Breast cancer is the most frequently diagnosed cancer among women, and the circulating tumor cell (CTC)-meditated distant metastasis is the leading cause of death. Thus, the detection of CTCs is of great importance for the early diagnosis of breast cancer and the prevention of metastasis. In this study, using human breast carcinoma BT474 cells as the model CTCs, a powerful assay platform is demonstrated by fluorescence spectrometry for the highly sensitive CTC detection by combining the dual-recognizing elements receptor-binding antibody and aptamer-mediated separation with double rolling circle amplification reactions (d-RCA, including RCA1 and RCA2). The aptamer-inserted RCA1 product (RCA1-p) exhibits the considerably improved affinity towards target cells originating from the multivalent binding effect. The immunomagnetic separation removes nontarget cells coexisting in complex biological milieu, while the centrifugal separation of cells/DNAs mixture eliminates the excess probes, thereby circumventing the unwanted interferences. The fluorescence spectrometric results show that a 34-fold enhanced fluorescence signal is achieved upon BT474 cells, and the target cells can be quantitatively detected down to 9 cells/200 μL with the linear range of five orders of magnitude, indicating a significantly enhanced detection performance. Even if BT474 cells are spiked in the fresh whole blood, no obvious fluctuation in the fluorescence signal is detected, demonstrating that the newly developed d-RCA assay system is suitable for screening CTCs in complex environments and is expected to be a promising tool for estimating distant metastasis and predicting the recurrence of tumors.

中文翻译：

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免疫磁分离与适体介导的双滚环扩增相结合，用于高度敏感的循环肿瘤细胞检测

乳腺癌是女性中最常被诊断出的癌症，循环肿瘤细胞（CTC）思考的远处转移是主要的死亡原因。因此，CTC的检测对于乳腺癌的早期诊断和转移的预防非常重要。在这项研究中，使用人乳腺癌BT474细胞作为模型CTC，通过结合双重识别元素受体结合抗体和适体介导的双滚环分离，通过荧光光谱法证明了强大的测定平台可用于高灵敏度CTC检测扩增反应（d-RCA，包括RCA1和RCA2）。插入适体的RCA1产物（RCA1-p）表现出对源自多价结合作用的靶细胞的显着改善的亲和力。免疫磁分离去除了复杂生物学环境中共存的非靶细胞，而细胞/ DNA混合物的离心分离则消除了多余的探针，从而避免了不必要的干扰。荧光光谱结果表明，BT474细胞可实现34倍增强的荧光信号，并且在5个数量级的线性范围内可定量检测低至9个细胞/ 200μL的靶细胞，表明检测性能显着增强。即使将BT474细胞掺入新鲜的全血中，也不会检测到明显的荧光信号波动，