Aptamer Recognition And DNA Nanotechnology For Tumor Cells Detection Research

Malignant tumor has become one of the diseases that seriously threaten human health and life.In recent years,the detection of tumor cells and tumor markers has attracted extensive attention of researchers.Developing new strategies for imaging and detection of tumor are of great significance for improving the survival rate of patients.A series of typical approaches,such as magnetic separation,flow cytometry and fluorescence measurement,have been performed for cancer cell detection.However,current approaches often fail to take into account the sensitivity and easy operation,or some of the detection methods demand for stringent instruments.Therefore,the development of tumor cell detection methods with high sensitivity,convenient operation and good biocompatibility is of great significance for the early diagnosis of tumors.In this thesis,aptamer was selected as molecular recognition element,and two DNA-based activatable detection probes were constructed in combination with DNA nanotechnology to carry out the studies of tumor cell-surface specific glycosylation imaging and highly sensitive detection of tumor cells.The specific research content is as follows:1.Imaging of tumor cell-surface specific glycosylation based on aptamer recognition and proximity-induced DNA hybridizationThe dynamic changes of cell-surface protein glycosylation act as an important indicator that reflects cellular physiological states and disease developments.The visualization of protein-specific glycosylation is of great value to elucidate its function and mechanism.However,the ubiquity of glycosylation makes probing the glycans on a certain protein as difficult as looking for a needle in the haystack.Hence,we developed a proximity-induced DNA hybridization strategy for visualization of protein-specific glycosylation.The strategy relies on three kinds of DNA probes:glycan anchor probes,protein recognition probes and signal probes.First,glycan anchored probes were anchored to glycans on cell surface by MOE and bioorthogonal reaction.The protein recognition probes targeted specific proteins by aptamer recognition.Finally,signal probes binded to the glycan anchor probes,followed by proximity-induced DNA hybridization with protein recognition probes on the same glycoprotein molecule,which results in a structural switch of the signal probe and subsequent generation of signal.The results showed that this strategy could successfully achieve fluorescence imaging of tyrosine protein kinase 7(PTK-7)-specific sialic acid on the surface of CCRF-CEM cells,and could further monitor dynamic changes of the glycosylation during drug treatment.Therefore,this strategy is expected to provide new ideas for studying the relationship between protein-specific glycosylation and tumorigenesis and development.2.Extracellular ATP-activated hybridization chain reaction for precise and highly sensitive detection of tumor cellsHighly sensitive detection of cancer cells is important for evaluating cancer development and improving survival rates.In view of the extremely high concentration of ATP in the tumor microenvironment,we developed an extracellular ATP-activated hybrid chain reaction(HCR)strategy for accurate and highly sensitive detection of tumor cells.The ATP-activated HCR system consists of an aptamer initiation probe(ZYsls-Trigger)and two pairs of hairpin DNA probes(H1 and H2).First,ZYsls-Trigger contained two components,the aptamer ZYsls region recognized tumor cells,and the trigger part serving as the HCR promoter.Next,The toehold of H1 is sealed by the ATP aptamer(ATP-a),which prevents the trigger-mediated HCR in the absence of ATP.In order to reduce background signal,AF488 fluorescence group and BHQ1 quenched group were modified on the stem of signal probe H2.In the presence of target tumor cells,ZYsls-Trigger probe recognized tumor cells,and extracellular ATP induces the dissociation of the ATP-a from the H1,exposing a toehold in H1,followed by trigger-induced HCR reaction.The results showed that this strategy could detect 25 cells in 200 μL of binding buffer,showing excellent detection sensitivity.In addition,the method showed good detection performance in10% fetal bovine serum and mixed cell samples.Benefiting from targeted recognition of aptamers and ATP-activated HCR amplification,this strategy can achieve precise and highly sensitive detection of tumor cells,which is expected to provide a novel approach for biomedical and tumor-related research.