IMMOBILIZATION OF LACTATE-DEHYDROGENASE ON A PYROLYTIC CARBON-FIBER MICROELECTRODE

An immobilized enzyme carbon fiber microelectrode (12 .mu.m i.d., 500 .mu.m) is perfected in this work. The principle of measurement is based on the voltammetric detection of NADH. This detection is improved by electrochemical treatment of the electrode. The oxidation current in proportional to NADH from 10-5 to 10-3 M. The detection limit is 10 .mu.M. The enzyme is immobilized by impregnation in an inert protein sheath that has first been electrochemically deposited around the active tip of the electrode. The lactate dehydrogenase (LDH) immobolization leads to an electrode sensitive to pyruvate. For each concentration of cofactor the response curve of the electrode exhibits a linear range for the substrate. With 3 .times. 10-5 M NADH the detection limit for pyruvate is lower than 1 .mu.M. This LDH-immobilized microelectrode allows us to estimate that pyruvate concentration in a rat cerebrospinal fluid sample of 50 .mu.L is 10-4 M.