Microflow cytometry was previously pursued to achieve high-throughput cell analyses. It has advantages of parallelization and miniaturization capabilities that allow fewer detecting elements to simultaneously observe multiple channels. However, the major disadvantage is that it is difficult to precisely control parallel cell positions in each channel when cells pass through the detection area. In this study, we developed a multilayer microfluidic device, which has one sample flow inlet, two sheath flow inlets, and one flow outlet, which can achieve parallel sheath 3D flow focusing and fluorescence detection in 32 channels. The sample flow in each channel is focused and allows the cells or particles to pass through the detection zone one by one. Optimized flow rate ratios of the sample flow, lateral sheath flow, and circular sheath flow were found. The highest throughput of 106 cells/s was obtained using mammalian cells. The current cytometer, which incorporates a microoptic-integrated microfluidic device and newly developed optical system, was proven to have multicolor detection ability.
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