A novel CRISPR/Cas14a system integrated with 2D porphyrin metal-organic framework for microcystin-LR determination through a homogeneous competitive reaction.

Selective and sensitive detection of microcystin-LR (MC-LR) is of vital importance because of its high toxicity and broad distribution. Herein, a novel and versatile fluorescence sensor (Cas14-pMOFs fluorescence sensor) was developed by combining the CRISPR/Cas14a system with a 2D porphyrin metal-organic framework nanosheets (2D-pMOFs) for MC-LR determination. The designed CRISPR/Cas14a system was activated by the unbound complementary DNA (cDNA), which was positively correlated with MC-LR concentration. Furthermore, the activated Cas14a protein was utilized to indiscriminately cleave the FAM-labeled single-stranded DNA (ssDNA-FAM), which was pre-absorbed on Cu-TCPP(Fe) nanosheets. Because of the desorption of the cleaved ssDNA-FAM, the pre-quenched fluorescence signal was recovered. Owing to the excellent performance in quantifying cDNA using this Cas14-pMOFs fluorescence sensor with a limit of detection (LOD) of 0.12 nM, this Cas14-pMOFs fluorescence sensor was able to detect MC-LR in a range from 50 pg/mL to 1 μg/mL with the LOD of 19 pg/mL. This work not only provided a new insight for the exploration of fluorescence sensors based on 2D-pMOFs coupled with CRISPR/Cas14a, but also, demonstrated its universality in both nucleic acid and non-nucleic acid targets determination.
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