Bienzyme Carbon Paste Electrodes for L-Glutamate Determination

playing an important role in various areas, such as amino acid biosynthesis, bioprocess monitoring, the food industry, and in medicine. It is widely used as a flavor enhancer in various foodstuffs and seasonings (1), as well as being connected to the Chinese restaurant syndrome (2). It has been associated with several diseases; hence, it is used as a clinical marker for general and protein metabolism (3), and for nutritional status (4). Glutamate is a principal neurotransmitter in the mammalian brain (5,6) playing a vital role in understanding the dynamics of the energy balance of the brain and evaluation of selective neuronal injury following various damages to the nervous system (7,8). Its quantification was found to be useful for the diagnosis of some myocardial and hepatic diseases (9), for the pathology of neurological (10) and psychiatric disorders (11). It is also often used in clinical laboratories for the determination of aminotransferase activities (12). Many different methods have already been proposed for L-glutamate determination, mainly: chromatographic techniques (13), kinetic potentiometry (14), or capillary electrophoresis with electrochemical detection (15). Since these methods involve complicated and time-consuming procedures, they are inadequate for rapid analysis of a large number of samples or for timely monitoring. In the past 10 years, attention was mainly focused on the use of amperometric enzyme electrodes, taking advantage of the inherent high substrate selectivity of the enzymes. Most often NAD+ dependent glutamate dehydrogenase (16-24) and GlOx (6,12,25-52) have been employed for the determination of L-glutamate in different systems. However, glutamate dehydrogenase based systems require the addition of soluble NAD+, complicating thus the sensor construction. This drawback can be eliminated by the use of GlOx. Most of the GlOx based systems detect glutamate by direct electrochemical reduction/oxidation of the co-substrate (O2) or the formed H2O2, both reactions requiring high overpotentials, thus, resulting in repressed electrochemical selectivity. Therefore, either a gas permeable or the combination of several sizes and charges excluding membranes is required, complicating the sensor design. Attempts to decrease the working potential for H2O2 detection were made modifying the electrode surface (12,25,33,44,45), incorporating various mediators into the sensor (32,34,40,41), or by using coupled GlOx-HRP systems (39,52). Bienzyme Carbon Paste Electrodes for L-Glutamate Determination