A droplet digital LAMP-based lab-on-a-disc system for multiplex allele-specific detection of tumor-derived DNA...

The absence of accurate, rapid, fully automated, and cost-effective detection methods has hindered the clinical translation of tumor-derived DNA mutations as cancer biomarkers. Here, we develop a sample-to-answer platform, termed multiplexed droplet digital loop-mediated isothermal amplification-based lab-on-a-disc (mddLAMP-LoD), for rapid and allele-specific detection of cancer-associated mutations. The system seamlessly combines automated microfluidics, an improved allele-specific LAMP assay with mismatch-engineered primer design for single-nucleotide discrimination, and artificial intelligence-assisted droplet imaging to enable precise mutation quantification. Full automation is realized through a combination of microfluidic passive valves, a gelatin-based thermosensitive active valve, a static magnetic field-driven on-chip nucleic acid extraction module, and polysaccharide additives that preserve water-in-oil droplet stability for over 1.5 h under thermal amplification. The mddLAMP-LoD platform demonstrates superior performance in detecting multiplex PIK3CA point mutations, achieving detection limits at the attomolar level or as low as100 cells, with a linear range of 0-6362 copies/μL. When coupled with deep learning, it exhibits high sensitivity, specificity and reproducibility, showing strong concordance with commercial kits in the analyses of breast cancer tissue and plasma samples. Using a 0.1 % mutation threshold, PIK3CA mutation frequency in adjacent tissue effectively predicts breast cancer prognosis, while serum PIK3CA profiles correlate with cancer staging and track disease progression. We validated that the mddLAMP-LoD system provides rapid, ultrasensitive mutation profiling suited for intraoperative molecular diagnosis and point-of-care liquid biopsy, representing a significant advance toward accessible precision oncology.