High-Speed Microfluidic Assays for Measuring In Vivo Neurotransmitter Dynamics

描述(由申请人提供)：我们目前对大脑功能的理解受到现有实验技术的限制。特别是，缺乏测量体内神经化学动力学的方法。考虑到正是这些动力学是神经元交流的主要机制，从而提高了大脑功能，这一点至关重要。微渗析是目前监测体内化学动力学的主要技术。不幸的是，微透析存在一些重大缺陷。时间分辨率通常在10-20分钟左右。采样区域很大(1-5毫米)，因此很难测量特定大脑区域的活动。植入过程中损伤严重，不可能进行严格的校准。在线微透析-毛细管电泳法(MD-CE)的引入使S的时间分辨率提高到10-30，但仍然存在许多问题。我们建议系统地检修现有的在线MD-CE仪器，以实现时间和空间分辨率的显著提高。微量自由流动电泳(u-FFE)将作为一种能够连续分析采样神经递质的分离技术而被引入。样品流将使用不相容的氟溶剂进行分隔，以防止在神经递质从探针通过在线标记反应并到达分离界面时时间信息的损失。将开发一种电驱动的直插式微泵，以极低的流速(&lt；50nL/分钟)直接从大鼠脑中取样。这种直接采样技术的空间分辨率仅受采样毛细管出口尺寸(40微米)的限制。我们预计，一种在线直接采样-UFFE仪器将能够在-100毫秒的时间尺度上监测&lt；100微米大脑区域中许多重要神经递质的体内动态，包括谷氨酸、GABA、甘氨酸、D-丝氨酸和牛磺酸。在这种时间和大小尺度上测量一系列神经递质将影响神经科学研究的所有领域，包括中风、癫痫、记忆、疼痛、成瘾等。作为一个额外的好处，本提案中描述的仪器比现有的高时间分辨率微透析分析简单得多，这表明它可能更容易被传统神经科学实验室的非专业人员采用。

DESCRIPTION (provided by applicant): Our current understanding of brain function is limited by available experimental techniques. In particular, methods for measuring in vivo neurochemical dynamics are lacking. This is critical considering that it is these dynamics that are the primary mechanism of the neuronal communication that gives rise to brain function. Microdialysis is currently the dominant technique for monitoring in vivo chemical dynamics. Unfortunately, microdialysis suffers from some significant drawbacks. Temporal resolution is often on the order of 10-20 minutes. Sampling regions are large (1-5 mm) making it difficult to measure activity in specific brain regions. Damage during implantation is significant and rigorous calibration is not possible. Introduction of online microdialysis - capillary electrophoresis (MD-CE) assays have improved temporal resolution to 10- 30 s but many issues remain. We propose to systematically overhaul existing online MD-CE instrumentation with the goal of achieving significant gains in temporal and spatial resolution. Micro-free flow electrophoresis (u-FFE) will be introduced as a separation technique capable of continuously analyzing sampled neurotransmitters. The sample stream will be compartmentalized using an immiscible fluorous solvent to prevent loss of temporal information as neurotransmitters travel from the probe, through the online labeling reaction and to the separation interface. An electrically actuated, inline micropump will be developed to directly sample fluid from the rat brain at very low flow rates (<50 nL/min). The spatial resolution of this direct sampling technique will only be limited by the outlet size of the sampling capillary (40 um). Combined we expect an online direct sampling - uFFE instrument will be capable of monitoring the in vivo dynamics of a number of important neurotransmitters, including glutamate, gaba, glycine, D-serine and taurine, on a -100 ms time scale in <100 um brain regions. Measurements on this time and size scale for a range of neurotransmitters would impact all areas of neuroscience research including stroke, epilepsy, memory, pain, addiction, etc. As an added benefit the instrumentation described in this proposal is much simpler than existing high temporal resolution microdialysis assays, suggesting that it may be more easily adopted by non-specialists in traditional neuroscience laboratories.