FIBEROPTIC BIOSENSOR WITH FLUORESCENCE DETECTION BASED ON IMMOBILIZED ALKALINE-PHOSPHATASE

A method for quantifying the concentration of an enzymatic substrate has been demonstrated that is based on competition between two substrates for the active site of an enzyme. This was accomplished by fixing the concentration of a substrate whose reaction can be monitored optically, and observing the effect of the addition of a second substrate whose concentration was to be determined. The model enzyme, alkaline phosphatase, was immobilized on a nylon mesh, which was placed in front of a bifurcated fiber-optic bundle. The concentration of 4-methylumbelliferyl phosphate in the sample solution was fixed. This substrate of alkaline phosphatase becomes a highly fluorescent molecule upon hydrolysis of the phosphate ester. The rate of fluorescence production as a result of this reaction was monitored. The addition of a second substrate caused a decrease in the rate of fluorescence production and, therefore, an apparent inhibition of the enzyme. This method was demonstrated for two different substrates of alkaline phosphatase.