Environmental microbial bioprospecting enabled by a Raman fingerprinting and functional sorting on a microfluidic...

Function-driven microbial bioprospecting has been hindered by persistent trade-offs among throughput, resolution, and cell viability in current single-cell analysis technologies. To overcome these limitations, we developed MicroSD-RFFS, a platform that seamlessly integrates a microfluidic static droplet array with Raman spectral fingerprinting and functional sorting. This platform achieves high-throughput single-cell encapsulation via blade-assisted droplet generation (∼3s, 720 droplets per chip), high-resolution spectral acquisition under low-power excitation (532 nm, 5 mW, 5 s per cell) by leveraging static droplet arrays on cost-effective aluminum foil-based chips, and culture-compatible sorting via substrate-induced replica plating. Applied to the screening of phosphorus-solubilizing bacteria (PSB), MicroSD-RFFS employs a dual-biomarker strategy that utilizes the C-D band ratio calibrated for D₂O tolerance (CDR_cal) to assess functional activity, and Raman fingerprinting for strain identification. By integrating an optimized dimensionality reduction method (a permutation-based singular value decomposition (SVD) low-rank approximation algorithm) along with discriminant models, the system efficiently isolated and identified six PSB strains from complex soil communities, achieving near-perfect discrimination probability (minimum Prob. = 0.979) and a reconstruction error on the order of 10^-26∼10^-28. These results are consistent with omics data and markedly outperform those obtained through conventional multivariate analysis. Their functional traits were accurately ranked based on CDR_cal, consistent with conventional phosphorus-solubilizing activity assays. Thus, MicroSD-RFFS provides an efficient and robust platform and method for function-driven microbial discovery at the single-cell level.