In designing an amperometric (L)-glutamate biosensor based on the electrochemical detection of enzymatically generated H2O2, the key challenges are selectivity against electrooxidizable interferents, micromolar detection limit, and response time of seconds or less. With these issues in mind, a glutamate microbiosensor consisting of Pt black (PtB1k) electrodeposited on a 125-mu m-dia. Pt wire with a permselective film of electropolymerized, overoxidized polypyrrole (OPP) and a top layer of (L)-glutamate oxidase ((L)-GluOx) crosslinked with glutaraldehyde was constructed. The OPP film permits passage of H2O2 to the electrode surface while rejecting the common interferents of brain extracellular fluid, ascorbate (AA) and dopamine (DA). The PtB1k treatment of Pt electrodes was adopted since the microscale roughness of PtB1k increases the effective surface area of the electrode and promotes electrochemical H202 oxidation at lower potential. This microbiosensor was highly sensitive (80 +/- 10 nA mu M-1 cm(-2)) to (L)-glutamate at 450 mV versus Ag/AgCl, a significantly lower potential than the similar to 700 mV used with most similar (L)-glutamate sensors prepared on polished Pt electrodes. Notably, microbiosensor response to both AA and DA was undetectable at concentrations exceeding physiological extracellular concentrations of these compounds. The described (L)-glutamate biosensor also showed excellent response time (similar to 1-2 s) and temporal stability (similar to 3 week half-life). In addition, this microbiosensor was robust enough for insertion into live brain tissue where it responded rapidly to (L)-glutamate, but was insensitive to dopamine. (c) 2006 Elsevier B.V. All rights reserved.