All-In-One OsciDrop Digital PCR System for Automated and Highly Multiplexed Molecular Diagnostics

Digital PCR (dPCR) holds immense potential for precisely detecting nucleic acid markers essential for personalized medicine. However, its broader application is hindered by high consumable costs, complex procedures, and restricted multiplexing capabilities. To address these challenges, an all-in-one dPCR system is introduced that eliminates the need for microfabricated chips, offering fully automated operations and enhanced multiplexing capabilities. Using this innovative oscillation-induced droplet generation technique, OsciDrop, this system supports a comprehensive dPCR workflow, including precise liquid handling, pipette-based droplet printing, in situ thermocycling, multicolor fluorescence imaging, and machine learning-driven analysis. The system's reliability is demonstrated by quantifying reference materials and evaluating HER2 copy number variation in breast cancer. Its multiplexing capability is showcased with a quadruplex dPCR assay that detects key EGFR mutations, including 19Del, L858R, and T790M in lung cancer. Moreover, the digital stepwise melting analysis (dSMA) technique is introduced, enabling high-multiplex profiling of seven major EGFR variants spanning 35 subtypes. This innovative dPCR system presents a cost-effective and versatile alternative, overcoming existing limitations and paving the way for transformative advances in precision diagnostics. This work unveils an innovative, chip-free digital PCR system, revolutionizing precision diagnostics by overcoming cost and complexity barriers. The OsciDrop technique, paired with advanced instrumentation and machine learning, enables highly sensitive, efficient nucleic acid quantification. This system streamlines dPCR, introduces digital stepwise melting analysis, and significantly boosts multiplexing and ease of use, paving the way for scalable, cost-effective diagnostics. image