Enthalpy arrays for thermodynamic characterization

描述（由申请人提供）：一种用于表征分子相互作用而无需开发检测方法的通用方法将大大有利于功能蛋白质组学研究和药物发现;然而，现有方法均无法提供大量测量所需的有效检测和低试剂消耗。该提案致力于开发一种灵敏的双分子测定工具，以检测涉及蛋白质、配体、RNA、DNA和其他感兴趣分子的双分子相互作用，而不需要任何特殊的测定开发。特别地，该测定不需要开发分子特异性抑制剂、探针、检测器、标记物的附着、在表面上的固定或分子的化学修饰。该测定基于直接检测目标物质相互作用期间释放的焓。该检测器，一个nanocalorimeter，将能够测量的相互作用的分子浓度在低微摩尔范围内使用200 nl的样品体积。这项工作的技术目标是开发一种探测器，可以在六秒的有效测量时间内探测纳卡路里范围内的热量。与传统的微量热法相比，这表示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个检测器组成的阵列，并使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，焓阵列还可用于进行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学和生物化学结合起来。我们通过多学科的努力，将微制造与几个工程学科（机械，电气化学和系统）以及应用物理学联系起来，开发了概念的初步证明。我们正在与几位高级生物化学家合作，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.