Hybrid Nanostructures for Wireless In Vivo Action Potential Sensing

描述（由申请人提供）：使用电化学探针在体外和体内检测神经元细胞的动作电位，使我们对神经细胞信号传导机制有了基本的了解。这些探针难以在体内使用，特别是当需要同时检测许多神经元细胞时，因为每个神经元需要连接到电化学探针，并且受试者必须被麻醉。我们的长期目标是开发一种侵入性更小的技术来监测体内单个神经细胞的动作电位，具有临床应用的潜力。提出的研究背后的具体假设是，半导体纳米颗粒荧光共振能量转移（FRET）染料在近红外光谱中发射和激发，可以增强先前开发的光学动作电位传感技术的信噪比，以允许来自单个神经细胞的电压的非侵入性体内映射。首先，在所提出的方案中，单神经元的空间分辨率是通过使用染料材料，荧光通过双光子激发过程。第二，量子点的光学性质具有优于通常采用的有机染料的许多已证明的优点，包括单光子和双光子吸收截面的数量级改进、增强的光稳定性、高量子效率和宽的带隙以上吸收。第三，这些纳米颗粒材料具有20-50 ns之间的短荧光寿命，表明该技术可用于通过光栅扫描仪器以毫秒时间分辨率绘制来自大区域的电压信号。该提案的实验重点是设计和体外表征的高度发光的纳米颗粒FRET供体与共价结合的移动的亲脂性受体对，可以刺激和发射近红外光。具体目标是：1.设计和合成基于束缚FRET的供体和受体染料。这将通过（i）合成由共价连接至亲脂性有机受体的纳米颗粒供体组成的各种染料，（ii）优化纳米颗粒表面化学以促进对人工磷脂双层的最大粘附，以及（iii）表征这些双层上的染料FRET特性来实现。2.使用哺乳动物神经细胞的体外光学信号传导和细胞毒性研究。（i）将评价染料表面官能化以促进与细胞膜的非特异性结合而无内吞作用。然后，我们将研究FRET染料（ii）在体外神经网络上的光学特性，通过同时光和电电压传感，和（iii）粘附动力学和细胞毒性。

DESCRIPTION (provided by applicant): The use of electrochemical probes for sensing the action potentials of neuron cells in vitro and in vivo has led to much of our basic understanding concerning the mechanisms of nerve cell signaling. These probes are difficult to employ in vivo, especially when the simultaneous detection of numerous neuron cells is required, as each neuron requires a connection to an electrochemical probe, and subjects must be anesthesized. Our long term goal is to develop a much less invasive technology to monitor action potentials, in vivo, from individual nerve cells, that has potential for clinical application. The specific hypothesis behind the proposed research is that semiconductor nanoparticle Fluorescence Resonance Energy Transfer (FRET) dyes which emit and are excited in the near IR spectrum, can enhance the signal to noise of previously developed optical action potential sensing techniques to allow the noninvasive in vivo mapping of voltages from individual nerve cells. First, in the proposed scheme, single-neuron spatial resolution is achievable by using dye materials that fluoresce via a two-photon excitation process. Second, the optical properties of quantum dots have numerous demonstrated advantages over the organic dyes that have been typically employed, including orders of magnitude improvent in the one and two-photon absorption cross- sections, enhanced photostability, high quantum efficiencies, and a broad above band-gap absorption. Third, these nanoparticle materials have short fluorescence lifetimes between 20-50 ns, indicating that this technique could be used to map out the voltage-signals from large areas with millisecond time resolution via raster-scanning instrumentation. The experimental focus of this proposal is on the design and in vitro characterization of highly luminescent nanoparticle FRET donors with covalently bound mobile lipophilic acceptor pairs that can be stimulated with and emit near IR light. The specific aims are to: 1. Design and synthesize tethered FRET based donor and acceptor dyes. This will be accomplished through the (i) synthesis of various dyes consisting of a nanoparticle donor covalently linked to a lipophilic organic acceptor, (ii) optimization of the nanoparticle surface chemistry to encourage maximum adhesion to artificial phospholipid bilayers, and (iii) characterization of the dye FRET properties on these bilayers. 2. In Vitro optical signaling and cytotoxicity studies using mammalian nerve cells, (i) The dye surface functionalization will be evaluated to encourage nonspecific binding to cell membranes without endocytosis. Then, we will investigate the FRET dye (ii) optical characteristics on in vitro neural networks via simultaneous optical and electrical voltage sensing, and (iii) adhesion kinetics and cytotoxicities.