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Möller, Friederike M.; Kieß, Michael and Braun, Dieter (2016): Photochemical Microscale Electrophoresis Allows Fast Quantification of Biomolecule Binding. In: Journal of the American Chemical Society, Vol. 138, No. 16: pp. 5363-5370

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Abstract

Intricate spatiotemporal patterns emerge when chemical reaction's couple to physical transport. We, induce electrophoretic transport by a confined photochemical reaction and use it to infer the binding strength of a second, biomolecular binding reaction under physiological conditions. To this end, we use the photoactive compound 2-nitrobenzaldehyde, which releases a proton upon 375 nm irradiation. The charged photoproducts locally perturb electroneutrality due to differential diffusion, giving rise to an electric potential Phi in the 100 mu V range on the micrometer scale. Electrophoresis of biomolecules in this field is counterbalanced by back-diffusion within seconds. The biomolecule concentration is measured by fluorescence and settles proportionally to exp(-mu/D Phi). Typically, binding alters either the diffusion coefficient D or the electrophoretic mobility mu. Hence, the local biomolecule fluorescence directly reflects the binding state. A fit to the law of mass action reveals the dissociation constant of the binding reaction. We apply this approach to quantify the binding of the aptamer TBA15 to its protein target human-alpha-thrombin and to probe the hybridization of DNA. Dissociation constants in the nanomolar regime were determined and match both results in literature and in control experiments using microscale thermophoresis. As our approach is all-optical, isothermal and requires only nanoliter volumes at nanomolar concentrations, it will allow for the fast screening of biomolecule binding in low volume multiwell formats.

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