Xie Tian Jin, Xie Jia Li, Luo Yu Jie, Mao Kai, Huang Cheng Zhi, Li Yuan Fang, Zhen Shu Jun
Key Laboratory of Luminescence Analysis and Molecular Sensing (Southwest University), Ministry of Education, College of Chemistry and Chemical Engineering, Southwest University, 400715 Chongqing, P. R. China.
Key Laboratory of Luminescent and Real-Time Analytical Chemistry (Southwest University), Chongqing Science and Technology Bureau, College of Pharmaceutical Sciences, Southwest University, 400715 Chongqing, P. R. China.
Anal Chem. 2023 May 9;95(18):7237-7243. doi: 10.1021/acs.analchem.3c00156. Epub 2023 Apr 25.
DNA nanosheets (DNSs) have been utilized effectively as a fluorescence anisotropy (FA) amplifier for biosensing. But, their sensitivity needs to be further improved. Herein, CRISPR-Cas12a with strong -cleavage activity was utilized to enhance the FA amplification ability of DNSs for the sensitive detection of miRNA-155 (miR-155) as a proof-of-principle target. In this method, the hybrid of the recognition probe of miR-155 (T1) and a blocker sequence (T2) was immobilized on the surface of magnetic beads (MBs). In the presence of miR-155, T2 was released by a strand displacement reaction, which activated the -cleavage activity of CRISPR-Cas12a. The single-stranded DNA (ssDNA) probe modified with a carboxytetramethylrhodamine (TAMRA) fluorophore was cleaved in large quantities and could not bind to the handle chain on DNSs, inducing a low FA value. In contrast, in the absence of miR-155, T2 could not be released and the -cleavage activity of CRISPR-Cas12a could not be activated. The TAMRA-modified ssDNA probe remained intact and was complementary to the handle chain on the DNSs, and a high FA value was obtained. Thus, miR-155 was detected through the obviously decreased FA value with a low limit of detection (LOD) of 40 pM. Impressively, the sensitivity of this method was greatly improved about 322 times by CRISPR-Cas12a, confirming the amazing signal amplification ability of CRISPR-Cas12a. At the same time, the SARS-CoV-2 nucleocapsid protein was detected by the strategy successfully, indicating that this method was general. Moreover, this method has been applied in the analysis of miR-155 in human serum and the lysates of cells, which provides a new avenue for the sensitive determination of biomarkers in biochemical research and disease diagnosis.
DNA纳米片（DNSs）已被有效地用作生物传感的荧光各向异性（FA）放大器。但是，它们的灵敏度需要进一步提高。在此，具有强切割活性的CRISPR-Cas12a被用于增强DNSs的FA放大能力，以灵敏检测作为原理验证靶标的miRNA-155（miR-155）。在该方法中，miR-155的识别探针（T1）与阻断序列（T2）的杂交体被固定在磁珠（MBs）表面。在miR-155存在的情况下，T2通过链置换反应被释放，从而激活CRISPR-Cas12a的切割活性。用羧基四甲基罗丹明（TAMRA）荧光团修饰的单链DNA（ssDNA）探针被大量切割，无法与DNSs上的手柄链结合，导致FA值较低。相反，在不存在miR-155的情况下，T2无法释放，CRISPR-Cas12a的切割活性无法被激活。TAMRA修饰的ssDNA探针保持完整，并与DNSs上的手柄链互补，从而获得高FA值。因此，通过明显降低的FA值检测到miR-155，检测限低至40 pM。令人印象深刻的是，CRISPR-Cas12a使该方法的灵敏度大幅提高了约322倍，证实了CRISPR-Cas12a惊人的信号放大能力。同时，该策略成功检测到了严重急性呼吸综合征冠状病毒2（SARS-CoV-2）核衣壳蛋白，表明该方法具有通用性。此外，该方法已应用于人体血清和细胞裂解物中miR-155的分析，为生物化学研究和疾病诊断中生物标志物的灵敏测定提供了一条新途径。
