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.