An amperometric biosensor was developed for the interference-free determination Of L-glutamate with a bienzyme-based Clark electrode. This sensor is based on the specific dehydrogenation by L-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with salicylate hydroxylase (SHL, EC 1.14.13.1). The enzymes were entrapped by a poly(carbamoyl) sulfonate (PCS) hydrogel on a Teflon membrane. The principle of the determination scheme is as follows: the specific detecting enzyme, GLDH, catalyses the specific dehydrogenation Of L-glutamate consuming NAD(+). The product, NADH, initiates the irreversible decarboxylation and the hydroxylation of salicylate by SHL in the presence of oxygen. This results in a detectable signal due to the SHL-enzymatic consumptions of dissolved oxygen in the measurement Of L-glutamate. The sensor has a fast steady-state measuring time of 20 s with a quick response (1 s) and a short recovery (1 min). It shows a linear detection range between 10 mu M and 1.5 MM L-glutamate with a detection limit of 3.0 mu M. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from other amino acids and clectroactive substances. (C) 2007 Elsevier Inc. All rights reserved.