A novel ISFET-based NAD+-dependent enzyme sensor for lactate

An integrated NAD+-dependent enzyme field-effect transistor (ENFET) device for the biosensing of lactate is described. The aminosiloxane-functionalized gate interface is modified with pyrroloquinoline quinone (PQQ) that acts as a catalyst for the oxidation of NADH. A synthetic amino-derivative of NAD+ is covalently linked to the PQQ monolayer. Faradaic impedance spectroscopy and chronopotentiometry are used as electrochemical methods for probing in situ bioaffinity interaction between NAD+-dependent enzyme, lactate dehydrogenase (LDH) (E.C. 1.1.1.27), and the NAD+-functionalized Au-electrode surface. The association constant of 1.5×105 M−1 was found by both techniques. The affinity complex formed between the NAD+/PQQ-assembly and the LDH is cross-linked and yields an integrated biosensor ENFET-device for the analysis of lactate. The device reveals the lower detection limit of 1×10−4 M for lactate and the sensitivity of 24±2 mV dec−1. The response time of the device is as low as 15 s.