The detection of epidermal growth factor receptor (EGFR) mutation L858R in circulating tumor DNA (ctDNA) is beneficial for the clinical diagnosis and personalized therapy of non-small cell lung cancer (NSCLC). Herein, for the first time, the combination of the primer exchange reaction (PER) and clustered regularly interspaced short palindromic repeats (CRISPR) and its associated nucleases (Cas) 14a was used in electrochemical biosensor construction for the detection of ctDNA EGFR L858R. EGFR L858R, as the target, induced the isothermal amplification of the PER reaction, and then the CRISPR/Cas14a system was activated; subsequently, the substrate ssDNA-MB was cleaved and the electron on the surface of the gold electrode transferred, resulting in the fluctuation of the electrochemical redox signal on the electrode surface, whereas the electrochemical signal will be stable when EGFR L858R is absent. Therefore, the concentration of EGFR L858R can be quantified by electrochemical signal analysis. The low detection limit is 0.34 fM and the dynamic detection range is from 1 fM to 1 μM in this work. The PER-CRISPR/Cas14a electrochemical biosensor greatly improved the analytical sensitivity. In addition, this platform also exhibited excellent specificity, reproducibility, stability and good recovery. This study provides an efficient and novel strategy for the detection of ctDNA EGFR L858R, which has great potential for application in the diagnosis and treatment of NSCLC.
Pian Wu,Xiaosheng Ye,Danqi Wang,Fangjie Gong,Xiaoqian Wei,Shan Xiang,Jingwen Zhang,Tianhan Kai,Ping Ding
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.
Xiaoya Ren,Xu Cui,Ying Zeng,Yimei Guo,Junhua Chen,Jiafeng Pan
In this research article, an intelligent clustered regularly interspaced short palindromic repeats (CRISPR)/Cas14a-based logic system was developed for microplastic monitoring on the basis of catalytic hairpin assembly (CHA) and DNA walkers on magnetic beads. Polyvinyl chloride (PVC) and polystyrene (PS) can activate the CHA-DNA walker sensing system to generate numerous single-stranded DNA fragments that can be recognized by Cas14a. After the cleavage reaction targeting the fluorophore-quencher reporter, a high Cy5 signal can be observed. The fluorescent biosensor is ultrasensitive, with a limit of detection of 3.4 ng/ml for PVC and 4.1 ng/ml for PS. Using PVC and PS as the two inputs, an OR logic gate was further developed. The input combinations of (10), (01), and (11) can give an output of 1. The sensing system is robust and can work in real water samples with good accuracy. Our proposed biosensor can provide an ultrasensitive and intelligent tool for microplastic monitoring.
危险品化学品经营许可证（带存储）