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可用实验技术的限制。特别是，缺乏用于测量体内神经化学动力学的方法。这是至关重要的，因为正是这些动力学是神经元通信的主要机制，从而产生大脑功能。微透析是目前监测体内化学动力学的主要技术。不幸的是，微透析具有一些显著的缺点。时间分辨率通常在10-20分钟的量级。采样区域很大（1-5 mm），因此难以测量特定大脑区域的活动。植入过程中损坏严重，无法进行严格校准。在线微透析-毛细管电泳（MD-CE）分析的引入已经将时间分辨率提高到10- 30秒，但是仍然存在许多问题。我们建议系统地检修现有的在线MD-CE仪器，目标是在时间和空间分辨率方面取得重大进展。微自由流电泳（u-FFE）将被引入作为一种分离技术，能够连续分析采样的神经递质。样品流将使用不混溶的含氟溶剂进行区室化，以防止神经递质从探针通过在线标记反应到达分离界面时的时间信息损失。将开发一种电致动的内联微型泵，以非常低的流速（<50 nL/min）直接从大鼠脑中取样流体。这种直接采样技术的空间分辨率将仅受采样毛细管的出口尺寸（40 μ m）的限制。结合起来，我们期望在线直接采样- uFFE仪器将能够在<100 μ m的脑区域中以约100 ms的时间尺度监测许多重要神经递质（包括谷氨酸、gaba、甘氨酸、D-丝氨酸和牛磺酸）的体内动力学。在这个时间和大小尺度上的一系列神经递质的测量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，在这个建议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通过非专家在传统的神经科学实验室。

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.