Advances in microfabrication technology can enable innovative tools for isothermal titration calorimetry (ITC), which enables direct, label-free determination of thermodynamic parameters of reaction systems. Currently existing microfabricated ITC devices, however, either do not yet allow quantitative thermodynamic characterization, or are not well suited to ITC characterization of fast chemical reactions. Aiming to address these limitations, we present a microfabrication-based ITC approach that uses a combined in-mixing and post-mixing titration approach for accurate characterization of thermodynamic parameters. By accounting for reaction heat produced both during and after the reagent mixing process, accurate, quantitative ITC measurements are achieved. The ITC device design in this approach features a pair of three-dimensional self-crossing channels that serve both as micromixers and reaction cells. A bismuth-antimony thermopile whose hot and cold junctions are aligned to the centers of the crossing channels, is used for reaction heat detection. The microfabricated ITC device has a low limit of detection of 45 nW, and a short thermal response time of 800 ms. The utility of the device has been demonstrated with quantitative ITC measurements of the model chemical reaction system of 18-Crown-6 and barium chloride. The thermodynamic parameters of the reaction system, including the stoichiometry, equilibrium binding constant, and enthalpy change were obtained and found to be in agreement with widely accepted values in the literature.
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