A CRISPR-Cas12a-mediated dual-mode luminescence and colorimetric nucleic acid biosensing platform based on upconversion nanozyme

In this study, a CRISPR-Cas12a-mediated dual-mode upconversion luminescence/colorimetric nucleic acid biosensing platform is developed based on UCNP@SiO₂/CeO₂ (UNSC) nanozyme. Here, UNSC is conjugated with single-stranded DNA (ssDNA) probes used as both peroxidase-like nanozyme and upconversion luminescence donors. When no target nucleic acid is present, ssDNA-conjugated UNSC attaches on magnetic graphene oxide (MGO) via pi-pi stacking force, resulting in upconversion luminescence quenching (OFF) and no color change after magnetic removal of nanozymes attached on the MGO. In the presence of target nucleic acid, Cas12a is specifically activated by targeted nucleic acid and indiscriminately cleaves the ssDNA probes on UNSCs. UNSCs then detach from the MGO surface due to the weakening of binding force, leading to upconversion luminescence recovery (ON) and colorimetric change due to the existence of free nanozyme in the 3,3’,5,5’-tetramethyl-benzidine assay. As a proof-of-concept, this biosensing platform shows a limit of detection of around 320 fM in the upconversion luminescence mode and ∼28.4 pM in the colorimetric mode for nucleic acid detection, respectively. This UNSC nanozyme-based CRISPR-Cas12a dual-mode biosensing system also demonstrates high selectivity, good repeatability, and facile operation, which allows easy adaption to other nucleic acid-based detection only by redesigning the sequence of CRISPR RNA.