Implantable Microarray Probe for Real-Time Glutamate and GABA Detection

项目摘要 非常需要开发一种多功能的新型神经递质(NT)传感器， 选择性、灵敏和可靠，以允许研究行为和疾病症状的神经生物学机制。 这项STTR计划将重点放在实现一种新型的用于体内实时检测谷氨酸(GLU)的微阵列探针 和γ-氨基丁酸(GABA)。谷氨酸和GABA是正常大脑功能、神经元必不可少的神经递质。 活动性、信息处理和可塑性、网络同步。谷氨酸是一种主要的兴奋性NT，GABA是 一种主要的抑制性NT，它们必须保持适当的平衡，才能使大脑正常运行。一种GLU-GABA 调节失调在几种大脑疾病中起着关键作用，包括癫痫(一种影响1.2%美国人的疾病)， 痴呆症(一种到2050年将影响全球1.3亿人的疾病)和帕金森氏症(一种影响150万人的疾病 今天的美国人)。对NT动态平衡的基本了解可能会导致新的有效的治疗方法 为病人制定的策略。现有的监测方法无法连续、实时地测量动态 时间到了。目前使用的主要方法是微透析法，这种方法的时间分辨率很低，因此 不适用于评估发生在秒或更短时间范围内的行为事件。相比之下，生物传感器更容易 小型化，适合活体研究；它们选择性地将GLU-GABA氧化成二次电活性产物 在有酶存在的情况下，然后用安培法检测。不幸的是，以前可用的生物传感器 非常繁琐，依赖于外部使用的试剂，并且由于它们的校准非常不准确 已经在体外进行，而不是在体内进行。我们用GLU-GABA检测克服了这些问题，并展示了一种 生物传感器阵列探头对GABA的敏感度提高26倍，对GLU的敏感度提高4倍，而且不需要 用于外部试剂。在这里，我们计划开发我们的下一代生物传感器微电极阵列(MEA)探针 这项技术将允许以最高的灵敏度、选择性和可靠性实时检测GLU-GABA。这个 探测器将具有以下新功能：至少2个GABA和2个GLU位置，用于测量不同环境中的浓度 大脑中的位置。此外，它还将配备一个微流控通道，用于立即引入化学品 微电极的附近。最后，我们将整合在现场运行的按需现场校准器(ODIC 传感器校准，以实现准确检测。该项目的具体目标是：(1)微制造和表征 铂在硅探针上的ODIC修饰，(II)GLU-GABA探针的体外表面修饰和优化 以及(Iii)在癫痫大鼠的大脑中实时检测GLU-GABA。拟议的研究还将使 该技术的替代应用，包括：用于神经毒素、活性氧物种和 疾病生物标志物。到2024年，基于神经刺激设备的市场估计将达到160亿美元。如果只有1% 虽然生物传感器探头技术可以进入市场，但这仍将是拟议工作的充分理由。

Project Summary 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.