DUAL ENZYME-BASED MICROBIOSENSOR TO MEASURE ATP RELEASE FROM THE CAROTID BODY

描述(申请人提供)：神经化学物质改变细胞或神经元的电活动，诱导基因和蛋白质表达，从而导致神经元或细胞表型的短期和长期改变。因此，确定特定细胞和神经元的神经化学特征对于了解构成健康和疾病正常发育和衰老的生理和病理功能至关重要。微型和纳米生物传感技术的最新进展为测量神经元和细胞释放的神经化学物质的实时释放提供了一条途径。ATP从中枢和外周神经系统的神经元中释放出来，并通过与P2X离子门控通道结合来介导快速突触传递。颈动脉小体中的外周动脉化学感受器在调节呼吸和循环对氧分压变化的反应中起关键作用，它们在发育早期提供必要的感觉输入，以稳定和维持生命中的呼吸。低氧化学敏感性的成熟发生在哺乳动物出生后发育的最初几周内。我们推测，外周动脉化学感受器中氧敏感细胞释放的ATP增加可能在介导低氧化疗敏感性的发展中起作用。为响应探索性/发展性生物工程研究拨款(EBRG)的项目公告(PA，03-058)，我们的目标是：1)进一步开发一种新型的基于双酶的安培微型生物传感器(顶端直径5-10微米)，该传感器可以测量多种生理条件下溶液中的ATP释放；以及2)测量超融合大鼠颈动脉小体的ATP释放，从而建立出生后成熟期间颈动脉小体释放的刺激反应曲线。这些研究将与佐治亚理工学院的同事合作完成，他们已经开发出了微生物传感器的原型，将用于本提案中概述的实验。由于突触传递是外周和中枢神经系统功能的核心基本过程，而ATP是参与这一过程的重要分子，我们相信这种微生物传感器的成功制造将使其能够广泛应用于多种实验范式，询问参与发育和衰老过程中突触传递的细胞和分子机制。三磷酸腺苷(ATP)是一种允许神经相互交流的基本分子。有了正确的工具，(微生物传感器)我们可以测量这种从组织和神经元释放出来的小分子。这项拨款建议开发一种微生物传感器来测量颈动脉体中三磷酸腺苷的释放，以了解新生儿如何对氧气浓度的变化产生足够的呼吸反应。

DESCRIPTION (provided by applicant): Neurochemicals modify the electrical activity of cells or neurons, induce gene and protein expression, which lead to short and long-term modifications in the phenotype of the neuron or cell. Thus, defining the neurochemical profile of specific cells and neurons is essential to understanding physiological and pathological functions that underlie normal development and aging in health and disease. Recent advances in micro and nanobiosensing technologies has provided an avenue to measure real-time release of neurochemical released from neurons and cells. ATP is released from neurons in the central and peripheral nervous system, and mediates fast-synaptic transmission via binding to P2X ion-gated channels. The peripheral arterial chemoreceptors in the carotid body are critical in regulating ventilation and circulatory responses to changes in oxygen tension, and they provide essential sensory input during early development to stabilize and maintain breathing throughout life. Maturation of hypoxic chemosensitivity occurs within in the first several weeks of mammalian postnatal development. We hypothesize that increase release of ATP from oxygen sensing cells in the peripheral arterial chemoreceptors may operative in mediating the development of hypoxic chemosensitivity. In response to the program announcement (PA, 03-058) for exploratory/developmental bioengineering research grants (EBRG), we aim to 1) further develop a novel dual enzyme-based amperometric microbiosensor (tip diameter 5-10 mcirons) that can measure ATP release across a wide range of physiological conditions in solution, and 2) measure ATP release from the superfused rat carotid body thereby establishing the stimulus-response profile of ATP release from the carotid body during postnatal maturation. These studies will be done in collaboration with colleagues at Georgia Institutute of Technology, who have already developed the prototype of the microbiosensor to be used in experiments outlined in this proposal. Since synaptic transmission is the core elementary process in the function of the peripheral and central nervous system, and ATP is an important molecule involved in this process, we believe that successful fabrication of this microbiosenor will allow for general application to multiple experimental paradigms that interrogates cellular and molecular mechanisms that meditate synaptic transmission during development and aging. Adenosine triphosphate (ATP) is an essential molecule that allows nerves to communicate with each other. With the correct tools, (microbiosensors) we can measure this small molecule as it is released from tissues and neurons. This grant proposes to develop a microbiosensor to measure the release of ATP from the carotid body to understand how the newborn develops an adequate breathing response to changes in oxygen concentration.