| Exploring novel diagnosis and prognostic monitoring methods for early-stage tumors holds great significance for the improvement of cancer patients cure and survival rate.Aptamer,as a "chemical antibody",can specifically recognize tumor associated biomarkers with high affinity.Being synthetic and stable,aptamer probes show great applicable potential in the basic research of tumor diseases.However,the repotred tumor cell-specific aptamers are generally long oligonucleotides containing 30-100 bases,which hard to be synthesized.Furthermore,long aptamers bear more secondary structures unfavorable for target binding and increase challenges in the designing of novel target responsive activated Aptamer probe.By comparison,the split aptamer,consisting of two brief fragments derived from parent aptamers,shows the characteristics of flexible design,simple preparation and price economy.Split aptamer not only owns the target specificity derived from the original aptamer,but also gets some special properties(such as temperature sensitivity).These properties greatly enrich its application in the field of tumor cells research.However,the development and application of split aptamer probes are still in their infancy,and some key issues,such as deficiencies functionality,weak affinity,limited sensitivity and single mode of signal conversion,remain to be resolved.In this thesis,aiming at the resolve of above key issus and the exploration of novel tumor cell detection techniques,a series of split aptamer probes with target driven self-assembly properties have been designed.By utilizing advantages of polymerase chain reaction(PCR),rolling circle amplification(RCA),hybridization chain reaction(HCR)and chain replacement reaction as signal conversion and amplification units,and using human hepatocellular carcinoma cells and human leukemia cells as the research object,carry out the system of tumor cells for high sensitive detection,control,real-time analysis and imaging research,a sensitive,maneuverable,real-time and in vivo study on tumor detection and characteriazation has been systematically performed.The detailed description is listed as follows:1.Split aptamers combined with PCR for label-free and sensitive detection of tumor cellsApta-PCR technology has been widely used in the field of tumor cell detection due to its advantages of high sensitivity,fast inspection speed.However,the reported Apta-PCR technology generally requires the destruction of cells in order to obtain aptamer fragments for PCR amplification,which is of harm to subsequent analysis of the cell.In addition,it needs special modification of the aptamer,which is expensive and complicated.In order to overcome these defects,this research establishes a label-free method for high sensitive detection tumor cell combined with PCR technology.That is to say,split aptamer,with thermosensitive properties,could specifically targets tumor cells.And then it disassembles into aptamer fragments through the regulation of temperature in a mild and simple way.At First,a split aptamer pairs,split apt-a and split apt-b,was designed with SMMC-7721 human hepatoma cell specific targeting ability and PCR amplification characteristic through connecting the PCR primer sequence to the ends of one split aptamer fragment.The split apt-a fragment used for PCR amplification could be isolated by increasing the temperature to 37? after incubated with cells on ice.Using a SYBR Green I dye-mediated PCR technique,this strategy held a good specificity and sensitivity for detecting SMMC-7721 cancer cells,which permitted the actually detection of 100 cells.The generality of the thermo-controlled Apta-PCR strategy was certified by using another split aptamer targeting CCRF-CEM cells.At the same time,the method for obtaining nucleic acid aptamer fragments was gentle and wouldn't destroy cell surface protein activity or cell viability,which was advantageous for subsequent analysis and research.The use of embedded DNA dyes achieves the fluorescence signal.It could overcome the defects of DNA labeling that was tedious and expensive and might be harmful to the recognition ability of aptamer.Thus,by effectively reducing the tumor cell detection cost and simplifying the experimental preparation steps,this strategy provided a new aptamer probe for tumor cell detection.2.Proximity-dependent hybridization chain reaction based on split aptamer for tumor cell detectionIn order to achieve direct detection of tumor cells and simple,enzyme-free signal amplification,a proximity-dependent hybridization chain reaction probe based on split aptamer was designed for sensitive detection of tumor cells by integrating the advantages including simple operation,flexible design,signal amplification ability of HCR technology and the characteristics that self-assembled on cell surface of split aptamer.This probe could realize self-assembly and signal