Microcantilever Biosensors Based on Ligand Gated Ion Channel Receptors

描述（由申请人提供）：该项目的主要目标是开发一种用于药物开发的模拟人脑受体的传感器。该传感器将能够在短时间内评估大量候选药物的结合，从而大大提高了识别潜在治疗剂的速度。该项目的主要目标是开发基于最近发现的乙酰胆碱结合蛋白（AChBP）的受体蛋白的改进的微生物传感器。AChBP是一种可溶性蛋白质，其结构和药理学与中枢和外周神经系统中存在的烟碱乙酰胆碱受体（nAChR）惊人相似。由于nAChR是配体门控离子通道（LGIC）的一个大的超家族的成员，该项目将最终开发传感器，这将是有用的高通量药物筛选和药物发现这类神经元受体。具体而言，我们的目标是：1）开发一种微悬臂梁生物传感器利用AChBP及其衍生物作为生物传感器蛋白。2)研究基本的表面共轭化学，并建立微悬臂梁传感的修改协议。该项目充分利用了我们在LGIC结构和功能（Schulte博士）以及微悬臂梁设计（Ji博士）方面的综合专业知识。目的1将评估AChBP本身作为潜在有用的生物传感器分子。我们将通过探索其与nAChR，GABAR，glycineR和5-HT 3R配体的相互作用，初步展示这种传感器在高通量药物筛选中的应用。由于AChBP可以突变以改变其配体特异性，因此还将开发通过定点诱变和/或嵌合构建获得的衍生物。由于AChBP通常被用作配体门控离子通道受体建模的模板，这项工作的另一个好处将是在这些重要受体的配体特异性的基础上扩展知识。目标2将利用AChBP和其他受体蛋白，通过改进的结合化学结合季博士实验室开发的改进的微悬臂梁阵列技术来改善传感器响应

DESCRIPTION (provided by applicant): The primary goal of this project is to develop a sensor for use in drug development that mimics human brain receptors. This sensor will be capable of evaluating the binding of large numbers of drug candidates in a short period of time; thus greatly enhancing the rate at which potential therapeutic agents can be identified. The primary goal of this project is to develop improved microcantilever-based biosensors using receptor proteins based on a recently discovered acetylcholine binding protein (AChBP). The AChBP is a soluble protein that displays structure and pharmacology that is strikingly similar to the nicotinic acetylcholine receptor (nAChR) present in the central and peripheral nervous systems. Since the nAChR is a member of a large super-family of ligand gated ion channels (LGICs), this project will ultimately develop sensors that will be useful in high throughput drug screening and drug discovery for this class of neuronal receptors. Specifically, we aim to: 1) Develop a microcantilever biosensor utilizing the AChBP and its derivatives as biological sensor proteins. 2) Investigate fundamental surface conjugation chemistries and establish modification protocols for microcantilever sensing. The project draws heavily on our combined expertise in LGIC structure and function (Dr. Schulte) and microcantilever design (Dr. Ji). Aim 1 will evaluate the AChBP itself as a potentially useful biosensor molecule. We will initially demonstrate the use of this sensor in high-throughput drug screening by exploring its interaction with nAChR, GABAR, glycineR and 5-HT3R ligands. Since the AChBP can be mutated to alter its ligand specificity, derivatives obtained via site directed mutagenesis and/or chimeric construction will also be developed. Since the AChBP is commonly used as a template for modeling of ligand gated ion channel receptors, an additional benefit of this work will be an expanded knowledge of the basis of ligand specificity at these important receptors. Aim 2 will utilize the AChBP and other receptor proteins to improve sensor response through improved conjugation chemistries in combination with improved microcantilever array technology developed in Dr. Ji's laboratory