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.
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