The Development of a Chip-Scale Nano-Calorimeter

描述(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中心方面，有许多分析方法可用于研究这些相互作用的各个方面。每一个都有自己的长处和短处。量热法目前被使用，不是作为一种筛选工具，而是作为一种工具来了解特定的反应，在结合作用的研究中非常重要。量热计测量反应在一定反应物浓度范围内释放或吸收的能量，以确定热驱动过程(与键的数量和类型有关)和熵驱动过程(与结合位置和配体的形状有关)的相对贡献。不幸的是，对大量蛋白质(0.5到5毫克)的需求限制了它的使用。此外，还有一些反应的热量太小，无法用当前一代的量热仪进行测量。我们正在开发一种基于通过纳米孔阵列的非凡光学传输(EOT)的芯片量热仪。斯塔克等人和布罗罗等人已经证明，这些纳米孔阵列器件可以用作亲和力传感器，其中一个结合伙伴被固定在纳米孔阵列器件的表面。对于这些纳米孔阵列传感器，由于缓冲液的介电功能改变了等离子体激发条件，因此信号是温度相关的。在纳米孔阵列表面正上方约100 nm厚的电介质层中保持浓度恒定，可以使用EOT作为快速而灵敏的温度传感器来测量结合事件的反应热(焓，？H)。在单个芯片上多路复用多个纳米孔阵列传感器设备的固有能力使得能够同时测量控制，以表征混杂效应(例如，缓冲液稀释、混合、缓冲液中存在二甲基亚砜)以及这些影响的去卷积，以确定真实的反应热。这种多路传输也表明了将其用于高通量筛选的可能性，以及扩展量热法的当前作用。早期结果表明，纳米孔阵列量热系统有可能将所需蛋白质的数量减少1000倍，将灵敏度提高100倍。这将扩大量热法在药物研发中的使用。我们的研究计划包括三个具体目标，以证明这项技术的原理。目标1和目标2探索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。目的3将这些结果集成到量热系统中，并对照量化里程碑评估由此产生的测量性能。在这项应用中，我们建议开发一种新的芯片级纳米量热仪，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的使用量减少至少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.