An automated modular microfluidic platform for end-to-end mRNA synthesis and purification.

Abstract

Manual, batchwise downstream operations remain a major bottleneck in translating microfluidic mRNA synthesis into practical production workflows. Building on an oscillatory microfluidic IVT module (Os(IVT)) previously established for intensified transcription, here we introduce an end-to-end, automated, modular lab-on-a-chip platform that integrates transcription with inline nucleic-acid processing and purification. The workflow couples Os(IVT) to a newly developed microfluidic digestion/clarification (μFD) unit, where DNase I immobilized on a biocatalytic membrane performs rapid template removal while a micro-tangential flow filtration (μ-TFF) step enables simultaneous buffer exchange/clarification without sacrificing full-length mRNA recovery. The processed stream is then routed to an automated multimodal chromatography (MMC) module governed by real-time pH and conductivity feedback to reproducibly gate binding, washing, and elution. Using firefly luciferase mRNA as a model, the fully integrated Os(IVT)-μFD-MMC workflow completed synthesis-to-purification in 2 h, delivered 1275 ± 1.7 μg mRNA per run <i>vs.</i> 641 ± 0.6 μg for the fully batch benchmark (∼2.0×), with ∼93.3% MMC recovery and an 83.3% reduction in dsRNA impurities relative to the batch workflow. This work advances oscillatory microfluidic IVT from a standalone intensified reactor to a closed-loop, end-to-end microfluidic manufacturing platform, enabling rapid, reproducible production of high-quality mRNA with substantially reduced manual handling.