Enthalpy arrays for thermodynamic characterization

描述（由申请人提供）：一种表征分子相互作用的通用方法，无需开发检测，将极大地有利于功能蛋白质组学研究和药物发现；然而，没有一种可用的方法提供大量测量所需的高效检测和低试剂消耗。本提案旨在开发一种灵敏的焓分析工具，用于检测涉及蛋白质、配体、RNA、DNA和其他感兴趣分子的双分子相互作用，而无需任何特殊的分析开发。特别是，该分析不需要开发分子特异性抑制剂、探针、检测器、标签的附着、表面固定或分子的化学修饰。该分析是基于在感兴趣的物种相互作用期间释放的焓的直接检测。该探测器是一种纳米量热计，可以使用200nl的样品体积测量低微摩尔范围内分子浓度的相互作用。这项工作的技术目标是开发一种探测器，可以在6秒的有效测量时间内检测纳米卡范围内的焓。与传统的微量热法相比，这意味着所需样品和测量时间减少了近三个数量级。我们建议开发96个检测器阵列，并使用它来研究HIV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.