Wide Dynamic Range Multiplex Digital Clustered Regularly Interspaced Short Palindromic Repeat Chip for Rapid Detection and Absolute Quantification of Nucleic Acids

Rapid detection and accurate quantification of multiplex nucleic acids are crucial for infectious diagnosis and pathogen identification, yet they still represent a significant challenge that deserves further attention and investigation. Herein, this research developed a wide dynamic range multiplex digital clustered regularly interspaced short palindromic repeat (CRISPR) (WDRM-dCRISPR) chip and established a rapid, sensitive, multiplexed nucleic acid detection (RSMND) platform that can achieve absolute quantification of multiplex nucleic acids with a limit of detection of 90 copies/mL within 30 min. The chip integrates the rapid amplification capability of RPA, the specific cleavage activity of CRISPR/Cas12a, and the portability of microfluidics, enabling precise and reliable digital absolute quantification of multiplex nucleic acids. As a proof of concept, this chip allows for multiplex absolute quantification of foodborne pathogens with a linear dynamic range of 9 × 10¹ to 1.8 × 10⁶ copies/mL (R² > 0.9999), high sensitivity, high specificity, and strong tolerance to background interference. The platform was successfully used for the analysis of foodborne pathogens in milk. Compared to qPCR, the RSMND platform exhibited superior accuracy and sensitivity in nucleic acid quantification. Overall, this study demonstrates that the platform provides a powerful tool for multiplex nucleic acid detection and holds significant potential for applications in point-of-care testing.