(142ap) Separation and Isolation of Biomolecules Using Motion of Magnetic Spheres in Microfluidic Channels

The use of microfluidic devices and magnetic beads for applications in biotechnology has been extensively explored over the last decade. This work presents a simple technique for isolation and separation of biomolecules using magnetic bead movement on a microchip with zero-flow conditions. The microchip consisted of two well reservoirs (W1 and W2) connected via a tapered microchannel. The bead motion was first analyzed on the microchip and a permanent magnet was moved alongside the microchip, dragging the beads at equivalent speed through the microchannel between the two wells. It was found that  greater than 95 % bead transfer from W1 to W2 occurred at an optimal velocity of 0.7 mm/s. Prior to testing our microchip with enzymatic reactions, we characterized our enzyme, alkaline phosphotase (AP), and substrate, 6-8-difluoro-4-methylumbelliferyl phosphate (DiFMUP), with an off-chip control. AP was used to dephosphorylate the non-fluorescent DiFMUP to fluorescent 6,8-difluoro-7-hydroxy-4-methylcoumarin (DiFMU). Next, three assays were performed using our microchip: non-specific adsorption of DiFMUP, isolation of AP, and separation of AP. Biotinylated AP was immobilized on streptavidin coated magnetic beads for the isolation and separation assays. Our non-specific adsorption assay indicated that we were able to transfer our beads with less than 0.002% carryover of DiFMUP. Our isolation assay indicated that we were able to efficiently isolate biotinylated AP with our streptavidin conjugated magnetic beads. 100% of DiFMUP was converted to DiFMU within 15 minutes. Our separation assay elucidated the binding capacity of our beads, wherein leftover unbound AP converted 100% of DiFMUP within 10 minutes and samples with less than full bead capacity (i.e. all AP was transferred) did not convert any of the substrate. The K_m value for the isolation assay was approximately 16% greater than our off-chip control indicating that the immobilization of AP onto the beads has a minor inhibitory effect on the reaction kinetics of the enzyme.