Construction Of Enzyme Assisted Signal Amplification Fluorescent Biosensor And Detection Of Tumor Markers

The formation,growth and metastasis of tumors in vivo are often accompanied by changes in tumor markers such as RNAs and proteins,which can be detected in blood,urine,bile,cerebrospinal fluid and other body fluids.Thus,highly sensitive detection of tumor markers in body fluids plays a vital role in the diagnosis,prognosis,treatment and etiology of cancer.Immunoassay is a common method for tumor marker detection,but it has some shortcomings such as cumbersome operation,long time consuming and limited detection accuracy.Therefore,it is urgent to develop new detection methods with low cost,sensitivity,simplicity and high specificity.In recent years,fluorescent biosensor has played an important role in disease diagnosis and biological analysis because of their simple operation,high sensitivity and trace detection.Based on this,this dissertation takes FEN1、microRNA-215 and microRNA-21 as research objects,and uses enzyme-assisted signal amplification strategy to construct three fluorescent biosensors to realize sensitive detection of the above nucleic acid and protein tumor markers.Specific research work is as follows:1.Lighting-up aptamer transcriptional amplification for highly sensitive and label-free FEN1 detectionSpecific and sensitive detection of flap endonuclease 1(FEN1),an enzyme biomarker involved in DNA replications and several metabolic pathways,is of high values for the diagnosis of various cancers.In this work,a fluorescence strategy based on transcriptional amplification of lighting-up aptamers for label-free,low background and sensitive monitoring of FEN1 is developed.FEN1 cleaves the 5’ flap of the DNA complex probe with double flaps to form a notched ds DNA,which is ligated by T4 DNA ligase to yield fully complementary ds DNA.Subsequently,T7 RNA polymerase binds the promoter region to initiate cyclic transcriptional generation of many RNA aptamers that associate with the malachite green dye to yield highly amplified fluorescence for detecting FEN1 with detection limit as low as 0.22 p M in a selective way.In addition,the method can achieve diluted serum monitoring of low concentrations of FEN1,exhibiting its potential for the diagnosis of early-stage cancers.2.Target recycling-triggered polymerization/isomerization amplification cascades for sensitive microRNA assayHighly sensitive quantification of microRNA biomarkers is useful for the prediction and diagnosis of various diseases.Here,a powerful and cascaded signal amplification strategy by integrating target recycling and polymerization/isomerization cyclic amplification(PICA)with the CRISPR/Cas12 a system is established for sensitive fluorescent microRNA-215 detection.The elaborately designed variable primer hybridizes with microRNA-215 to trigger the target recycling process for the formation of many hairpins with the assistance of the reverse transcriptase.Aided by the DNA polymerase and betaine,subsequent PICA reaction of the hairpins is initiated for the yield of many long ss DNAs with multiple repeated sequences,which bind and activate the trans-cleavage activity of Cas12a/cr RNA to digest fluorescently quenched ss DNA reporter sequences for the production of drastically amplified signals,enabling the detection of microRNA-215 with a low detection limit of 2.1 p M.In addition,the sensing method can selectively distinguish microRNA-215 against other interfering substances and achieve the monitoring of low levels microRNA-215 in diluted serums,indicating its promising potential for sensing various microRNA biomarkers at trace levels.3.Cascaded and auto-cycling primer extension reaction for highly sensitive and palindromic sequence-based detection of microRNA-21MicroRNA-21,a valuable biomarker,is involved in major cellular processes.The abnormal expression of microRNA-21 in cells is desperately related with the beginning and development of various diseases.Accurate monitoring of its activity is hence vital for biomedical research and disease diagnosis.In this article,a highly sensitive fluorescent sensor based on cascaded and auto-cycling primer extension(APE)amplification strategy is constructed for the detection of microRNA-21.In our design,two toehold-mediated strand displacement reactions(TSDRs)can be activated in the presence of microRNA-21 and primer,enabling the cyclically reused of target microRNA and stable binding of primer to DNA-based copy-and-release hairpin(CRH)to initiate APE and strand displacement amplification(SDA)reactions generation of numerous G-quadruplexes.The formed G-quadruplexes can interact with the Th T dye resulting in a remarkably magnified fluorescent signal for realizing highly sensitive detection of microRNA-21.Due to the three-stage signal amplifications,the detection limit of this method reaches 0.32 p M.In addition,the sensing method can selectively distinguish microRNA-21 against other interfering substances and achieve the monitoring of low-level microRNA-21 in diluted serums.With features of wide linear range and excellent selectivity,our signal amplification method can be a reliable means for early diagnosis of disease.