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^-4M, and a sensitivity of 23±2mV per decade, and of NADP⁺ with a detection limit of 1×10^-4 and sensitivity that corresponds to 35±2mVper decade. The PQQ/LDH-functionalized ISFET device allows the kinetic analysis of the hydrolysis of NAD⁺ by NADase (k=2.0×10^-3s^-1) and by the cholera toxin, subunit A (k=4.2×10^-4s^-1).