Selective enzyme immobilization on arrayed microelectrodes for the application of sensing neurotransmitters

• Selective immobilization of glutamate oxidase on closely arrayed microelectrodes.
• Optimized chitosan electrodeposition for enzyme immobilization.
• Optimized permselective polymer coatings for rejecting charged interferents.
• No cross-talk effect or diffusional interaction among closely arrayed microelectrodes.
• Near-real time glutamate detection.

The method for selective enzyme immobilization on arrayed microelectrodes has been investigated in order to fabricate a near-real time biosensor for potential ex vivo or in vivo applications (e.g. in fermentation processes, in neuroscience studies, etc.). In this study, chitosan is used as the enzyme immobilization material, permselective polymers (polypyrrole and Nafion®) are used as the exclusion layers for charged interferents, and glutamate oxidase is used as the model enzyme for immobilization. To fabricate a glutamate sensor in the array, glutamate oxidase is selectively immobilized on a microelectrode site which is closely arrayed to other microelectrodes (platinum microelectrodes with ∼100 μm separation) with the electrode dimension of ∼30 μm × 140 μm by adsorbing the enzyme on the electrodeposited chitosan matrix on the electrode surface. When testing the glutamate sensor with glutamate at physiological concentrations, it has a linear detection range up to 217 μM, a response time ∼1 s, a limit of detection 2.5 ± 1.2 μM, and a sensitivity 38.1 ± 5.4 nA/μM cm2 (n = 8); on the other hand, the closely arrayed control sensor without the chitosan film showed no signal upon the addition of glutamate. Little interferent current was observed. The sensor can retain up to 70% of its initial sensitivity for at least 9 days and no observable decrease in sensor sensitivity was detected after 20 times of continuous operations. Successful selective glutamate oxidase immobilization on closely packed microelectrodes for monitoring glutamate is demonstrated.