SBIR-STTR Award

There is an enormous need for the development of a new class of neurotransmitter (NT) sensors that are versatile, selective, sensitive and reliable to allow investigation of the neurobiological mechanisms of behavior and disease symptoms. This STTR proposal will focus on implementing a novel microarray probe for in vivo, real-time sensing of glutamate (GLU) and gamma-aminobutyric acid (GABA). GLU and GABA are NTs that are essential for normal brain function, neuronal activity, information processing and plasticity, and network synchronization. GLU is a major excitatory NT and GABA is a major inhibitory NT, and they must maintain a proper balance for the brain to operate normally. A GLU-GABA dysregulation plays a critical role in several brain disorders, including epilepsy (a disorder affecting 1.2% of Americans), dementia (a disorder that will affect 130 million worldwide by 2050) and Parkinson’s (a disorder affecting 1.5 million Americans today). A fundamental understanding of NT homeostasis would likely lead to new and effective therapeutic strategies for patients. Existing monitoring methods suffer from the inability to measure dynamics continuously, in real time. Currently, the primary method being used is microdialysis, which has very poor temporal resolution and is therefore not suitable to evaluate behavioral events that occur on a timescale of seconds or less. In contrast, biosensors are easy to miniaturize and are suitable for in vivo studies; they selectively oxidize GLU-GABA into a secondary electroactive product in the presence of enzymes, which is then detected by amperometry. Unfortunately, previously available biosensors have been cumbersome, have relied on externally applied reagents, and have been grossly inaccurate because their calibration has been carried out in vitro and not in vivo. We overcame these problems with GLU-GABA detection and demonstrated a biosensor array probe capable of a 26-fold higher sensitivity to GABA, a four-fold higher sensitivity to GLU, and no need for external reagents. Here, we propose to develop the next generation of our biosensor microelectrode array (MEA) probe technology that will allow GLU-GABA detection in real-time with the highest sensitivity, selectivity, and reliability. The probes will have the following new features: At least 2 GABA and 2 GLU sites for measuring concentrations at different positions within the brain. Also, it will feature a microfluidic channel for the introduction of chemicals in the immediate vicinity of the microelectrodes. Finally, we will incorporate an On-Demand In-situ Calibrator (ODIC) that runs in-situ sensor calibration for accurate detection. The specific aims of this project are: (i) microfabrication and characterization of platinum MEAs with ODIC on a silicon probe, (ii) surface modification and optimization of GLU-GABA probe in vitro and (iii) demonstrate real-time GLU-GABA detection in an epileptic rat brain. The proposed research would also enable alternative applications for the technology, including: point-of-use sensors for neurotoxins, reactive oxygen species, and disease biomarkers. The estimated market based on neurostimulation devices is $16 billion by 2024. If only 1% of that market is accessible by biosensor probe technology, it would still be sufficient justification for the proposed work.

Public Health Relevance Statement:
Project Narrative This project will advance basic neuroscience research tools by developing a novel implantable microarray probe for real-time glutamate and gamma-aminobutyric acid detection. The multiplexing and in vivo capabilities of the probe will allow researchers to develop more effective animal models and therapeutics for brain diseases and certain psychological disorders. These tools address critical healthcare issues with costs due to mental disease exceed $1.5 trillion annually, Alzheimer’s Disease affecting 15 million Americans, 1.2% of Americans suffering from Epilepsy, and 1.5 million suffering from Parkinson’s disease.

