CRISPR-Cas-Mediated Ultrasensitive Detection of Viral Nucleic Acids via Singlet Oxygen-Activated Chemiluminescence

作者：Fei Chen, Daian Chen, Ting Deng, Jishan Li, Jian‐Hui Jiang · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c03994 · 被引用次数：2 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、bioluminescence and chemiluminescence research

Herein, we report a novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-mediated chemiluminescence assay (CRISPR-Cas-CLA) for the ultrasensitive detection of viral nucleic acids of HPV18 and HPV16. The CRISPR-Cas-CLA comprises a CRISPR/Cas12a system that specifically recognizes the target nucleic acid, a signal-conducting nanoconjugate (MB-ssDNA-PSNP) formed by coupling singlet-oxygen ( 1 O 2 )-generating photosensitive nanoparticles (PSNPs) to magnetic beads (MBs) via a single-stranded DNA (ssDNA) linker, and 1 O 2 -activated chemiluminescence nanoparticles (CLNPs). In the presence of the target nucleic acid, the ssDNA linker of the nanoconjugate is cleaved by the target-activated CRISPR/Cas12a system, and the PSNPs are dissociated from the MBs. The PSNP-containing supernatant obtained by magnetic separation is added to the CLNP-coated detection plate. Upon light irradiation of the CLNP–PSNP mixture in the well, strong chemiluminescence is generated with the subsequent addition of hydrogen peroxide, enabling the detection of the target nucleic acids. The proposed CRISPR-Cas-CLA system offers ultrahigh sensitivity (∼1.04 aM), simple operation, and low cost, providing a new direction for the development of PCR-free detection strategies for ultralow abundance nucleic acids.