Biophotonics: Gold Superparticles as Raman-active Nanoprobes of Molecular Transport Across Cell Membranes

[00:86804]本提案的目标是开发一种微创的、基于拉曼的传感器，用于监测细胞内离子和神经递质浓度的变化。这些将用于解决转运体功能中的突出问题。金-二氧化硅“超粒子”（围绕二氧化硅核的金粒子壳）直径为100- 200nm，将通过自组装技术制备，并用作表面增强拉曼散射（SERS）的衬底。超粒子的表面将被功能化与非荧光受体设计，以识别无机或有机阳离子。这些纳米探针将通过皮升注射进入细胞，并将使用近红外激光激发监测SERS。采样将使用高光谱分辨率的微型拉曼仪器与倒置显微镜相结合。振动光谱将通过直接分配分析物群频率或通过调制受体光谱剖面来检测特定的结合事件。检测限可以在低纳摩尔范围内，分辨率为毫秒级。拉曼活性纳米探针将用于研究对神经元活动具有重要意义的两种传输系统。首先，SERS将以一种时间依赖性的方式监测Zn2+离子的流入，以深入了解Ca2+离子通道运输Zn2+的机制。这将与通过膜片钳记录技术的总离子流入的电生理测量相关联。其次，将通过监测细胞内血清素水平和苯丙胺类药物MDMA来研究血清素转运体（SERT）的新型反向转运活性。第三，通过将细胞内神经递质浓度与Na+ co-运输引起的离子电流变化相关联，再通过同时使用SERS和贴片夹紧技术来阐明SERT的运输模式。这可能会揭示安非他明和相关精神活性药物背后的作用模式的宝贵见解。这些研究代表了细胞内监测可以解决的无数分子运输问题，例如碳水化合物和氨基酸的主动偶联运输，多药耐药细胞的外排泵和病毒感染。传统上不相关的学科之间的思想交流将为培养学生提供肥沃的土壤。学生将接触到超分子和材料化学（有机合成、胶体科学、分子识别中的设计原理）、光子学（显微镜和激光光学、拉曼理论和应用光谱学、信号处理）、分子和细胞神经科学（细胞转染和培养、转运蛋白功能、电生理学）等方面的问题。他们对拟议项目的密切参与将使他们成为其中一个或多个领域的专家，并通过积极参加与项目有关的讨论小组和会议而对其他领域具有工作知识。

0086804WeiThe objective of this proposal is to develop a minimally intrusive, Raman-based sensor for monitoring changes in intracellular ion and neurotransmitter concentrations. These will be used to address outstanding questions in transporter function. Gold-silica 'superparticles' (shell of gold particles around a silica core) 100-200 nm in diameter will be prepared by self-assembly techniques and used as substrates for surface-enhanced Raman scattering (SERS). The surfaces of the superparticles will be functionalized with non-fluorescent receptors designed to recognize inorganic or organic cations. These nanoprobes will be introduced into cells via picoliter injection and will be monitored for SERS using near-infrared laser excitation. Sampling will be performed using a micro-Raman instrument with high spectral resolution coupled to an inverted microscope stage. The vibrational spectra will enable specific binding events to be detected by direct assignment of analyte group frequencies or by modulation of receptor spectral profiles. Detection limits can be in the low nanomolar range with resolutions on the order of milliseconds.Raman-active nanoprobes will be employed to investigate two transport systems of fundamental importance to neuron activity. First, the influx of Zn2+ ions will be monitored by SERS in a time-dependent fashion to provide insight into the mechanism of Zn2+ transport by Ca2+ ion channels. This will be correlated to electrophysiological measurements of total ion influx by patch clamp recording techniques. Second, novel reverse transport activity by the serotonin transporter (SERT) will be investigated by monitoring intracellular levels of serotonin and the amphetamine--like drug MDMA. Third, the mode of SERT transport will be elucidated by correlating intracellular neurotransmitter concentrations with changes in ion current caused by Na+ co--transport, again by the concurrent use of SERS and patch clamping techniques. This may reveal valuable insights into the mode of action behind amphetamine and related psychoactive drugs. These studies are representative of the myriad molecular transport issues that can be addressed by intracellular monitoring, such as actively coupled transport of carbohydrates and amino acids, efflux pumps in multidrug resistant cells, and viral infections.The mutual exchange of ideas from traditionally unrelated disciplines will provide fertile ground for student training. Students will be exposed to issues in supramolecular and materials chemistry (organic synthesis, colloid science, design principles in molecular recognition), photonics (microscope and laser optics, Raman theory and applied spectroscopy, signal processing), and molecular and cellular neuroscience (cell transfection and cultures, transporter function, electrophysiology). Their intimate involvement in the proposed project will make them experts in one or more of these areas, and have a working knowledge of the others by their active participation in project-related discussion groups and conferences.