Zhang Kai, Fan Zhenqiang, Yao Bo, Ding Yuedi, Zhao Jing, Xie Minhao, Pan Jianbin
NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China.
NHC Key Laboratory of Nuclear Medicine, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, Jiangsu, 214063, China.
Biosens Bioelectron. 2021 Apr 15;178:113019. doi: 10.1016/j.bios.2021.113019. Epub 2021 Jan 26.
Sialic acid-binding immunoglobulin (Ig)-like lectins (Siglecs) is a type I transmembrane receptor on the cell surface. Siglec-5, as one of the Siglecs family, play an important role as an inhibitory receptor for leukocytes in the human body. The development of novel siglec-5 assays can help to study the pathogenesis of related diseases as well as to develop novel therapeutic drugs. We use catalytic hairpin assembly (CHA) amplification strategy combined with CRISPR-Cas12a's side-cutting feature to build a 2D ultra-thin TiCT (MXene) based electrochemiluminescence (ECL) biosensor for the detection of Siglec-5. By using this ECL biosensor, the cleavage of CRISPR-Cas12a is reasonably combined with CHA-mediated isothermal amplification, thereby realizing the sensitive amplification assay Siglec-5 with 20.22 fM sensitivity. By introducing pairs of sites that are not in the same double-stranded DNA into the DNA duplex, the hybridization sequence of CRISPR-Cas12a complements the targeting mechanism to enhance indirect Siglec-5 amplification assay. Also, the double-strand DNA (dsDNA) design based on CRISPR-Cas12a amplification allows the same CRISPR RNA (crRNA, also known as guide RNA (gRNA)) to detect the output of DNA duplexes from different intermediate DNAs, which provides a common way for biomarker detection based on the conversion of protein analytes to intermediate DNA strategy. This work extends the application scope of CRISPR-Cas12a to the construction of ECL biosensors, evaluates the role of lectins, which can be used for the biochemical research and clinical diagnosis of protein markers. This is the first investigative work exploring the Trans-Cleavage activity of CRISPR-Cas12a for Mxene-based ECL biosensor establishment to the best of our knowledge.
唾液酸结合免疫球蛋白（Ig）样凝集素（Siglecs）是细胞表面的I型跨膜受体。Siglec-5作为Siglecs家族的一员，在人体中作为白细胞的抑制性受体发挥重要作用。新型Siglec-5检测方法的开发有助于研究相关疾病的发病机制以及开发新型治疗药物。我们采用催化发夹组装（CHA）扩增策略并结合CRISPR-Cas12a的侧切特性，构建了一种基于二维超薄TiCT（MXene）的电化学发光（ECL）生物传感器用于检测Siglec-5。通过使用这种ECL生物传感器，CRISPR-Cas12a的切割与CHA介导的等温扩增合理结合，从而实现了灵敏度为20.22 fM的Siglec-5灵敏扩增检测。通过将不在同一条双链DNA中的位点对引入DNA双链体，CRISPR-Cas12a的杂交序列与靶向机制互补，以增强间接Siglec-5扩增检测。此外，基于CRISPR-Cas12a扩增的双链DNA（dsDNA）设计允许相同的CRISPR RNA（crRNA，也称为引导RNA（gRNA））检测来自不同中间DNA的DNA双链体的输出，这为基于蛋白质分析物转化为中间DNA策略的生物标志物检测提供了一种通用方法。这项工作将CRISPR-Cas12a的应用范围扩展到ECL生物传感器的构建，评估了凝集素的作用，可用于蛋白质标志物的生化研究和临床诊断。据我们所知，这是第一项探索CRISPR-Cas12a的反式切割活性用于基于MXene的ECL生物传感器构建的研究工作。
