Rapid detection of tumor cells in serous effusion by multivalent aptamers based on rolling circle amplification

Abstract

Objective To establish a simple and rapid method for detecting tumor cells based on multivalent aptamers. Methods Rolling circle amplification was used to form a long single stranded DNA scaffold with repeat sequences of poly T, then the obtained product was hybridized with the fluorescence signal chain of aptamer AS1411 (with poly A bases) which was nucleolin targeting. Then, a Multi-AS1411-LS recognition probe was constructed and characterized. The feasibility of probe Multi-AS1411-LS for cell line identification was analyzed by flow cytometry and confocal microscopy, and the recognition conditions were further optimized. Finally, the malignant tumor cells in clinically collected serous effusions were detected by our constructed probe Multi-AS1411-LS, and the specificity and sensitivity of our probe were evaluated. Results Our constructed probe Multi-AS1411-LS could recognize tumor cells in 5 min at room temperature, with the mean fluorescence intensity of cells higher by about 100 times than that of monovalent formats, and the minimum detection limit of 3 cells in 500 μL samples. The probe distinguished benign and malignant cells in 70 clinical specimens, with a sensitivity of 87.80% and a specificity of 93.10%. Conclusion This detection method is rapid and simple to operate, and has good specificity and sensitivity in the detection of malignant tumor cells in clinical practice.

Keywords

• multivalent aptamers
• rolling circle amplification
• as1411
• detection of tumor cells