Analysis of NAD(P)+-cofactors by redox-functionalized ISFET devices

Functional ISFET devices for the specific analysis of the NAD(P)+-cofactors are described. The functional ISFET devices consist of a pyrroloquinoline quinone (PQQ)-modified gate, to which the NAD(P)+-dependent enzymes, lactate dehydrogenase, LDH (for NAD+ analysis), or alcohol dehydrogenase, AlcDH (for NADP+ analysis), are covalently linked. In the presence of NAD+ and lactate, or NADP+ and ethanol, the biocatalyzed generation of NADH or NADPH occurs. The catalyzed oxidation of the generated NAD(P)H cofactors by PQQ and O2 yields a steady-state concentration of PQQ/PQQH2 on the gate interface. The ratio PQQ/PQQH2 is controlled by the concentration of NAD(P)H, or by the parent oxidized NAD(P)+-cofactors. The functional ISFET devices allow the potentiometric analysis of NAD+ with the lower detection limit of 2×10−4 M, and a sensitivity of 23±2 mV per decade, and of NADP+ with a detection limit of 1×10−4 and sensitivity that corresponds to 35±2 mV per decade. The PQQ/LDH-functionalized ISFET device allows the kinetic analysis of the hydrolysis of NAD+ by NADase (k=2.0×10−3 s−1) and by the cholera toxin, subunit A (k=4.2×10−4 s−1).