Bifunctional Aptamer-Mediated Catalytic Hairpin Assembly For The Detection Of Cancer Cells

Background and objectiveCancer cells spread into body fluids in the process of invasion and metastasis of malignant tumors.Research shows that,cancer cells play an important role in metastasis.Measuring the specific cellular levels in body fluids provides clinicians with information to find tumor micrometastasis early,monitor the therapeutic effect,drug resistance,prognosis and individual therapy.Compared with the traditional tumor diagnosis,cancer cells detection is noninvasive,more convenient and easy to real-time monitoring.Thus,to construct a rapid,accurate,simple method has extensive application prospect.The traditional cancer cells detection assays,such as flow cytometry,immunohistochemistry,and the polymerase chain reaction,can solve some related matters.Unfortunately,they are often associated with a high cost and require advanced equipment,prolonged periods,and tedious experimental protocols.Cellsearch(?),considered as gold standard of CTCs detection in recent years,provides the new cancer cell detection platform,but still has the disadvantage of limited biomarkers,false negative and expensive equipment.Meanwhile,more and more new technology and novel nanomaterial have been applied for building cancer cell detection assay.These methods determine the cancer cells through different biomarkers,but isn't sensitive or specific enough.Aptamer has the unique advantages for high specificity,ease of synthesis and conversation,etc.And DNA nanostructure technology can highly amplify the signals through simple reaction between nucleic acids.Herein,we made good use of aptamer with high specificity for cancer cells and the character of nucleic acid,and catalytic hairpin assembly for exponential amplification to develop novel cancer cells detection assay.To take full advantage of aptamer technology,the aptamer was transformed into a bifunctional aptamer by extending the specific sequence.The bifunctional aptamer contained a cell-specific domain and an initiator domain designed for initiating the CHA reaction.The bifunctional aptamer was not only able to bind to the target cell,but also triggered the cascading amplification reaction.The strategy developed herein achieved homogenous detection of cancer cells while avoiding complicated sample separation and multi-step washing.The assay showed excellent analytical performance for cancer cell detection and reveals great potential to adopt DNA nanotechnology for cancer cell detection.Methods1.Construction of catalytic hairpin assembly reaction system1.1 All kinds of nucleotide sequences and hairpin probes were designed and synthesized,and the physical parameters of the hairpin structure were calculated and assessed.1.2 The analyses were performed through nondenaturating polyacrylamide gel electrophoresis(PAGE)analysis to analyze the reaction products of CHA reaction,and thus verify the response and stability of the reaction.1.3 The catalytic hairpin assembly were further verified with the fluorophore labeled hairpins to ensure the feasibility for further cancer cells detection assay.2.Analytical performance of bifunctional aptamer-mediated catalytic hairpin assembly for cancer cells detection and real sample analysis2.1 With different concentrations of cancer cells,we have monitored the fluorescence intensity and collect the signal at the end point to verify the feasibility of the proposed assay for cancer cells.2.2 To achieve optimal analytical performance,the incubating time and the reaction temperature were investigated by comparing the fluorescence intensity with each other.2.3 To evaluate the sensitivity of this proposed biosensor,assays were applied to detect cancer cells at various concentrations under the optimal conditions.After that,we calculate the detection limit and the linear range of the proposed method.2.4 To investigate the detection specificity of our strategy,the human bronchial epithelial cell line 16 HBE and leukocytes were selected as control cells.We have collected the signal for the cell suspension of the three cell lines,the mixture of control cells and A549 cells respectively.2.5 To further evaluate the performance of the strategy,four types of human clinical samples including serum,urine,CSF and hydrothrax were processed to detect different numbers of cancer cells respectively.Results1.With theoretical analysis,electrophoretic characterization and fluorescence signal analysis,we have obtained the same results that the substrates of catalytic hairpin assembly have the stable secondary structure,and the reaction can be specifically triggered and efficiently amplify the signals quickly.Thus,the designed hairpins and catalytic hairpin assembly can be further applied to construct the novel assay for cancer cells detection.2.The proposed strategy can specifically recognize the target cancer cells and produce the corresponding fluorescence signals.Under the optimal experimental conditions that the incubation time is 45 minutes and the reaction temperature is 37?,the increase in fluorescence intensity of A549 cells was proportional to their concentrations within the range of 10?103 cells/mL,and the linear regression equation was F=74.47 + 7.399log10C with a correlation coefficient of 0.9791.Also,the fluorescence signal clearly appeared for A549 cells(105 cells/mL),whereas negligible changes were observed with the addition of equivalent concentrations of control cells in comparison with the blank.The mixed cell suspension showed similar fluorescence intensities as that generated by A549 cells.And the fluorescence intensity of four kinds of clinical samples including serum,urine,CSF and hydrothrax with cancer cells was similar to that of PBS with the same number of A549 cells with various amounts of cancer cells.ConclusionA simple,rapid,and homogeneous CHA-based fluorescence aptasensor was designed for the detection of cancer cells.The developed method takes full advantage of aptamer and CHA reactions and was characterized by simplicity,rapid responses,and improved efficiency in single phase solutions.The bifunctional aptamer not only showed satisfactory selectivity against interference cells,but also mediated CHA signal amplification to obtain a high detection sensitivity with a detection limit of 10 cells/mL.This strategy was successfully applied to the detection of cancer cells in various clinical samples.Furthermore,the method was general and could be expanded for the analysis of other cells with available affinity ligands.Therefore,the assay can be applied for the detection of other biomarkers with the corresponding aptamers,such as exosomes,bacteria,and metal ions among others.The aptasensor is potentially useful for cancer cell analysis in different clinical samples for early prevention and treatment.The proposed biosensor also offers a new platform for the determination of circulating tumor cells during patient follow-up.The introduction of nanomaterials to the aptasensor can be the next research direction to refine the performance of the method.The present simple,enzyme-free,and low cost assay is also available for POCT.