Cheng Liangfen, Yang Fuhan, Tang Longfei, Qian Lelin, Chen Xu, Guan Feng, Zhang Juan, Li Genxi
Center for Molecular Recognition and Biosensing, School of Life Sciences, Shanghai University, Shanghai 200444, China.
Department of Urology, Shanghai Tenth People's Hospital, School of Medicine, Tongji University, Shanghai 200092, China.
Research (Wash D C). 2022 Apr 6;2022:9826484. doi: 10.34133/2022/9826484. eCollection 2022.
Evaluating tumor development is of great importance for clinic treatment and therapy. It has been known that the amounts of sialic acids on tumor cell membrane surface are closely associated with the degree of cancerization of the cell. So, in this work, cellular interface supported CRISPR/Cas trans-cleavage has been explored for electrochemical simultaneous detection of two types of sialic acids, i.e., N-glycolylneuraminic acid (Neu5Gc) and N-acetylneuraminic acid (Neu5Ac). Specifically, PbS quantum dot-labeled DNA modified by Neu5Gc antibody is prepared to specifically recognize Neu5Gc on the cell surface, followed by the binding of Neu5Ac through our fabricated CdS quantum dot-labeled DNA modified by agglutinin. Subsequently, the activated Cas12a indiscriminately cleaves DNA, resulting in the release of PbS and CdS quantum dots, both of which can be simultaneously detected by anodic stripping voltammetry. Consequently, Neu5Gc and Neu5Ac on cell surface can be quantitatively analyzed with the lowest detection limits of 1.12 cells/mL and 1.25 cells/mL, respectively. Therefore, a ratiometric electrochemical method can be constructed for kinetic study of the expression and hydrolysis of Neu5Gc and Neu5Ac on cell surface, which can be further used as a tool to identify bladder cancer cells at different development stages. Our method to evaluate tumor development is simple and easy to be operated, so it can be potentially applied for the detection of tumor occurrence and development in the future.
评估肿瘤发展对于临床治疗具有重要意义。已知肿瘤细胞膜表面唾液酸的含量与细胞的癌变程度密切相关。因此，在本研究中，探索了细胞界面支持的CRISPR/Cas反式切割用于电化学同时检测两种唾液酸，即N-羟乙酰神经氨酸（Neu5Gc）和N-乙酰神经氨酸（Neu5Ac）。具体而言，制备了用Neu5Gc抗体修饰的PbS量子点标记DNA，以特异性识别细胞表面的Neu5Gc，随后通过我们制备的用凝集素修饰的CdS量子点标记DNA结合Neu5Ac。随后，活化的Cas12a无差别地切割DNA，导致PbS和CdS量子点释放，两者均可通过阳极溶出伏安法同时检测。因此，细胞表面的Neu5Gc和Neu5Ac可以分别以1.12个细胞/mL和1.25个细胞/mL的最低检测限进行定量分析。因此，可以构建一种比率电化学方法用于细胞表面Neu5Gc和Neu5Ac表达和水解的动力学研究，其可进一步用作识别不同发育阶段膀胱癌细胞的工具。我们评估肿瘤发展的方法简单且易于操作，因此未来有可能应用于肿瘤发生和发展的检测。
