We demonstrate spontaneous separation of active enzyme molecules from a mixture based on their chemotactic response towards an imposed substrate concentration gradient. The separation is observed within a two-inlet-five-outlet microfluidic network, designed to allow mixture of active and inactive enzymes, each labeled with different fluorophores, to flow through one of its inlets. Substrate solution prepared in buffer was then introduced through the other inlet at the same flow rate. The steady state concentration profiles of the enzymes at specific positions along the length of the micro-channel were measured using a highly sensitive fluorescence microscope. In presence of a substrate concentration gradient, active enzyme molecules migrated more within the substrate channel compared to the inactive ones. For each of the outlets, the separation was quantified in terms of a separation coefficient. The experiments were repeated with different combinations of enzymes at different experimental conditions. Coupling the physics of laminar flow of liquid and diffusion of species in fluid, multiphysics simulations were developed to estimate the maximum possible separation for the given set of experimental conditions. Our results show that with appropriate microfluidic arrangement, molecular chemotaxis may lead to spontaneous, inexpensive, rapid and stress free separation protocols for active biomolecules

Chemotactic separation of enzymes

248th National Meeting (2014)

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