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Molecular & Cellular Analysis Technologies
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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.