2025/11/13 by Kartik Totlani, Essa Ahsan Khan, Husnain Ahmed +2 · 1 voice
Engineering · #Innovative Microfluidic and Catalytic Techniques Innovation #Electrowetting and Microfluidic Technologies #Microfluidic and Bio-sensing Technologies
paper · doi:10.1101/2025.11.13.688266
openalex publication_date 2025/11/13 · openalex created_date 2025/11/14 · openalex updated_date 2026/07/15
Abstract Precise characterization of regulatory sequence performance is fundamental to synthetic biology and next-generation gene therapies, driving the need for scalable and quantitative screening of genetic libraries. While droplet-based microfluidics offers the ultra-high throughput required to scale these assays, it often depends on complex, custom fluorescence-activated droplet sorting platforms. To address this limitation, we introduce an integrated microfluidic workflow that enables cell-free transcription in water-in-oil-in-water double emulsion picoreactors compatible with commercial flow cytometers. The core innovation is an integrated device that combines emulsion reinjection, electric-field-mediated step-injection of in vitro transcription (IVT) reagents, and downstream double emulsification, thereby reducing manual handling and preserving droplet integrity across multistep workflows. We validate the system by coupling isothermal rolling circle amplification (RCA) of DNA templates with on-chip IVT and Mango III aptamer-based fluorescence readout, demonstrating robust detection, binning, and sorting of transcription-active droplets. This workflow provides an accessible and modular platform for quantitative, high-throughput functional screening of regulatory sequences without the need for specialized optical sorting instrumentation.