Project Summary This STTR proposal will focus on developing and testing a novel, first-on-the-market implantable biosensor for in vivo, real-time sensing of gamma-aminobutyric acid (GABA) and glutamate (GLU) for animal studies. GABA and GLU are neurotransmitters (NTs) that are essential for information processing and plasticity, memory, and other functions. GLU is the major excitatory NT and GABA is the major inhibitory NT; a proper balance between them is vital for normal brain function. GLU-GABA dysregulation plays a critical role in several brain disorders, including epilepsy (a disease affecting 1.2% of Americans), dementia (which will affect 130M worldwide by 2050) and Parkinson’s (which affects 1.5M Americans today). A fundamental understanding of NT homeostasis including its temporal components and its role on behavioral events within and across brain areas would lead to a better understanding of human brain function and to new and more effective treatments. Existing NT sensing methods measure only one NT at a time, suffer from poor spatiotemporal resolution, are unable to measure NT dynamics at the circuit level, continuously in real time. Our goal is to develop an ultra-small, flexible (50µm) neural probe for chronic, direct and simultaneous amperometric detection of GLU&GABA, with sub-second temporal resolution and with no externally applied reagents. Phase I focused on manufacturing a prototype Si penetrating shank-type probe with 4 micropatterned sensors and one microfluidic on-demand in-situ calibrator (ODIC), optimizing the enzyme functionalization process, and performing a feasibility study on the measurement of physiologically-relevant changes in the levels of GLU&GABA in real time for freely moving rats for up to 2 weeks. The objectives of Phase II are an upgrade of the probe into a brain micromotion-resistant hybrid Si- flexible polymer probe of higher functionality (octrode + 2 ODIC micro-channels), improvements in selective functionalization, and validation of the probes in a rat model of temporal lobe epilepsy. The multifunctional ODICs will be applied to perform in-situ calibrations for chronic measurements and to investigate the circuit activity of 3 adjacent cortical layers in the whisker barrel cortex via chemical modulation. Upon completion, we expect to deliver a unique-on-the-market dual NT probe of excellent reliability and superior sensitivity, selectivity, and stability, with all performance parameters equal to or better than those offered by current technologies. To achieve this, Alcorix will partner with experts in amperometric NT sensing research from Louisiana Tech U. and experts in the manufacture of advanced neuroprobes from NeuroNexus, who will assist with hybrid Si-flexible polymer integration, in vitro and in vivo evaluation, and eventual market entry. The proposed research will also enable alternative uses such as point-of-use sensors for neurotoxins or disease bio-markers, and neural signal recording or ...