Nanobiosensing Neural Probes for Traumatic Brain Injury Applications

描述 拟议工作的主要目标是迈出独立研究计划的第一步，该计划侧重于基于独立优化材料的生物医学微/纳米系统的开发和应用，用于研究，监测和治疗神经系统疾病，包括创伤性脑损伤（TBI）和中风。具体而言，该项目涉及开发一种平台技术，该技术涉及整合最佳电极、酶固定化和电化学传感器的底物材料，以连续监测细胞外谷氨酸水平，具有高时空分辨率、灵敏度、选择性、稳定性和宽线性范围。这种多尺度材料系统将包括生物适应性聚合物纳米复合材料基底、金和石墨烯电极以及用于酶固定的聚合物纳米线。这种材料的组合将提供具有长期植入所需特性的传感器，用于在正常活动期间进行神经化学监测。虽然谷氨酸是这项工作的重点，但开发这种传感器的方法可以应用于许多不同的生物分析物，这些生物分析物将集成在这种生物传感器的后续实现中。 一种在医院环境外连续监测神经化学状态的方法将1）提供关于与创伤性脑损伤在损伤部位的长期影响相关的病理生理学的增强的理解2）描绘 神经化学和临床功能障碍之间的关系，3）允许长期监测康复和药物干预，4）允许新的药物治疗， 在高度控制的情况下发展。 总体目标的第一个目标是提供具有高灵敏度（>500 nA）的稳定神经接口。M-1.cm-2）通过有效的谷氨酸氧化酶固定来改变体内谷氨酸浓度。 这可以使用为实现这一目标而提出的独特材料组定制的微制造工艺来实现。一种新型的刺激响应性聚合物纳米复合材料，聚（醋酸乙烯酯）（PVAc-NC），基板将实施，以尽量减少炎症反应的植入物，从而最大限度地提高生物/非生物界面的稳定性。PVAc-NC具有高弹性， 在其干燥状态下的弹性模量（Edry ~ 4GPa），允许针状插入脑组织中，但在吸收生理流体后弹性模量（Ewet ~ 12 MPa）显示出三个数量级的降低，大大减少了与皮质组织的机械失配（Ecortex ~ 10 kPa）。高谷氨酸敏感性和选择性将通过用纳米结构的导电聚合物纳米线层（例如聚吡咯）涂覆金电极位点来建立，所述纳米结构的导电聚合物纳米线层将用于抑制谷氨酸氧化酶，谷氨酸氧化酶是一种选择性地与谷氨酸反应以形成包括过氧化氢在内的几种产物的酶。然后，过氧化氢将通过经由外部电子器件施加到电极的电势被氧化，并且将测量氧化电流。该电流的大小与电极附近的谷氨酸浓度线性相关。 第二个目的是扩大谷氨酸传感器的动态线性范围，以确保在正常和受损大脑中预期的整个范围内保持测量电流和实际谷氨酸浓度之间的线性。为此，薄膜金电极将被碳基石墨烯电极取代。 将需要转移过程以将石墨烯整合到PVAc-NC上，因为PVAc-NC与石墨烯生长温度不相容。然后，这些电极将使用与针对第一个目的开发的方法相当的方法用谷氨酸氧化酶官能化，但适用于基于石墨烯的电极。这两种类型的电化学传感器将在体外进行表征和校准，然后在TBI的啮齿动物模型中进行体内测试，以评估电极稳定性和体内行为与确定的体外校准参数的对应性。 公共卫生相关性： 创伤性脑损伤（TBI）和其他神经系统疾病是退伍军人健康方面的重要问题，特别是对于那些参与最近作战行动的退伍军人。严重神经创伤的许多症状和影响持续存在，并在最初受伤后持续多年，导致永久性严重残疾。这些后遗症与神经化学调节的改变有关，但由于缺乏可用于在医院重症监护室范围外连续监测神经化学水平的工具而未得到很好的理解。所提出的生物传感器将在正常活动期间以非常高的空间和时间分辨率连续监测神经化学水平。通过增强用于检测和解释电神经信号的成熟传感器的功能，我们将对神经创伤的长期影响、行为与神经化学之间的关系获得新的理解，并开发治疗神经创伤和调节神经化学的新方法。

DESCRIPTION The primary goal of the proposed work is to take the first steps toward an independent research program that focuses on the development and application of biomedical micro/nanosystems based on independently optimized materials for the study, monitoring, and treatment of neurological conditions, including traumatic brain injury (TBI) and stroke. Specifically, this project concerns the development of a platform technology involving the integration of optimal electrode, enzyme immobilization, and substrate materials for an electrochemical sensor to continuously monitor extracellular glutamate levels with high spatiotemporal resolution, sensitivity, selectivity, stability, and wide linear range. This multi-scale materials system will incorporate a bio-adaptive polymer nanocomposite substrate, gold and graphene electrodes, and polymer nanowires for enzyme immobilization. This combination of materials will provide a sensor with the requisite properties for long-term implantation for neurochemical monitoring during normal activity. Though glutamate is the focus of this work, the methods to develop this sensor can be applied to many different bioanalytes, which will be integrated in later implementations of this biosensor. A means to continuously monitor the neurochemical state outside of the hospital environment will 1) provide enhanced understanding regarding the pathophysiology associated with the long-term effects of TBI at the site of the injury 2) delineate the relationship between neurochemistry and clinical dysfunction, 3) allow for long term monitoring of rehabilitation and drug interventions and 4) allow for the novel drug therapies to be developed with highly controlled delivery. The first aim toward the overall goal is to provide a stable neural interface with high-sensitivity (>500 nA.