Development of microfluidic lab-on-a-chip platforms for high-throughput single-cell biochemical analysis and sorting applications

Abstract

Conventional cell analysis methods pool thousands of cells together, masking the biochemical heterogeneity that drives differences in drug response, immune activation, and disease progression. This research describes the design, fabrication, and performance testing of a polydimethylsiloxane (PDMS)-based microfluidic lab-on-a-chip platform built for high-throughput single-cell biochemical analysis and fluorescence-activated sorting. The device integrated hydrodynamic flow focusing for single-cell encapsulation in aqueous droplets, on-chip reaction chambers for biochemical assays, dual-channel fluorescence detection, and dielectrophoretic sorting into collection reservoirs. Four channel configurations were tested with Jurkat T-cells and primary human peripheral blood mononuclear cells (PBMCs). The hybrid configuration (30/50 µm stepped channel) achieved 93.4% sorting purity at 743 cells per minute with 93.7% post-sort viability—outperforming conventional FACS in viability (84.6%) while maintaining comparable purity. On-chip bioluminescence detection of intracellular ATP reached a limit of detection of 0.14 fmol per cell with 6.8% coefficient of variation. These results show that microfluidic platforms can match or exceed traditional sorting technologies for single-cell biochemical profiling when channel geometry is optimized for the specific cell type and assay requirements.