A digital microfluidics-based single-cell ddPCR platform for high-throughput gene copy number analysis applied to...

Digital polymerase chain reaction (dPCR) is a powerful tool for absolute quantification of nucleic acids, offering higher sensitivity than quantitative PCR (qPCR). However, current dPCR platforms struggle to detect gene copy number variations (CNVs) at the single-cell level due to limited droplet control and visualization. To address these challenges, we developed a digital microfluidic (DMF)-based platform that integrates droplet manipulation, sample processing, and nucleic acid amplification into a single, fully controllable workflow. The DMF chip, featuring 16,384 addressable electrodes, enables precise manipulation of nanoliter droplets containing single cells, supporting on-chip cell lysis, droplet merging, and splitting without complex workflows. Using cell lines, we validated the platform's capability for both qPCR and dPCR, achieving single-cell resolution. To demonstrate clinical relevance, we applied the platform to primary chimeric antigen receptor T (CAR-T) cells and successfully quantified vector copy numbers (VCNs) at the single-cell level, with performance comparable to commercial dPCR systems. This fully integrated, high-throughput platform minimizes sample loss and contamination while offering real-time visualization and scalability. Our DMF-based dPCR system provides a robust tool for single-cell genetic analysis, with broad potential applications in clinical diagnostics, personalized medicine, and research.