The ability to rapidly, sensitively, and selectively monitor changes in neurotransmitter concentrations in vivo can significantly aid in elucidating the mechanisms behind neurodegenerative diseases as well as many behaviors. For this reason, we designed, manufactured, and implemented multi-functional implantable microprobes, capable of detecting neurotransmitter concentration changes in anesthetized and awake laboratory rodents.
Our silicon-based multi-electrode arrays (MEAs) were fabricated using micro-electro-mechanical-systems (MEMS) technologies, allowing for highly reproducible array dimensions, a flexible design, and relatively low manufacturing costs due to mass production. Approximately 150 MEAs were micromachined simultaneously on thin, four-inch silicon wafers with two to five, micron-size electrodes per probe. Each electrode site was chemically modified for specific function.
For amperometric glutamate biosensors, electrode sites were modified with permselective polymers, polypyrrole and Nafion, to prevent interfering chemicals from reaching the electrode surface. The enzyme, glutamate oxidase, was immobilized via cross-linking agents to allow for the enzymatic detection of glutamate. These glutamate biosensors were able to detect physiological concentrations of glutamate with a ∼1 s response time in the presence of the electroactive interferents, dopamine and ascorbic acid. On another MEA site, an on-probe reference electrode (RE) was created by electrochemically depositing a biocompatible iridium oxide layer. Using the on-probe RE, the glutamate sensors could still detect physiological concentrations of glutamate in the presence of interferents, while reducing signal noise both in vitro and in vivo.
In the anesthetized rat, the glutamate biosensors detected glutamate release in the striatum by cortical electrical stimulation. In the freely moving rat, stress-induced glutamate release was detected in the striatum, and in trained rats, the biosensors detected glutamate during reward-seeking behavior. The MEAs may also be functionalized to simultaneously detect multiple analytes (e.g., glutamate and dopamine) in vivo. To do this, low enzyme loading and low spatial resolution must be overcome during enzyme immobilization. Methods for selective enzyme immobilization are explored in the recommendations section of this thesis.