Study On Construction Of Highly Sensitive Fluorescent Sensing Methods For Nucleic Acid And ATP

With the rapid development of social economy,people pay more and more attention to physical health.However,major diseases such as cancer still seriously threaten human life and health.Although medical treatments are becoming more developed and the cure rate of diseases is getting higher,the total death toll is still rising year by year.Therefore,the early diagnosis of the disease plays a vital role in the entire diagnosis and treatment process.Nucleic acid is the most basic material of life.However,if the nucleic acid is mutated or expressed abnormally,it may cause serious diseases such as malignant tumors,cardiovascular disease and Alzheimer’s disease.Adenosine triphosphate（ATP）is a universal energy currency in organisms,and it plays an irreplaceable role in the synthesis,replication and transcription of nucleic acids.However,the abnormal expression of ATP can also cause the aforementioned diseases.From this point of view,accurate detection of nucleic acid and ATP will provide a valuable reference for clinical diagnosis and treatment of diseases.Biosensors are device that can accurately detect biomolecules.Among them,fluorescent biosensor plays an important role.Fluorescent biosensor is a kind of detection device that specifically recognizes biomarkers and converts the recognition event into fluorescent signal output,and it is widely used in biomedicine,environmental science,food science and many other fields.This thesis aims to simplify the experimental operation and improve the detection sensitivity.Therefore,with the help of isothermal biological enzyme-assisted and DNAzyme-assisted signal amplification technology,fluorescence sensing methods have been developed and successfully applied to the highly sensitive detection of nucleic acids and ATP.The particularly constructed fluorescence sensing methods are summarized into the following parts:1.Target-dependent dual strand extension recycling amplifcations for non-label and ultrasensitive sensing of serum micro RNAMicroRNAs（miRNAs）have been used as a new type of tumor biomarkers for early detection of cancer.However,their high family sequence homology,low abundance and shorter sequence pose a major challenge for the analysis of miRNAs.In the present work,we developed a dual strand extension recycling signal amplifcation strategy for non-label detection of miRNA-155 with high sensitivity on the basis of DNA polymerase and the G-quadruplex/thioflavin T（Th T）complexes.The miRNA-155 target sequences could initiate two strand extension-based independent recycling cycles under the function of the DNA polymerase,resulting in the production of many active G-quadruplex segments.The dye of Th T further bound the G-quadruplexes to induce greatly enhanced fluorescent intensity for ultra-sensitively detecting miRNA-155 sequences at the low concentration of35 f M in the dynamic range from 0.1 p M to 100 p M.This method with a high selectivity could also be used for diluted serum samples.The demonstration of our new method for miRNA detection thus offers it with high potentials for miRNA biomarker analyses with the purpose of clinical diagnostics and biomedical applications.2.Target recycling transcription of lighting-up RNA aptamers for highly sensitive and label-free detection of ATPATP plays a key role in the synthesis,replication and transcription of nucleic acids in life processes.However,abnormal changes of concentration in the living organisms can cause major diseases such as malignant tumors.Therefore,the highly sensitive detection of ATP may provide a reliable basis for the clinical treatment of ATP-related diseases.We describe here a target recycling transcription of lighting-up aptamer strategy for label-free and sensitive detection of ATP in human serums.ATP specifically binds with its aptamer sequence and leads to the structure switching of the DNA assembly probes to trigger the recycling of the target ATP molecules via the toehold-mediated strand displacement reaction,which results in the formation of many ds DNAs containing the RNA promoter sequences for subsequent transcription generation of large amounts of lighting-up aptamers.The malachite green organic dye then associates with these lighting-up aptamers to produce significantly enhanced fluorescence signals for sensitively detecting ATP in the dynamic range of 10-500 n M with a detection limit of 7.3 n M in a label-free way.The sensing approach also has an excellent selectivity for ATP analogues molecules.In addition,this method can achieve sensitive detection of ATP molecules in diluted human serums,which proves its potential for early diagnosis of ATP-related diseases.3.Target-mediated assembly formation of four-way DNAzyme junctions for sensitive and accurate discrimination of SNP in K-ras geneDue to its high pathogenicity rate,DNA single nucleotide polymorphisms（SNPs）have always received widespread attention.However,it is a major challenge in the field of modern medical analysis to accomplish accurate and rapid screening single nucleotide polymorphisms（SNPs）in the genome because of the high degree of sequence similarity.Therefore,we have developed a four-way junction（4WJ）DNA biosensor with the participation of DNAzyme,which used toehold-mediate strand displacement reaction（TSDR）to achieve highly sensitive detection of K-ras gene point mutation（GGT（?）GTT）.After Mt DNA was assembled into 3WJ-DNA,it was the only key that can start TSDR and integrate the split DNAzyme fragements into an intact Mg²⁺dependent-DNAzyme.Mg²⁺dependent-DNAzyme bind with hairpin to continuously release G-quadruplex to bind with Th T,which greatly enhanced the specificity and sensitivity.Target DNA could be detected with a concentration as low as 3.63 f M in the dynamic range of 10 f M to 5 n M.In addition,the strand displacement process possessed a high fidelity because the toehold could be locked by frame-shift basepairing stably,so the positive signal detection of mutant gene could be achieved with a ratio of wild type to mutant type of 5000:1.We provided a cheap and efficient method for DNA single base polymorphism detection,which possessed great potential for the analysis of cancer biomarkers.