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

该提案的目的是开发一种侵入性最小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未决问题。 金-硅“超粒子”（金颗粒围绕二氧化硅核的外壳）直径为100-200 nm，将通过自组装技术制备，并用作表面增强拉曼散射（Sers）的基底。 超微粒的表面将被设计成识别无机或有机阳离子的非荧光受体功能化。 这些纳米探针将通过皮升注射引入细胞，并将使用近红外激光激发来监测Sers。 将使用具有高光谱分辨率的显微拉曼仪器与倒置显微镜载物台耦合进行采样。 振动光谱将使得能够通过分析物基团频率的直接分配或通过受体光谱分布的调制来检测特异性结合事件。 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两个对神经元活动至关重要的运输系统。 首先，通过Sers以时间依赖的方式监测Zn 2+离子的流入，以提供对Ca 2+离子通道的Zn 2+转运机制的深入了解。 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。 第二，新的反向转运活动的血清素转运蛋白（SERT）将通过监测细胞内水平的血清素和苯丙胺类药物MDMA的研究。 第三，SERT运输的模式将阐明相关的细胞内神经递质浓度与离子电流的变化所造成的Na+共运输，再次通过同时使用Sers和膜片钳技术。 这可能揭示出对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转运问题，如碳水化合物和氨基酸的主动偶联转运，多药耐药细胞中的外排泵，以及病毒感染。传统上不相关学科的相互交流将为学生培训提供肥沃的土壤。 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），以及分子和细胞神经科学（细胞转染和培养，转运功能，电生理学）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，并通过积极参与与项目有关的讨论小组和会议，对其他领域有工作知识。

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.