An integrated NAD(+)-dependent enzyme-functionalized field-effect transistor (ENFET) system: development of a lactate biosensor

An integrated NAD(+)-dependent enzyme field-effect transistor (ENFET) devic e for the biosensing of lactate is described. The aminosiloxane-functionali zed gate interface is modified with pyrroloquinoline quinone (PQQ) that act s as a catalyst for the oxidation of NADH. Synthetic amino-derivative of NA D(+) is covalently linked to the PQQ monolayer. An affinity complex formed between the NAD(+)/PQQ-assembly and the NAD(+)-cofactor-dependent lactate d ehydrogenase (LDH) is crosslinked and yields an integrated biosensor ENFET- device for the analysis of lactate. Biocatalyzed oxidation of lactate gener ates NADH that is oxidized by PQQ in the presence of Ca2+-ions. The reduced catalyst, PQQH(2), is oxidized by O-2 in a process that constantly regener ates PQQ at the gate interface. The biocatalyzed formation of NADH and the O-2-stimulated regeneration of PQQ yield a steady-state pH gradient between the gate interface and the bulk solution. The changes in the pH of the sol ution near the gate interface and, consequently, the gate potential are con trolled by the substrate (lactate) concentration in the solution. The devic e reveals the detection limit of 1 x 10(-4) M for lactate and the sensitivi ty of 24 +/- 2 mV dec(-1). The response time of the device is as low as 15 s. (C) 2000 Elsevier Science S.A. All rights reserved.