Design optimisation and characterisation of an amperometric glutamate oxidase-based composite biosensor for...

A polymer/enzyme composite biosensor for monitoring neurochemical glutamate was performance optimised in vitro for sensitivity, selectivity and stability. This first generation Pt/glutamate oxidase-based sensor displayed appropriate sensitivity (90.4 ± 2.0 nA cm^-2 μM^-1). It also has ideal stability/biocompatibility with no significant decrease in response observed for repeated calibrations, exposure to electron beam sterilisation, or following storage at 4 °C either dry (28 days) or in ex-vivo rodent brain tissue (14 days). Potential non-glutamate contributing signals, generated by extracellular levels of the principal endogenous electroactive interferents, were typically <5% of the basal (10 μM) glutamate response. Changes in molecular oxygen (the natural enzyme mediator) over the normal brain tissue range of 40-80 μM had minimal effect on the glutamate signal for concentrations of 10 and 100 μM (Mean K_MO₂ = 1.86 ± 0.74 μM, [O₂]_90% = ca. 15 μM). Additionally, a low μM calculated limit of detection (0.44 ± 0.05) and rapid response time (ca. 1.67 ± 0.06 s), combined with no effect of pH and temperature changes over physiologically relevant ranges (7.2-7.6 and 34-40 °C respectively), collectively suggest that this composite biosensor should reliably detect l-glutamate when used for neurochemical monitoring. Preliminary experiments involving implantation in the striatum of freely moving rats demonstrated stable recording over several weeks, and reliable detection of physiological changes in glutamate in response to behavioural/neuronal activation (locomotor activity and restraint stress).