Combination of Immunomagnetic Separation with Aptamer-Mediated Double Rolling Circle Amplification for Highly Sensitive Circulating Tumor Cell Detection

Abstract

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.