The Development of a Chip-Scale Nano-Calorimeter

描述（由申请人提供）：结合相互作用的研究是基础生物学研究和药物研发的一个核心方面，有许多分析方法可用于研究这些相互作用的各个方面。每个都有自己的长处和短处。量热法目前不是作为一种筛选工具，而是作为一种了解特定反应的工具，在结合相互作用的研究中非常重要。量热计测量在一定反应物浓度范围内反应释放或吸收的能量，以确定焓驱动过程（与键的数量和类型有关）和熵驱动过程（与结合位点和配体的形状有关）的相对贡献。不幸的是，对大量蛋白质（0.5到5mg）的需求限制了它的使用。此外，还有一些反应的热量太小，当前一代的量热计无法测量。我们正在开发一种基于通过纳米孔径阵列的超光传输（EOT）的芯片级量热计。Stark等人和Brolo等人已经证明，这些纳米孔阵列器件可以作为亲和传感器，其中一个结合伙伴固定在纳米孔阵列器件的表面。由于缓冲介质的介电函数改变了等离子激元的激发条件，因此这些纳米孔阵列传感器的信号与温度有关。在纳米孔阵列表面上方约100nm厚的介电层中保持浓度恒定，可以使用EOT作为快速灵敏的温度传感器来测量结合事件的反应热（焓，¿H）。在单个芯片上复用多个纳米孔阵列传感器设备的固有能力使控制的同时测量能够表征混淆效应（例如缓冲液稀释，混合，缓冲液中DMSO的存在）和这些效应的反卷积以确定真正的反应热。这种多路复用也表明了将其用于高通量筛选以及扩展量热法当前作用的可能性。早期结果表明，纳米孔阵列量热系统有可能将所需的蛋白质量减少1000倍，灵敏度提高100倍。这将扩大量热法在药物研发中的应用。我们的研究计划由三个具体目标组成，以演示该技术的原理验证。目的1和2探讨纳米孔阵列器件设计和样品交付中涉及的基本设计选项和权衡。Aim 3将这些结果整合到一个量热系统中，并根据定量里程碑评估由此产生的测量性能。在本应用中，我们建议开发一种新的芯片级纳米热量计，以解决当前量热技术的关键限制（化合物使用，灵敏度和分析时间）。该项目的两个主要性能目标是将化合物使用量减少至少1000倍，将灵敏度提高至少100倍，同时确保与现有液体处理设备的兼容性。

DESCRIPTION (provided by applicant): The study of binding interactions is a central aspect of basic biology research and pharmaceutical R&D and there are numerous analytical methods available to study various aspects of these interactions. Each has its own strengths and weaknesses. Calorimetry is currently used, not as a screening tool, but as a tool to understand a specific reaction and is very important in the study of binding interactions. A calorimeter measures the energy released or absorbed by a reaction over a range of reactant concentrations to determine the relative contributions of enthalpically driven processes (related to the number and types of bonds) and entropically driven processes (related to the shapes of the binding site and the ligand). Unfortunately, the need for a large amount of protein (0.5 to 5mg) limits its usage. Additionally, there are some reactions where the amount of heat is too small for the current generation of calorimeters to measure. We are developing a chip scale calorimeter based on extraordinary optical transmission (EOT) through an array of nanometric apertures. Stark et al and Brolo et al have shown that these nanohole array devices can be used as affinity sensors where one of the binding partners is immobilized on the surface of the nanohole array device. With these nanohole array sensors the signal is temperature dependent due to the dielectric function of the buffer changing the plasmon excitation conditions. Holding the concentration constant in an approximately 100nm thick layer of dielectric directly above the nanohole array surface enables the use of EOT as a fast and sensitive temperature sensor to measure the heat of reaction (enthalpy, ¿H) from binding events. The inherent ability to multiplex many nanohole array sensor devices on a single chip enables the simultaneous measurement of controls to characterize confounding effects (e.g. buffer dilution, mixing, presence of DMSO in the buffer) and deconvolution of these effects to determine the true heat of reaction. This multiplexing also indicates the possibility of using this for high throughput screening as well as expanding on the current role of calorimetry. Early results indicate that a nanohole array calorimetry system has the potential to reduce the amount of protein required by 1000-fold and increase sensitivity by 100-fold. This will expand the use of calorimetry in pharmaceutical R&D. Our research plan consists of three specific aims to demonstrate proof-of-principle for this technology. Aims 1 and 2 explore the fundamental design options and tradeoffs involved in nanohole array device design and sample delivery. Aim 3 integrates these results into a calorimetry system and assesses the resulting measurement performance against quantitative milestones. In this application we propose to develop a new chip-scale nanocalorimeter that addresses the key limitations (compound usage, sensitivity, and analysis time) of current calorimetry technologies. The two primary performance goals for this project are to decrease compound usage by at least 1000-fold and to increase sensitivity by at least 100-fold while ensuring compatibility with existing liquid handling equipment.