DETECTION OF HEAVY-METAL SALTS WITH BIOSENSORS BUILT WITH AN OXYGEN-ELECTRODE COUPLED TO VARIOUS IMMOBILIZED OXIDASES AND DEHYDROGENASES

Immobilized oxidases were bound on the surface of an affinity membrane and mounted on an oxygen electrode. These biosensors were used for heavy metal salt measurements. After immobilization of the enzymes, first order kinetics of inactivation were observed. Surface immobilization increases the sensitivity by a factor of 10, compared to reticulation of the enzyme in a gelatin matrix. After immobilization, 50% inactivation was observed with 20 muM HgCl2 for L-glycerophosphate oxidase and 50 nm for pyruvate oxidase. Restoration of activity after HgCl2 treatment is feasible, but neither complete nor reproducible. To reuse the biosensor, L-lactate dehydrogenase (LDH) from rabbit muscle in solution was coupled to immobilized L-lactate oxidase (insensitive to heavy metal salts). LDH (particularly inexpensive) was replaced after each measurement. The I50 in phosphate buffer was 1 muM for HgCl2 and 0.1 muM for AgNO3; with other heavy metal salts, no inhibition was observed below 500 muM. In Tris buffer, the I50 was 10 muM for CdCl2 and ZnCl2, 50 mum for Pb-acetate and 250 muM for CuSO4. The use of different enzymes and buffers may allow measurement of specific heavy metal salts.