Fabrication of a microfluidic enzyme reactor utilizing magnetic beads

An enzyme-catalyzed microfluidic assay using magnetic micro-beads is described. Here, diaphorase (DI) (E.C. 1.6.99) is covalently attached to the magnetic micro-beads (2.7 mum) and integrated into a short section of a microchip fabricated from PDMS. DI converts non-fluorescent resazurin to fluorescent resorufin in the presence of nicotinamide adenine dinucleotide phosphate (NADH). In this work, an embedded magnet holds the micro-beads in place within the microchannel while a solution of resazurin and NADH in buffer is flowed through the beads. Incorporation of the micro-beads into the microchannel requires only a few minutes and offers well-defined spatial resolution and reproducibility. At a flow rate of 41.2 microL/h, a stable state for the enzyme reaction in the microfluidic format was achieved within 50 s. The maximum conversion of the reaction was obtained at a concentration of 1.25 mM NADH. The reaction yield is affected by ZnCl(2) and at concentrations in excess of 90.0 mM, the activity of DI was almost double without ZnCl(2). At 5.2 mM potassium chloride, the activity of DI reached its maximum value. Overall, the conversion of resazurin in microfluidic format was more than twice than that in a batch assay.