Integrated microdroplet workflow for high-throughput cell-free transcription in double emulsion picoreactors.

Precise characterization of regulatory sequence performance is fundamental to synthetic biology and next-generation gene therapies, driving the need for scalable gene expression assays. 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 stepinjection 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 biosensing, demonstrating robust detection, binning, and sorting of transcription-active droplets using a positive-control template. Rather than performing library screening, this work establishes and validates the underlying workflow, providing an accessible and modular platform that lays the foundation for functional screening of regulatory sequences without the need for specialized optical sorting instrumentation.