3D printed, multi-material microfluidic calorimetry: Critical tools to study protein stability

项目摘要 该提案旨在通过将绝热的微热量测量方法应用于微热量测量领域， 扫描量热仪（ASC），以提高蛋白质去折叠的热力学数据的准确性。 与现有的数千种显微DSC（差示扫描量热仪）不同， ASC在相变期间不必牺牲灵敏度的准确性，实际上 随着扫描接近相变，精度提高。这一点尤其重要，因为 微量量热法是提供焓、结合、结合和/或结合的实验测量的黄金标准。 亲和力和热容来计算蛋白质结构变化的熵和吉布斯能。 这项工作的重要性在于，它将产生一套工具， 药理学和生物化学的发现，通过了解 热力学在疾病发生、诊断和治疗中的作用。 微流体设备3D打印的最新进展可以消除 在研究蛋白质折叠/解折叠时， 稳定Aim 1将建立在我们之前在3D打印和注射液体方面的经验基础上 材料，以通过制造微ASC装置来改善微流体装置的能力。我们 我将设计一系列印刷热量计部分使用我们的定制数字光项目 立体光刻（DLP-SL）3D打印机。每个打印部分可以在打印后进行修改 以实现不同的功能，包括但不限于：（1）铸造模具，以使金属 外壳，由可注射材料组成的热电发电机，以及（3）低热 导电气凝胶浸渍树脂。然后，由于打印机可以将内部特征打印为 小到7厘米，我们可以确保每个部分都有一个倒钩和一个交配受体， 不同的打印部分组装成一个热量计。目标2将使用这些 功能化的部分，以创建绝热条件，低热导率，低热 噪声，以及微型ASC和微型ITC（等温滴定）所需的高灵敏度 量热计）在相同的微流体平台上。Aim 3将使用微型ASC/ITC设备， 测量两种淀粉样蛋白，淀粉样蛋白酶（Aβ）和溶菌酶的解折叠动力学。𝛽这将 表明该技术适用于改进的热力学测量， 适用于其他蛋白质系统。本研究的总体目标是开发一系列设备 可以被广泛使用，然后使用这些工具来测量基本的 热力学行为决定了可导致疾病的关键淀粉样蛋白的稳定性。

Project Summary This proposal aims to revolutionize the micro-calorimetry field by miniaturizing an adiabatic scanning calorimeter (ASC) to improve the accuracy of thermodynamic data of protein unfolding. Unlike the thousands of existing microscopic-DSCs (differential scanning calorimeters), a micro- ASC does not have to sacrifice accuracy for sensitivity during phase transitions, and actually improves in accuracy as the scan approaches a phase transition. This is particularly important as micro-calorimetry is the gold standard to provide experimental measurements of enthalpy, binding affinity, and heat capacity to calculate the entropy and Gibbs energy of protein structural changes. The significance of the proposed work is it will produce a suite of tools that can increase the pace of pharmacological and biological chemistry discoveries, by understanding the fundamental role thermodynamics has in disease occurrence, diagnosis, and treatment. Recent advancements in 3D printing of microfluidic devices can remove the roadblocks that have prevented taking advantage of ASC’s benefits when studying protein folding/unfolding and stability. Aim 1 will build on our previous experience with both 3D printing and injecting liquid materials to improve the capabilities of microfluidic devices by making micro-ASC devices. We will design a series of printed calorimeter sections using our custom digital light project stereolithography (DLP-SL) 3D printer. Each printed section can then be modified after printing to achieve a different function, including but not being limited to: (1) casting molds to make metallic enclosures, a thermoelectric generator composed of injectable materials, and (3) low thermal conductivity aerogel impregnated resins. Then, because the printer can print internal features as small as 7𝜇𝑚, we can make sure each section has a barb and a mating receptor to allow the different printed sections to be assembled together into a calorimeter. Aim 2 will use these functionalized sections to create the adiabatic conditions, low thermal conductance, low thermal noise, and high sensitivities needed for both a micro-ASC and a micro-ITC (isothermal titration calorimeter) on the same microfluidic platform. Aim 3 will use the micro-ASC/ITC devices to measure the unfolding dynamics of two amyloid proteins, amyloid-𝛽 (A𝛽) and lysozyme. This will show that the technology is suitable for improved thermodynamic measurements and can be applied to other protein systems. The overall objective of this study is develop a series of devices that can be widely accessible, and then use those tools to measure the fundamental thermodynamic behavior that dictates the stability of key amyloid proteins that can cause disease.