amplification on the cell surface,and then achieved quantitative detection of tumor cells.At first,a split HCR probe,split HCR—a and split HCR-b,consisting of a split nucleic acid aptamer sequence and a split type HCR trigger chain sequence were designed.The split aptamer probes could be self-assembled on the surface of target cell,leading to the two split HCR trigger sequence closed to each other.Then the H1 hairpin chain was opened,triggering the HCR reaction,and finally achieved amplified fluorescence signal.This probe could realize the high sensitive detection of tumor cells,permitting the actually detection of 20 cells in buffer.At the same time,the probe could identify the SMMC-7721 cells in mixed cell samples,and realize the quantitative detection of SMMC-7721 in 10%serum fluid.Using split aptamer and HCR technology,the probe realizes the self-assembly and signal amplification on cell surface with high specificity and sensitivity.This design greatly promotes the application process of aptamer molecular probes in tumor detection field,and may be used as a new technique for high sensitivity and high selectivity detection of tumor cells.3.Split aptamer combined RCA technology to build DNA nanoensemble for tumor cell detection and manipulationIn order to solve the shortcomings of the affinity and single function of the monovalent aptamer probes for tumor cell detection,a DNA nanoensemble was constructed for highly sensitive detection,capture and release of tumor cells by using RCA technology combined with temperature sensitive split nucleic acid aptamer,.A long chain DNA(named nanosolo)was constructed by RCA technology.This nanosolo structure containing split-b probe repeated units could multivalently bind to target cancer cell with split-a fragment,forming a nanoensemble structure on the cell surface,and then realize the detection and capture of tumor cell.The release of the captured cells could be realized by increasing the temperature.Results showed that nanoensemble held a good specificity and greatly strong affinity,leading to a?2.8-time increase in signal to back ratio(S.B.R).Moreover,nanoensemble greatly accelerated the target interaction,which could realize almost 50%binding in 1 min.Also,nanoensemble extended the tolerance range of aptamer-target binding from 4 ? to room temperature.These advantages then supported the convenient and effective use of nanoensemble,such as cancer cell detection in biofluids and mixed cell samples,as well as temperature-controlled cancer cell catch/release on the microplate well surface.The probe not only greatly improved the defects of low affinity,faint sensitivity,narrow suited temperature range of the monovalent split aptamer,but also introduces DNA nanomaterials with the potential as an effective drug carrier for tumor treatment.In sum,as a versatile and intelligent aptamer melocular probe,DNA nanoensemble might be expected to play an important role in cancer-related studies.4.Split aptamer combined DNA strand displacement for tumor cell real-time assay and in vivo imagingThe detection and real-time analysis of tumor cells under physiological conditions is of great significance in the study of tumor diseases.In order to solve the problem that the current split aptamer probes can not be used effectively for tumor cell detection under physiological conditions and the slower rate of related signal transformation,a novel probe was constructed for cancer cell real-time assay and in vivo imaging combined with split aptamer by using DNA strand displacement with the characteristics of fast and sensitive signal conversion.The split aptamer pairs,ZYlla and ZY1 lb probe,specifically targeting of SMMC-7721 tumor cells,were selected as the model.By adding the split HCR trigger sequence into the end of the two probes sequence and then connecting to be one single stand using polyT linker,a new split aptamer(split-ZY11-SD probe)was developed.This probe could specifically recognize target cells leading to structural changes and initiating strand displacement reactions,thereby achieving rapid and sensitive detection of tumor cells.Flow cytometric analysis showed that the split-ZY11-SD probe could achieve highly sensitive detection of SMMC-7721 cells within 1 min,leading to a S.B.R of about 15.The probe could achieve real-time analysis of SMMC-7721 cells by confocal laser scanning microscope imaging,and be able to quantitatively detect SMMC-7721 cells in mixed cell samples.The analysis of SMMC-7721 cells in serum showed that the probe had good anti-interference ability.By directly injected into the tumor site of mice,the probe could be used in vivo imaging of SMMC-7721 cancer cell.This probe design is simple and convenient,which holds great potentials to be used as a new molecular probe for the real-time detection of cancer cell and in vivo imaging. |
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