Construction Of Multivalent Aptamers And Its Application In Tumor Cell Detection

Early detection and early diagnosis of cancer and timely precise treatment can help curb the development of tumors,increase the life span of patients,and reduce mortality.Therefore,the rapid detection of tumor cells in clinical specimens plays a key role in the occurrence,treatment and prognosis of malignant tumors.However,the complex environment of clinical samples makes it difficult for many methods to detect rare tumor cells in samples,which greatly limits the sensitivity and specificity in early diagnosis.Nucleic acid aptamer-based tumor cell detection methods have the advantages of flexible design and low cost,but when monovalent aptamers recognize targets,they still have the disadvantages of poor selectivity and low targeting efficiency.In this study,the significance of constructing multivalent aptamers is to overcome the shortcomings of monovalent aptamers and make the target recognition more sensitive and efficient.ObjectiveA multivalent aptamer probe is constructed based on rolling circle amplification to improve the specificity and sensitivity of the detection of tumor cells in clinical specimens.And building a cell capture model based on multivalent aptamers to achieve tumor cell enrichment,which can lay the foundation for accurate and rapid clinical detection of malignant tumor cells.Methods1.To construct multivalent recognition probe(Multi-AS1411-LS),the DNA scaffold with repeat sequences of poly T was formed by rolling circle amplification,and then hybridized with the fluorescence signal chain of aptamer(with Poly A bases)targeted nucleolin.And the PAGE,fluorescence measurement,and Zeta potential were performed to characterize the process of probe construction.The feasibility of Multi-AS1411-LS for cell line identification were analyzed by flow cytometry and confocal microscopy.Finally,70 cases of clinical serous cavity effusion specimens were detected with Multi-AS1411-LS probe and observed by fluorescence microscope to evaluate its specificity and sensitivity.2.Through the specific binding between streptavidin and biotin,the Multi-AS1411-LS probe was bound to the surface of the microplate and fabricated for capturing tumor cells,we evaluated its capture efficiency,and investigate the enrichment of malignant tumor cells in clinical specimens.Results1.The multivalent aptamer probe Multi-AS1411-LS constructed based on rolling circle amplification has the following characteristics:(1)PAGE electrophoresis confirmed the successful synthesis of the Multi-AS1411-LS scaffold;(2)The Multi-AS1411-LS scaffold did not aggregate after thermal activation,and after hybridization,it can produce high-intensity fluorescence when combined with Evagreen;(3)It has the activity of G-quadruplex;(4)the zeta potential charge of Multi-AS1411-LS increases significantly.The above confirmed the successful preparation of the probe of Multi-AS1411-LS.2.The Multi-AS1411-LS probe was able to recognize tumor cells in 5min at room temperature due to the multivalent effect.The mean fluorescence intensity of cells labelled by the Multi-AS1411-LS probe was around 100 times higher than that of monovalent formats.The Multi-AS1411-LS probe can distinguish benign and malignant cells in clinical specimens,with sensitivity of 87.80% and specificity of 93.10%according to the pathological results.3.The Multi-AS1411-LS cell capture model has a high capture efficiency of tumor cell,which can be captured efficiently within 30 minutes,and the capture efficiency can reach about 85%.In the investigation of the tumor cell enrichment effect of clinically positive specimens,we found that this method can increase the proportion of tumor cells in clinically positive specimens.ConclusionsWe successfully constructed the multivalent aptamer probe Multi-AS1411-LS and cell capture model,which have good sensitivity,specificity and capture effect,can efficiently enrich tumor cells in the mass fluid,and provide a new application prospect for precision medicine.