AN AMPEROMETRIC NADH BIOSENSOR BASED ON NADH OXIDASE FROM THERMUS-AQUATICUS

A biosensor for the determination of the reduced cofactor nicotinammide adenine dinucleotide (NADH) has been developed using the enzyme NADH oxidase from Thermus aquaticus immobilized on an Immobilon AV membrane. A hydrogen peroxide electrode,was used as the detection system. The NADH electrode showed a monotonous response with near linearity in the 5.10(-7) to 2.10(-5) M range, with a detection limit of 2.10(-7) M. Analytical parameters such as pH, response time and lifetime,vere characterized. The probe showed no change in sensitivity between pH 4.5 and 9.5 and retained 70% of its initial activity after 50 days of storage in buffer. A method for the measurement of lactic dehydrogenase (LDH) activity was developed using the biosensor. The response was linear in the 10-1000 U l(-1) range. The addition of NADH and LDH to serum gave recovery values between 94 and 107%. The determination of LDH in two control sera and in three serum samples was performed with a standard spectrophotometric procedure and the biosensor. The results correlated well.