Development of an l-glutamate biosensor using the coimmobilization of l-glutamate dehydrogenase and p-hydroxybenzoate hydroxylase on a Clark-type electrode

A bienzyme-based Clark electrode was developed for the interference-free determination of l-glutamate. This sensor is based on the specific dehydrogenation by l-glutamate dehydrogenase (GLDH, EC 1.4.1.3) in combination with p-hydroxybenzoate hydroxylase (HBH, EC 1.14.13.2). The enzymes were entrapped by a poly(carbamoyl) sulfonate 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 NADP+. The product, NADPH, initiates the irreversible hydroxylation of p-hydroxybenzoate by HBH in the presence of oxygen. This results in a detectable signal due to the HBH-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 (2 s) and a short recovery (1 min). It shows a linear detection range between 10 μM and 1.5 mM l-glutamate and a detection limit of 5 μM. A Teflon membrane, which is used to fabricate the sensor, makes the determination to avoid interferences from different amino acids and electroactive substances.