Near-infrared light activated and hybridization chain reaction cascaded CRISPR/Cas12a system under the enhancement of Mn²⁺ for intracellular biosensing

While CRISPR/Cas12a system is a potent in vitro fluorescence biosensing toolbox, several difficulties including collective biological delivery, potential pre-activation and inadequate sensitivity make it challenging to further perform intracellular application. Herein, we raise a series of solving strategies. First, MnO2 nanoflower is used to conjointly carry the biomolecular modules comprising CRISPR/Cas12a system through a simple physisorption to actualize an efficient endocytosis, after which the MnO2 matrix will be reduced by the widespread intracellular biothiols to not only release the surface attached biomolecules but also produce abundant Mn2+ to enhance transcleavage activity. Besides, a light activation conception realized by inserting the biosensing frame with a facile photocleave group is applied to achieve a controllable spatiotemporal behavior, for which the target recognition process will be manually initiated by illuminating with an ultraviolet upconversion luminescence converted from 808 nm near-infrared photons. By making usage of hybridization chain reaction to construct a final cascaded CRISPR/Cas12a system, the additional signal amplification design renders this newly-raised CRISPR/Cas12a system-mediated fluorescent biosensor has an ultra-sensitive detection ability for microRNA-21. Moreover, this special CRISPR/Cas12a system is also equipped with a high-efficiency imaging performance in live cells and even bodies, impelling the development of CRISPR system based biosensing techniques.