In vivo chemical monitoring using capillary separations

描述（由申请人提供）：我们的总体目标是开发新的生物分析方法来探索体内脑化学动力学。大脑中的神经元和神经胶质通过释放神经递质与邻近细胞上的受体相互作用来进行交流。监测体内神经化学物质和代谢物的浓度动态是了解脑功能、疾病和治疗的重要工具。在体内监测化学信息的一种通用而有效的方法是将采样方法（如微透析）与分析测量相结合。尽管这种方法已被证明是无价的，但其效用受到时间分辨率、空间分辨率差、神经肽监测结果差以及仅适用于急性测量的限制。在这个项目中，我们将开发技术和方法来解决这些问题。时间分辨率很重要，因为在行为和实验操作过程中，发射器的浓度会迅速变化。当浓度脉冲传输到分析系统时，时间分辨率常常受到色散的限制。我们将开发一种微流体采样系统，将含水样品流分割成油流中的液滴，然后通过基于芯片的快速电泳分析液滴。样本流分割将防止弥散在质量运输和允许时间分辨率为10秒或更好的许多神经递质。该系统将与小型化的采样探针相结合，以提高空间分辨率，并允许进入小的大脑区域。神经肽调节许多脑功能；然而，在体内监测它们受到当前方法灵敏度的限制，因此样品必须收集约30分钟，导致时间分辨率差。我们将开发基于毛细管液相色谱和微流控免疫分析的高灵敏度神经肽检测方法。检测限为1 pM / 1 ？L样品允许在神经肽监测的时间分辨率前所未有的10倍改进。体内化学测量几乎总是精确地进行；然而，如果能够在几周内监测神经化学，以监测与疾病（如成瘾）或正常功能（如学习）相关的渐进变化，那将是非常有用的。反应性神经胶质瘤通常会阻止长期监测，这是一种组织反应，会导致探针被封装，并阻止从活跃的神经组织中取样。我们将探索使用已知的抑制反应性胶质瘤和支持神经再生的化合物的药理学干预来延长体内监测。最后，我们将进行基础神经科学研究，作为测试方法的一种手段，并向更广泛的神经科学界展示它们的实用性。这些应用包括确定：1)瘦素受体在调节多巴胺和摄食行为中的作用；2)精神兴奋剂对阿片肽的影响；3)不同行为表型背后的神经化学差异是药物成瘾易感性的一个模型。

DESCRIPTION (provided by applicant): Our overall objective is to develop new bioanalytical methods for exploring brain chemistry dynamics in vivo. Neurons and glia in the brain communicate by releasing neurotransmitters that interact with receptors on neighboring cells. Monitoring the concentration dynamics of neurochemicals and metabolites in vivo is a vital tool in the effort to understand brain function, diseases, and treatments. A versatile and effective approach for in vivo monitoring of chemical messages is to couple sampling methods, such as microdialysis, to analytical measurements. Although this approach has proven invaluable, its utility is limited by poor temporal resolution, poor spatial resolution, poor results for neuropeptide monitoring, and application to only acute measurements. In this project, we will develop technology and methods to solve these problems. Temporal resolution is important because concentrations of transmitters can change rapidly during behavior and experimental maneuvers. Temporal resolution is often limited by dispersion of concentration pulses as they are transported to the analytical system. We will develop a microfluidic sampling system whereby the aqueous sample stream is segmented into droplets within a stream of oil and the droplets subsequently analyzed by rapid chip-based electrophoresis assays. Sample stream segmentation will prevent dispersion during mass transport and allow temporal resolution of 10 s or better for many neurotransmitters. This system will be coupled to miniaturized sampling probes to improve spatial resolution and allow access to small brain regions. Neuropeptides regulate many brain functions; however, monitoring them in vivo is limited by the sensitivity of current methods so that samples must be collected for ~30 min resulting in poor temporal resolution. We will develop high sensitivity neuropeptide assays based on capillary liquid chromatography and microfluidic immunoassays. The assays will have detection limits of 1 pM for 1 ?L samples allowing an unprecedented 10-fold improvement in temporal resolution for neuropeptide monitoring. In vivo chemical measurements are nearly always performed acutely; however, it would be extremely useful to be able to monitor neurochemistry over a period of weeks to monitor progressive changes associated with diseases, like addiction, or normal function, like learning. Long term monitoring is typically prevented by reactive gliosis, a tissue reaction that results in encapsulation of the probe and prevents sampling from active neural tissue. We will explore the use of pharmacological interventions with compounds known to suppress reactive gliosis and support neuroregeneration to prolong in vivo monitoring. Finally, we will perform fundamental neuroscience studies as a means of testing the methods and demonstrating their utility to the broader neuroscience community. These applications include determining: 1) the role of leptin receptors in regulating dopamine and feeding behavior; 2) the effect of psychostimulants on opioid peptides, and 3) neurochemical differences underlying distinct behavioral phenotypes that are a model for vulnerability to drug addiction. Mental illnesses and neurological diseases comprise some of the most devastating and expensive to treat disorders in modern society. Determining the neurochemical imbalances underlying such disorders is a key step in developing appropriate therapies; however, in most cases the neurochemistry is not well understood. In this project, we are developing novel instrumentation and techniques that enable neurochemicals to be monitored in the living brain. These new methods will enable important questions to be addressed relating to underlying causes of diseases involving the brain as diverse as addiction, Huntington's disease, and obesity.