CRISPR-Cas12a-mediated dual-enzyme cascade amplification for sensitive colorimetric detection of HPV-16 target and ATP.

The establishment of sensitive and facile colorimetric platform based on the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is of great significance for in vitro diagnosis. Herein, we develop a dual-enzyme cascade amplification strategy based on CRISPR-Cas12a and glucose oxidase (GOx) for instrument-free and sensitive detection of target analytes. HPV-16 DNA as the model nucleic acid target directly initiated CRISPR-Cas12a-based signal transduction, resulting in the enzymatic cleavage of ssDNA linker and the release of GOx from magnetic nanoparticles 1 (MNPs1). Following simple magnetic separation, the supernatant containing GOx was taken out and used to catalyze the substrate, resulting in a visually detectable color change. The detection limit (LOD) of HPV-16 DNA was as low as 1 pM, and the entire process could be completed within 70 min without the need for expensive equipment. Notably, the dual-enzyme cascade amplification strategy was successfully applied to the detection of non-nucleic acid targets, such as ATP, via a simple signal transduction process. The visual LOD for ATP detection reaches 2.5 μM. The approach provides a robust, sensitive and reliable point-of-care biosensing platform for the detection of target analytes.