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

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.

Target-catalyzed hairpin structure-mediated padlock cyclization for ultrasensitive rolling circle amplification.

Because STAT3 is a potent proto-oncogene, screening STAT3 gene has potential for use in tumor diagnosis, classification of subtypes, and molecular target therapy. Thus, in this study, using STAT3 gene as the model molecule, we developed a novel amplification strategy, ultrasensitive rolling circle amplification (THP-RCA) based on target-catalyzed hairpin structure-mediated padlock cyclization, for the ultrasensitive detection of human proto-oncogenes in a homogenous solution. In this system, HP1 was designed as the cyclization template and RCA reaction primer, while HP2 was the padlock probe. The two probes can fold into a hairpin structure via the self-hybridization and thus lock the signaling process in the absence of target species. The hybridization of HP2 with HP1 in an end-to-end fashion occurs with the help of target DNA. Subsequently, HP2 is cyclized by ligase on HP1 template. Interestingly, during the hybridization and enzymatic cyclization of HP2, the target DNA only serves as the catalytic probe and is not exhausted. The cyclized HP2 enables the rolling circle amplification, generating a long tandem single-stranded (ss) DNA product that is capable of hybridizing with considerable quantity of molecular beacons (MBs). As a result, the dramatically amplified fluorescence value is achieved for the ultrasensitive detection of the STAT3 gene. As a result, target DNA is able to be quantified down to 100 fM with a high specificity towards wild-type target DNA. Moreover, the sensing system is suitable for the target detection in human serum. The novel sensing strategy shows tremendous prospect for application in tumor diagnosis and clinical therapy guidance.