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A microfluidic device was implemented to detect the enzymatic reaction of botulinum toxin A (BTA) using Förster resonance energy transfer (FRET). The microfluidic device comprised a main channel having two loading zones, a reaction chamber and a side channel perpendicular to the main channel. The reaction chamber defined by weir in the main channel was packed with microbeads. The movement of the peptide substrate and the BTA in the microfluidic device was controlled by electrophoresis, and the enzymatic reaction of the BTA was detected through the changes of the fluorescence intensity in the reaction chamber. As a result, it was observed that the enzymatic reaction was affected by the electric voltage applied for the movement of the BTA and the peptide and improved by packing the microbeads in the reaction chamber. The microfluidic device provides the tool to investigate the proteolysis of the substrate by the BTA

FRET-Based Detection of Enzymatic Reaction of Botulinum on Microfluidic Device