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

Area of Science:

• Biotechnology
• Analytical Chemistry
• Medical Diagnostics

Background:

• Digital polymerase chain reaction (dPCR) offers high sensitivity for absolute quantification.
• Traditional dPCR imaging systems face limitations in speed and accuracy due to image stitching.

Purpose of the Study:

• To develop an enhanced chip-based dPCR (cdPCR) device with a large-field imaging system.
• To overcome limitations of traditional dPCR imaging, improving absolute detection limits and accuracy.

Main Methods:

• Designed a cdPCR device with 21,384 microreaction chambers.
• Developed a large-field fluorescence imaging system (28 mm × 20 mm) for one-shot image acquisition.
• Implemented Köhler illumination and image processing modules for enhanced image quality and precision.

Main Results:

• Achieved one-shot fluorescence image acquisition of the entire cdPCR chip in 1 second.
• Demonstrated 90% illumination uniformity for high-quality imaging.
• Validated the system with lung cancer DNA, showing excellent correlation (R² > 0.9997) between measured and theoretical values.

Conclusions:

• The developed system provides an automated, rapid, and accurate platform for cdPCR fluorescence image processing.
• Elimination of image stitching errors enhances the precision of absolute quantification.
• The system advances dPCR analysis for improved diagnostic applications.