Project Terms:
3D Print; Acetylcholine; Address; Affect; Alzheimer's Disease; American; Animal Model; Area; base; Behavioral; Behavioral Mechanisms; Biological Markers; Biosensor; Brain; Brain Diseases; Buffers; Caliber; Calibration; Chemicals; Chronic; Communities; cost; Dementia; Detection; Development; Devices; Disease; Dopamine; Enzymes; Epilepsy; Equilibrium; Event; experimental study; falls; gamma-Aminobutyric Acid; Glucose; Glutamates; Goals; Healthcare; Homeostasis; Hydrogen Peroxide; Implant; implantation; In Situ; In Vitro; in vivo; information processing; interest; Investigation; Knowledge; Lead; Liquid substance; Literature; Louisiana; Measurement; Measures; Mechanics; Mental disorders; Methods; Microdialysis; Microelectrodes; Microfabrication; Microfluidics; Microscopy; miniaturize; Modification; Monitor; multimodality; nervous system disorder; neurobiological mechanism; Neurologic; Neurons; Neurosciences; Neurosciences Research; Neurotoxins; Neurotransmitters; next generation; novel; Optics; Oxidases; Oxides; Parkinson Disease; Patients; Pattern; Performance; Peroxides; Phase; Phenylenediamines; Platinum; Play; Positioning Attribute; Process; Psyche structure; Rattus; Reactive Oxygen Species; Reagent; Research; Research Personnel; Role; Running; sensor; Serotonin; Side; Signal Transduction; Silicon; Site; Small Business Technology Transfer Research; success; Surface; Symptoms; Technology; temporal measurement; Testing; Therapeutic; Thick; Time; tool; Treatment Efficacy; Work

This STTR proposal will focus on developing and testing a novel, first-on-the-market implantable biosensor forin vivo, real-time sensing of gamma-aminobutyric acid (GABA) and glutamate (GLU) for animal studies. GABAand GLU are neurotransmitters (NTs) that are essential for information processing and plasticity, memory, andother functions. GLU is the major excitatory NT and GABA is the major inhibitory NT; a proper balance betweenthem 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 homeostasisincluding its temporal components and its role on behavioral events within and across brain areas would lead toa better understanding of human brain function and to new and more effective treatments. Existing NT sensingmethods measure only one NT at a time, suffer from poor spatiotemporal resolution, are unable to measure NTdynamics 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-secondtemporal resolution and with no externally applied reagents. Phase I focused on manufacturing a prototype Sipenetrating 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 measurementof physiologically-relevant changes in the levels of GLU&GABA in real time for freely moving rats for up to 2weeks. 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 selectivefunctionalization, and validation of the probes in a rat model of temporal lobe epilepsy. The multifunctional ODICswill be applied to perform in-situ calibrations for chronic measurements and to investigate the circuit activity of 3adjacent cortical layers in the whisker barrel cortex via chemical modulation. Upon completion, we expect todeliver a unique-on-the-market dual NT probe of excellent reliability and superior sensitivity, selectivity, andstability, with all performance parameters equal to or better than those offered by current technologies. Toachieve this, Alcorix will partner with experts in amperometric NT sensing research from Louisiana Tech U. andexperts in the manufacture of advanced neuroprobes from NeuroNexus, who will assist with hybrid Si-flexiblepolymer integration, in vitro and in vivo evaluation, and eventual market entry. The proposed research will alsoenable alternative uses such as point-of-use sensors for neurotoxins or disease bio-markers, and neural signalrecording or neurostimulation in conjunction with specific locally-injected drugs. The 2023 world market forelectrophysiological neuro-probes (Grand View Research) is about $266M, CAGR of 3.1%. If only 1% of thatmarket can be captured by this technology it would be more than sufficient justification for the proposed work.

Public Health Relevance Statement:
Project Narrative This project will advance basic neuroscience research tools by developing a novel implantable octrode probe, to be the first on the market for real-time, simultaneous direct measurement of the critical neurotransmitters, gamma-aminobutyric acid (GABA) and glutamate. The multiplexing and in vivo capabilities of the probe will allow researchers to develop more effective animal models and therapeutics for brain diseases and mental disorders, which affect millions of Americans such as Alzheimer's Disease (15M), Epilepsy (4M), and Parkinson's disease (1.5M), and costs over $1.5 trillion in healthcare spending annually.

Project Terms:
<µfluidic><4-Aminobutanoic Acid><4-Aminobutyric Acid><4-amino-butanoic acid><γ-Aminobutyric Acid>