High-Throughput Dual-Optical Droplet Screening for Quantitative Functional Bacterial Selection

Abstract Droplet microfluidics enables ultra-high-throughput biological screening, yet most platforms rely on single-parameter optical detection, limiting the information available for selection and analysis. Here, we present a high-throughput droplet screening and sorting platform that integrates simultaneous fluorescence and absorbance (scattering) detection with acoustic actuation for robust, kHz-rate operation. By combining complementary optical modalities within a single device, we enable multi-parameter characterization and sorting of droplets based on both molecular and cellular properties. We first validate the system using model droplets containing defined combinations of fluorophores and absorbers, achieving on-demand sorting of multiple sub-populations with efficiencies exceeding 97% at kilohertz rates. We then demonstrate biological functionality by quantifying bacterial growth via optical density (scattering/absorbance) and protein expression via green fluorescent protein (GFP) fluorescence within the same droplets. This dual readout enables simultaneous assessment of cell proliferation and gene expression and allows normalization of fluorescence signals to cell number, providing single-cell-level insight within droplets. Importantly, we observe a temporal delay of ∼42 min between growth and fluorescence signals, highlighting the dynamic relationship between cell proliferation and protein expression. The ability to resolve and exploit such multi-parameter dynamics enables more precise selection of functional phenotypes, including differentiation of sub-populations based on growth and expression characteristics. This integrated acoustofluidic platform provides a versatile and scalable approach for multi-parameter droplet analysis and sorting, with applications in microbiology, diagnostics, and directed evolution, where simultaneous measurement of the cellular state and function is critical.