Efficient detection of pathogens is essential for the development of a reliable point-of-care diagnostic device. Magnetophoretic separation, a technique used in microfluidic platforms, utilizes magnetic microbeads (mMBs) coated with specific antigens to bind and remove targeted biomolecules using an external magnetic field. In order to assure reliability and accuracy in the device, the efficient capture of these mMBs is extremely important. The aim of this study was to analyze the effect of an electroosmotic flow (EOF) switching device on the capture efficiency (CE) of mMBs in a microfluidic device and demonstrate viability of bacteria capture. This analysis was performed at microbead concentrations of 2 × 106 beads/mL and 4 × 106 beads/mL, EOF voltages of 650 V and 750 V, and under constant flow and switching flow protocols. Images were taken using an inverted fluorescent microscope and the pixel count was analyzed to determine to fluorescent intensity. A capture zone was used to distinguish which beads were captured versus uncaptured. Under the steady-state flow protocol, CE was determined to range from 31% to 42%, while the switching flow protocol exhibited a CE of 71–85%. The relative percentage increase due to the utilization of the switching protocol was determined to be around two times the CE, with p < 0.05 for all cases. Initial testing using bacteria-bead complexes was also performed in which these complexes were captured under the constant flow protocol to create a calibration curve based on fluorescent pixel count. The calibration curve was linear on a log-log plot, with R2-value of 0.96. The significant increase in CE highlights the effectiveness of flow switching for magnetophoretic separation in microfluidic devices and prove its viability in bacterial analysis.

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