循环肿瘤细胞（CTC）已成为乳腺癌诊断有价值的生物标志物，而叶酸受体是一种细胞表面受体糖蛋白，在乳腺癌中过度表达。在这项工作中，我们设计并制造了一种基于轻度还原辅助CRISPR/Cas系统的电化学生物传感器，用于灵敏检测叶酸受体阳性CTC。具体而言，由于叶酸与细胞表面叶酸受体之间的强亲和力，首先制备叶酸功能化磁珠来捕获CTC。然后，对细胞膜进行轻度还原处理，暴露出大量游离的巯基，可以与马来酰亚胺-DNA偶联，引入信号放大的CRISPR/Cas12a系统。 CRISPR/Cas12a的反式切割活性被激活后，电活性分子亚甲蓝修饰的长链DNA可以随机切割成短DNA片段，然后通过葫芦的主客体识别将其捕获在石墨电极上[7 ]uril，产生与 CTC 数量相对应的高度放大的电化学信号。该电化学生物传感器不仅展现了2个细胞/mL的低检测限的灵敏度，而且凸显了其在复杂环境中优异的选择性和稳定性。因此，我们的生物传感器可以为 CTC 分析提供替代工具。版权所有 © 2024 Elsevier B.V. 保留所有权利。

Circulating tumor cell (CTC) has been a valuable biomarker for the diagnosis of breast cancer, while folate receptor is a kind of cell surface receptor glycoprotein which is overexpressed in breast cancer. In this work, we have designed and fabricated an electrochemical biosensor for sensitive detection of folate receptor-positive CTCs based on mild reduction assisted CRISPR/Cas system. Specifically, folate functionalized magnetic beads are firstly prepared to capture CTCs owing to the strong affinity between folate and the folate receptors on the surface of cells. Then, the cell membranes are treated by mild reduction so as to expose a large number of free sulfhydryl groups, which can be coupled with maleimide-DNA to introduce the signal amplified CRISPR/Cas12a system. After the trans-cleavage activity of CRISPR/Cas12a is activated, the long chain DNA modified with electroactive molecules methylene blue can be randomly cleaved into short DNA fragments, which are then captured on the graphite electrode through the host-guest recognition with cucurbit [7]uril, generating highly amplified electrochemical signal corresponding to the number of CTCs. The electrochemical biosensor not only demonstrates the sensitivity with a low detection limit of 2 cells/mL, but also highlights its excellent selectivity and stability in complex environment. Therefore, our biosensor may provide an alternative tool for the analysis of CTCs.Copyright © 2024 Elsevier B.V. All rights reserved.