Rapid And Sensitive Detection Of MicroRNA Based On Water-soluble Fluorescent Polymers

MicroRNA(miRNA),a kind of endogenous non-coding small molecule single-stranded RNA with 19-23 base,abounds in animals,plants and virus.A large number of researches have shown that miRNA is closely related to the physiological processes of organisms,including growth and development,cell proliferation and apoptosis,fat metabolism,tumor growth and so on.Abnormal expression of certain miRNA directly results in the occurrence of human diseases.Therefore,it is of important significance to sensitive detection of miRNAs with specific sequences,because of the low abundance of miRNA in the cells and the high homology of the miRNAs sequences.Nucleic acid amplification technology is one of the most used methods to amplify the detection signal,and then enhance the sensitivity in miRNA detection.It is well know that the above method need expensive enzymes,design of complex sequenses and harsh conditions,resulting in high professional requirements,long dtection cycle and high cost.Thus,it is urgent to development of a novel method for highly sensitive detection of miRNA with high applicational value.The contents of this paper are as follows:The first chapter shows the background of miRNA study,including the origin of miRNA,mechanism and relationship between miRNA and disease.And the method of detection of miRNA based on nucleic acid hybridization and signal amplification technology was discussed.The research background and contents are also presented.In the second chapter,by taking advantage of signal amplication of water-soluble fluorescent polymer,a novel method for directly detection of target miRNA was reported.The method has the apparent advantage of simple,rapid,low cost,without the requirement of target miRNA amplification and low professional.The water-soluble fluorescent polymer has a number of fluorescein and can specific identify target miRNA,which was prepared by the copolymerization of the acrylic acid fluorescein,acryloyl-modified DNA(Acrydite-DNA)sequence and acrylamide.The polymer chain labeled with 86 fluorophore benefits the signal amplification.Because of the fluorescent reporter moiety was encapsulated by the polymer,the collision quenching reduced,which leaded to the further signal amplification.The target miRNA can be selectively separated by the self-assembly of the thiolated DNA(SH-DNA)sequence on the surface of the gold-magnetic particles,which greatly simplifies the detection procedure.The proposed method has the detection limit of 1.44 fM,which is comparable to the mostly reported method.Otherwise,the fluorescence was investigated when the target miRNA sequence was single base mismatch,three base mismatches,and arbitrary sequence.The experimental results indicated that the proposed method has high selectivity for target miRNA.In the third chapter,a novel fluorescent protein stain is synthesized.The fluorescent protein stain has aldehyde group reacted with the amino group of the protein to form covalent bond,which help to staining and resist to fading.This is also favorable for fluorescent or naked eye recognition and determination.Bovine serum albumin and lysozyme were stained with the fluorescent protein stain.Compared with the traditional method of coomassie bright blue and silver staining,the fluorescent protein stain is mixed with the protein samples before electrophoresis,and then the protein band is directly observed through electrophoresis.The fluorescent protein stain has the advantage of convenient,simple,time saving and high sensitivity.In the fourth chapter,a novel method for directly detection of miRNA based on water-soluble fluorescent polymers was established.The miRNA with different base sequences were detected,indicating the method has high selectivity.And,we synthesized a new kind of fluorescent protein stain agent,and it was successfully applied in protein stain.