Liu Yansheng, Liu Haoran, Wang Guofu, Jia Xiaobo, Zhou Jin, Dong Xinwei, Li Hongqi, Chang Haixin, Li Zhaoxu, Jin Zhicheng, Li Kang
School of Electronic Engineering, Guangxi University of Science and Technology, No.2, Wenchang Road, Liuzhou City, 545006, Guangxi, China.
Quantum-Nano Matter and Device Lab, State Key Laboratory of Material Processing and Die and Mould Technology, School of Materials Science and Engineering, Huazhong University of Science and Technology, Wuhan City, 430074, Hubei, China.
Mikrochim Acta. 2025 Jul 11;192(8):497. doi: 10.1007/s00604-025-07365-w.
This study presents a convenient and efficient dual-check strategy for detecting Monkeypox DNA utilizing the SERS (Surface-enhanced Raman Spectroscopy)-CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system. The Monkeypox plasmid DNA (mpDNA) is recognized by the CRISPR RNA (crRNA)-Cas12a protein complex, where crRNA encompasses a targeting complementary sequence. Upon recognition, the trans-cleavage activity of Cas12a is activated and trans-cleaves the probe DNA (Cy3-ssDNA) which is modified on Au nanoparticles (AuNPs). As the ssDNA strand is cleaved, Cy3 molecules are released in the solution, while the amount of Cy3 modified on the AuNPs decreases. The free Cy3 molecules are collected from the supernatant, and their SERS intensities are measured using the silver nanopillar (AgNRs) substrate. The mpDNA with varying concentration from 5 nM to 0.5 fM can be quantitatively determined based on the SERS signals of free Cy3 and the collected nanotag. This strategy allows the detection of mpDNA with a concentration of 50 fM within 60 min. Moreover, the strategy can successfully detect single-base mutated mpDNA. Owing to the non-specific trans-cleavage activity of the protein, this strategy can be adapted to various nucleic acid detection scenarios by designing complementary RNA and DNA sequences.
本研究提出了一种利用表面增强拉曼光谱（SERS）-成簇规律间隔短回文重复序列（CRISPR）系统检测猴痘病毒DNA的便捷高效的双重检测策略。猴痘病毒质粒DNA（mpDNA）由CRISPR RNA（crRNA）-Cas12a蛋白复合物识别，其中crRNA包含靶向互补序列。识别后，Cas12a的反式切割活性被激活，并反式切割在金纳米颗粒（AuNPs）上修饰的探针DNA（Cy3-ssDNA）。随着单链DNA（ssDNA）链被切割，Cy3分子释放到溶液中，而AuNPs上修饰的Cy3数量减少。从上清液中收集游离的Cy3分子，并使用银纳米柱（AgNRs）底物测量其SERS强度。基于游离Cy3的SERS信号和收集到的纳米标签，可以对浓度从5 nM到0.5 fM的mpDNA进行定量测定。该策略能够在60分钟内检测出浓度为50 fM的mpDNA。此外，该策略能够成功检测单碱基突变的mpDNA。由于该蛋白的非特异性反式切割活性，通过设计互补的RNA和DNA序列，该策略可适用于各种核酸检测场景。
