processes it is undesirable to allow H.sub.2 0 to be present in the gas chromatograph carrier gas since it enters, in sensitive measurements, in a large, unfavorable ratio to the molecule to be measured.
Furthermore, attempts to increase resolution of a mass spectrometer to improve selectivity in measurements results in an undesirable reduced transmission of the material being measured.
It would be highly desirable to provide biochemical sensing apparatus which could be used to detect the concentration of a wide variety of molecules. Furthermore, it would be highly desirable to provide a biochemical sensing apparatus which does not require the present of an electrolyte and for which small volumes of samples such as physiological fluids can be tested to determine the concentration of molecules.
Furthermore, it would be highly desirable to provide a chemical sensing apparatus and a method for its use to be used to measure, in a selective manner and with extreme sensitivity, the concentration of relatively high molecular weight molecules. Also, it would be desirable to provide a means for measuring the concentration and/or quantity of molecules wherein the presence of water does not adversely affect the sensitivity of the measurement.