| Cancer is a type of disease that is seriously harmful for human health.So far,the early diagnosis and treatment of cancer remains the hottest issue in the world.Numerous of researches have shown that certain nucleic acids or proteins in cells,tissues and organs of cancer patients have large differences in expression(generally up-regulated or down-regulated)compared with healthy people,which can be regarded as tumor markers in clinical diagnosis.Traditional methods for tumor markers often suffer from the shortcomings of sophisticated operations,low sensitivity and high instrument cost.Therefore,developing new highly sensitive and selective methods for the detection of tumor markers such as nucleic acids and proteins is thus of great importance for cancer research and diagnosis,which is also the hotspot in the field of bioanalysis.Recently,for highly sensitive detection of tumor markers,various signal amplification technologies have been introduced for the construction of novel biosensing methods.In this thesis,three highly selective and sensitive fluorescence assays based on nucleic acid enzyme-assisted signal amplification have been developed for quantitative analysis of miRNA and apurinic/apyrimidinic endonuclease 1(APE1),which are the important potential tumor makers.The research contents are as follows:1.MiRNA is closely associated with a variety of diseases including cancers.In the second chapter,using miRNA-141 as a model analyt,we developed a fluorescence polarization method for highly sensitive detection of miRNA based on polystyrene nanospheres and T7exonuclease-assisted double-cycle signal amplification.In the absence of miRNA,the hairpin-structured recognition probe could not hybridize with the single-stranded signal probe which had a FAM modification at 5’-end and a biotin at 3’-end.The signal probe could be linked with streptavidin-modified polystyrene nanospheres through biotin-streptavidin interaction,thus greatly increased the mass/volume of FAM fluorophers and significantly enhanced the fluorescence polarization signal.After adding the target into the detection system,miRNA hybridized with hairpin probe to form a double-stranded structure,and the 5’end of hairpin probe was then recognized and cleaved by T7 exonuclease,releasing the miRNA and non-degraded fragment from hairpin probe.The released target miRNA could trigger the next cycle of hybridization-cleavage-release,producing more fragments from hairpin probe.The generated fragments could hybridize with the signal probe,starting the degradation of signal probes by T7 exonuclease,releasing the fragments and free FAM.The next cycle of hybridization and cleavage with signal probe was induced by the released fragment at same time.The free FAM could not connect with streptavidin-modified polystyrene nanospheres.Thus,the final system exhibited a small fluorescence polarization signal.Therefore,the assay of target miRNA-141 was achieved by recording the changes in fluorescence polarization values.Taking advantages of T7 exonuclease-assisted double-cycle signal amplification and polystyrene nanospheres enhanced fluorescence polarization signal,this method exhibited a high sensitivity(detection limit is 0.001 nM).In addition,it was also applicable to determine the total miRNA extract and compare the expression of miRNA-141 in human prostate cancer cells(22Rv1)and cervical cancer cells(Hela).Consequently,the proposed assay is expected to be used for the detection of miRNA-related tumor disease and biomedical research.2.In the third chapter,using miRNA-155 as a model target,we constructed another fluorescence polarization method for highly sensitive detection of miRNA,based on the double-strand specific nuclease-assisted target circulation and catalyzed-hairpin-assembly aggregation enhanced fluorescence polarization.This assay designed three hairpin probes HP2,HP3 and HP4 as the assembling elements of the nucleic acid nanostructure.The terminal of HP2was labeled with a FAM fluorophore and the terminals of HP3 and HP4 were labeled with biotins.In the presence of miRNA,the target could form a HP1/miRNA heteroduplex with the hairpin probe HP1,which initiates the degradation of HP1 in the HP1/miRNA by a double-strand specific nuclease,thereby releasing the target miRNA for the next cycle of hybridization and degradation.Simultaneously,an undegraded fragment form HP1 was also generated which could act as the primer to trigger the self-assembling of HP2,HP3 and HP4 into a Y-junction structure.Under the assistance of streptavidin,a nucleic acid/protein nanocomposite with a large mass/volume was formed through the biotin-streptavidin interaction,resulting in an enhanced fluorescence polarization signal.But,in the absence of the target,HP2,HP3,and HP4 could not be opened by each other,and the final system exhibited a small fluorescence polarization signal.Thus,a sensitive analysis of miRNA could be achieved by recording the signal change of the fluorescence polarization signal.In addition,this method was also applicable to determine the total miRNA extract extracted from human cervical cancer cells(Hela).Therefore,the fluorescence polarization miRNA assay is expected to be used for the miRNA-related clinical diagnosis and research.3.Apurinic/apyrimidinic endonuclease 1(APE1)is an important endonuclease in the process of DNA base excision repair,which is closely relevant to tumor development and the sensitivity of anti-tumor drugs.In the fourth chapter,we developed a fluorescence strategy for highly sensitive assay of APE1 activity based on the terminal deoxynucleotidyl transferase-assisted amplification.In this study,a hairpin probe with a tetrahydrofuran(as an abasic site)modification in the stem was designed as the substrate for APE1.In the absence of APE1,the intact hairpin probe(3’end modified inverted base)could not be extended by the terminal deoxynucleotidyl transferase due to the block of 3’-end with invert-base modification.In the present of APE1,the abasic site in the hairpin probe could be cleaved by APE1,producing a fresh 3’hydroxyl and 5’phosphate terminus.At the same time,the short single-stranded fragment with 3’hydroxyl terminus was released due to the decrease of melting temperature of the stem part.Using the short single-stranded fragment as the primer,the terminal deoxynucleotidyl transferase could catalyze the polymerization in the presence of dATP/dGTP.And a long single-strand DNA with repeated G-quadruplex was produced.In the presence of K~+,thioflavin T inserted into the G-quadruplex structure and an enhance the fluorescence signal was then obtained.The determination of APE1 activity could be achieved by monitoring changes of fluorescence intensity.This method had the advantages of simple,rapid,and highly sensitive,which could also be applied to study the inhibition of APE1 activity by drugs.Thus,this strategy has great potential application in APE1-related biomedical research. |
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