DRUG ASSAYS USING ELECTROPHORESIS WITH CELL BIOSENSORS

了解滥用药物对中枢神经系统影响的能力 神经系统在很大程度上取决于对化学物质的理解 神经元之间的平衡。理想情况下，化学分析技术必须是 能够探测细胞内化学成分的变化 (细胞质分析)以及来自明确定义的基因座 细胞外间隔，如突触裂隙。两大主打 用传统的分析方案实现这一目标的障碍是 一直：(L)无法处理超小样本量(在订单上 10(-15)到10(-12)L)，以及(2)无法灵敏地检测到 缺少强生色团或易氧化基团的生物分子。 例如，多肽就属于这一类，其中许多是 神经递质和神经调节剂，以及乙酰胆碱和 谷氨酸。我们通过耦合毛细管克服了这些障碍 电泳法是一种小型化的高效分离技术， 处理亚皮升样本体积，使用活细胞生物传感器 几乎可以在单分子上检测到任何神经活性化合物 水平。该系统使用配体-受体结合和信号转导 电泳后扩增分析物存在的途径 分离。使用两种方法测量转换后的信号：(1) 荧光显微镜下细胞内游离钙离子的变化 用Fluo-3，a培养的PC12和N6108-15细胞的浓度 结合时使其荧光量子产率增加几倍的染料 与Ca~(2+)结合，(2)非洲爪哇跨膜电流的测定 显微注射编码特定受体的信使核糖核酸的卵母细胞。这个 这些生物传感器系统相对于传统分析方案的优势是 以无与伦比的选择性检测分馏的能力 分子处于其自然状态。有了这种技术，我们已经能够 检测复杂生物混合物中的缓激肽和乙酰胆碱。 此外，通过对激活的受体使用选择性拮抗剂，我们已经 表明可以毫不含糊地分配由 毛细管电泳法作为该受体的激动剂 推断该激动剂是否仅通过一个受体亚类起作用。它 我们相信这种方法论可以帮助模型的开发 解释药物(或其作用耦合的内源性物质)如何 通过药物)扰乱正常的神经化学通讯并影响 神经元的可塑性。这项研究计划的长期目标是 实现毛细管电泳联用单细胞生物传感器 探索单个神经元之间的化学连接，并 识别新的神经递质。

The ability to understand the effects of abused drugs on the central nervous system is critically dependent on understanding the chemical balance between neurons. Ideally, a chemical analysis technique must be capable of probing variations in the chemical profile within the cell (cytoplasmic analysis) as well as from well-defined loci in the extracellular compartments such as the synaptic cleft. The two main obstacles to achieving this goal with traditional analysis schemes have been: (l) the inability to handle ultrasmall sample volumes (on the order of 10(-15) to 10(-12) L), and (2) the inability to sensitively detect biomolecules that lack strong chromophores or easily oxidized groups. Falling into this category are peptides, for example, many of which are neurotransmitters and neuromodulators, as well as acetylcholine and glutamate. We have overcome these obstacles by coupling capillary electrophoresis a miniaturized highly efficient separation technique that handles sub-picoliter sample volumes, with a living cell biosensor that can detect virtually any neuroactive compound at the single-molecule level. The system uses ligand- receptor binding and signal-transduction pathways to amplify the presence of an analyte after electrophoretic separation. The transduced signal is measured using two approaches: (1) fluorescence microscopy that images changes in intracellular free Ca2+ concentrations in PC12 and N6108-15 cultured cell lines using fluo-3, a dye that increases its fluorescence quantum yield severalfold upon binding with Ca2+, and (2) measurement of transmembrane currents in Xenopus laevis oocytes microinjected with mRNA that encodes a specific receptor. The strength of these biosensor systems over conventional analysis schemes is the ability to detect, with unsurpassed selectivity, a fractionated molecule in its native state. With this technique, we have been able to detect bradykinin and acetylcholine in complex biological mixtures. Further, by using selective antagonists to the activated receptor, we have shown that it is possible to assign unambiguously a band separated by capillary electrophoresis as an agonist to that receptor and also to deduce whether the agonist solely operates via one receptor subclass. It is our belief that this methodology can aid in the development of models explaining how drugs (or endogenous substances whose effects are coupled through drugs) disrupt normal neurochemical communication and affect neuronal plasticity. The long-term objective of this research program is to implement capillary electrophoresis coupled to single-cell biosensors to probe the chemical connectivity between individual neurons and to identify novel neurotransmitters.