BIOSENSORS FOR DETECTION OF ENZYMES IMMOBILIZED IN MICROVOLUME REACTION CHAMBERS

A biosensor system is presented in which a small quantity of enzyme placed in a very small volume produces a measurable chemical change. The enzyme used is urease, and the pH changes it causes are measured with a light-addressable potentiometric sensor (LAPS). A model is presented which prediocts the sensitivity of the system as a function of chamber volume, specific activity of the enzyme, and the amount of pH buffering present. Under our usual conditions, the detection limit for urease was 4.4 × 107 molecules, which corresponds to 40 pg or (in a 300 μl sample volume) a concentration of 2.4 × 10-13 M. Much lower detection limits have been achieved by using smaller chambers and lower buffering. The urease is immobilized on a porous membrane which is then placed into the reading chamber. By making conjugates in which urease is linked to antibodies or binding proteins to a desired analyte, an assay for any analyte can be constructed. Results on an assay for total single-stranded DNA show a detection limit of 2 pg. © 1990.