¿M-1.cm-2) to changes in in vivo glutamate concentration through efficient glutamate oxidase immobilization. This with be achieved using microfabrication processes customized for the unique materials set proposed to achieve this goal. A novel stimuli-responsive polymer nanocomposite, poly (vinyl acetate) (PVAc-NC), substrate will be implemented to minimize the inflammatory response to the implant, thus maximizing the stability of the biotic/abiotic interface. PVAc-NC has a high elastic modulus (Edry ~ 4 GPa) in its dry state, permitting needle- like insertion into brain tissue, but displays a three order-of-magnitude reduction in elastic modulus (Ewet ~ 12 MPa) after absorption of physiological fluids, greatly reducing mechanical mismatch with cortical tissue (Ecortex ~ 10 kPa). High glutamate sensitivity and selectivity will be established by coating the gold electrode site with a nanostructured conductive polymer nanowire layer, such as polypyrrole, which will serve to immobilize glutamate oxidase, an enzyme that selectively reacts with glutamate to form several products, including hydrogen peroxide. The hydrogen peroxide will then by oxidized by a potential applied to the electrode via external electronics and the oxidation current will be measured. The magnitude of this current is linearly-related to the concentration of glutamate near the electrode. The second aim is to expand the dynamic linear range of the glutamate sensor to ensure linearity between measured current and actual glutamate concentration is maintained through the entire range expected in normal and injured brains. Toward this end, thin-film gold electrodes will be replaced with a carbon-based graphene electrode site. A transfer process will be required to integrate graphene onto PVAc-NC, as PVAc-NC is incompatible with graphene growth temperatures. These electrodes will then be functionalized with glutamate oxidase using methods comparable to those developed toward the first objective, but adapted for use on a graphene-based electrode. The two types of electrochemical sensors will be characterized and calibrated in vitro, then tested in vivo in a rodent model of TBI to assess electrode stability and the correspondence of in vivo behavior with the determined in vitro calibration parameters. PUBLIC HEALTH RELEVANCE: Traumatic brain injury (TBI) and other neurological disorders are important concerns with regard to veterans' health, especially for those veterans involved with recent combat operations. Many of the symptoms and effects of severe neurotrauma persist and progress for years beyond the initial injury, causing permanent severe disability. These sequelae are related to alterations in neurochemical regulation, but are not well understood due to the lack of tools available to monitor neurochemical levels continuously outside of the realm of an intensive care unit in a hospital. The proposed biosensor will continuously monitor neurochemical levels during normal activity with very high spatial and temporal resolution. By augmenting the functionality of well-established sensors for detecting and interpreting electrical neural signals, we will gain new understanding of the long-term effects of neurotrauma, the relationship between behavior and neurochemistry, and develop new ways to treat neurotrauma and regulate neurochemistry.