Chip-based digital PCR with large-field one-shot imaging for high-sensitivity nucleic acid quantification

To enhance the absolute detection limit of digital polymerase chain reaction (dPCR) by accommodating more templates in a single loading, we developed a chip-based dPCR (cdPCR) device with dimensions of 26 mm × 16 mm, incorporating 21,384 microreaction chambers. Traditional fluorescence imaging systems cannot capture the entire chip in a one-shot, necessitating image stitching, which slows detection and introduces the risk of stitching errors. To overcome this limitation, we designed a large-field fluorescence imaging system with an effective imaging area of 28 mm × 20 mm, enabling one-shot acquisition of the entire cdPCR fluorescence image in 1 s. A Köhler illumination system was employed to achieve 90 % illumination uniformity, enhancing image quality. Additionally, image processing modules, including rotation and brightness correction, were employed to ensure precise detection. Validation experiments using lung cancer DNA demonstrated a strong correlation (R² > 0.9997) between measured and theoretical values. This system provides an automated, rapid, and accurate platform for cdPCR fluorescence image processing, eliminating stitching errors and advancing absolute quantification in dPCR analysis.