Enthalpy arrays for thermodynamic characterization

描述(由申请人提供)：一种在不进行分析的情况下表征分子相互作用的通用方法将对功能蛋白质组学研究和药物发现都大有裨益；然而，现有的方法都不能提供大量测量所需的有效检测和低试剂消耗。这项建议旨在开发一种灵敏的焓分析工具来检测涉及蛋白质、配体、RNA、DNA和其他感兴趣的分子的双分子相互作用，而不需要任何特殊的分析开发。特别是，该分析不需要开发特定于分子的抑制剂、探针、检测器、标记的附着、表面固定化或分子的化学修饰。该分析是基于对感兴趣物种相互作用过程中释放的热的直接检测。该探测器是一种纳米量能器，将能够使用200毫升的样品体积测量低微摩尔范围内的分子浓度相互作用。这项工作的技术目标是开发一种探测器，可以在纳米卡路里范围内探测热，有效测量时间为6秒。与传统的微量热法相比，这意味着所需的样品和测量时间减少了近三个数量级。我们建议开发一个由96个探测器组成的阵列，并用它来研究艾滋病毒感染和生命周期的关键方面。除了检测相互作用外，焓阵列还可用于滴定以确定结合常数。这项工作将几个工程学科与应用物理和生物化学结合起来。我们通过将微制造与几个工程学科(机械、电气、化学和系统)以及应用物理联系起来的多学科努力，开发了初步的概念验证。我们正在与几位资深生物化学家合作，以确保该系统满足他们的科学需求。

DESCRIPTION (provided by applicant): A generic method to characterize molecular interactions with no assay development would substantially benefit both functional proteomics research and drug discovery; however, none of the available approaches offers the efficient detection and low reagent consumption needed for the large number of measurements. This proposal addresses developing a sensitive enthalpic assay tool to detect bimolecular interactions involving proteins, ligands, RNA, DNA and other molecules of interest without requiring any special assay development. In particular, the assay does not require development of molecule-specific inhibitors, probes, detectors, attachment of labels, immobilization on surfaces, or chemical modifications of the molecules. The assay is based on direct detection of the enthalpy released during interaction of the species of interest. The detector, a nanocalorimeter, will enable measurement of interactions for molecular concentrations in the low micro-molar range using sample volumes of 200 nl. The technical goal of this work is to develop a detector that can detect enthalpies in the nanocalorie range with an effective measurement time of six seconds. This represents nearly three orders of magnitude reduction in the required sample and measurement time compared with conventional microcalorimetry. We propose to develop an array of 96 detectors and use it to study key aspects of HIV infection and life cycle. In addition to detecting interactions the enthalpy array can be used to perform titrations to determine binding constants. The effort integrates several engineering disciplines with applied physics and biochemistry. We have developed the initial proof of concept through a multidisciplinary effort bridging microfabrication with several engineering disciplines (mechanical, electrical chemical and system) as well as applied physics. We are collaborating with several senior biochemists to ensure that the system meets their scientific needs.