Hybrid Nanostructures for Wireless In Vivo Action Potential Sensing

描述（由申请人提供）：利用电化学探针来感知体外和体内神经元细胞的动作电位，使我们对神经细胞信号传导的机制有了许多基本的了解。这些探针很难在体内使用，特别是当需要同时检测多个神经元细胞时，因为每个神经元都需要连接到电化学探针，并且受试者必须麻醉。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，这具有临床应用的潜力。提出的研究背后的具体假设是，半导体纳米粒子荧光共振能量转移（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，从而允许来自单个神经细胞的无创体内电压映射。首先，在提出的方案中，通过使用通过双光子激发过程发出荧光的染料材料，可以实现单神经元的空间分辨率。其次，量子点的光学特性比通常使用的有机染料有许多优点，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，以及宽的带隙吸收。第三，这些纳米颗粒材料的荧光寿命在20- 50ns之间，这表明该技术可以通过光栅扫描仪器以毫秒级的时间分辨率绘制来自大面积的电压信号。本提案的实验重点是设计和体外表征具有共价结合的可移动亲脂受体对的高发光纳米颗粒FRET供体，可以用近红外光激发并发射。具体目标是：1。设计和合成系缚FRET为基础的供体和受体染料。这将通过(i)合成由纳米粒子供体与亲脂性有机受体共价连接而成的各种染料，（ii）优化纳米粒子表面化学以促进与人工磷脂双层的最大粘附，以及（iii）表征染料在这些双层上的FRET特性来实现。2. 在哺乳动物神经细胞的体外光信号传导和细胞毒性研究中，(i)染料表面功能化将被评估以促进非特异性结合细胞膜而不发生内吞作用。然后，我们将研究FRET染料（ii）通过同步光学和电压传感在体外神经网络上的光学特性，以及（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.