Sensitive Detection Of Proteins Based On Terminal Protection Of Small-molecule-linked DNA Combining With Isothermal Amplification

Protein,an important component of all cells and tissues of the human body,is the material basis of life.It is closely associated with all kind of human life activities,such as the transport of material,the transmission of energy,the metabolism of reaction,the invasion of foreign objects,inheritance of genes and so on.But due to the wide variety of proteins,different functions and various ways of action,these pose a challenge to protein detection.Therefore,enriching assay methods and achieving sensitive detection for proteins are of great significance for the study of protein physiological function,the early diagnosis and treatment of protein-related diseases as well as the corresponding drug research and development.The physiological function of many proteins is dependent on the involvement of small molecules,such as interactions of treptavidin and biotin,folic acid receptor and folic acid.Terminal protection based on the specific interaction between small molecule and the target protein,which prevents the degradation of DNA linked to small molecule by exonuclease.The terminal protection mechanism provides a new idea for protein detection.In this thesis,using streptavidin-biotin as the protein-small molecule model,two new methods for protein fluorescence analysis have been established based on terminal protection of molecule-linked DNA combing with branched rolling ring amplification(BRCA)and catalytic hairpin assembly(CHA)amplification.(1)Sensitive detection of streptavidin based on small terminal protection of molecule-linked DNA combing with branched rolling circle amplification.In this experiment,In the presence of the target protein-SA,SA specifically binds to the small molecule of biotin attached to the 3’end of the DNA probe to form terminal protection,which prevents the degradation of the DNA probe by exonuclase I(Exo I).As the ligation DNA probe,the protected DNA probe hybridizes with two ends of the DNA probe.Under the action of DNA ligase,with the ligation DNA probe as the template,the DNA probe forms a closed ring.Then,under the action of DNA polymerase,with the closed ring DNA probe as the temlate,primer 1 and reverse primer 2 are extented,forming the branched rolling circle amplification.Due to the special circular structure of the amplification template,the final extension product is very long double-stranded DNA with many repetitive DNA.Finally,the fluorescent dye SYBR Green I(SG),which is specific to double stranded DNA,is used for fluorescence detection.The amplification efficiency of BRCA can reach 10~5～10~9times,which can effectively improve the sensitivity of protein detection.The method is sensitive and the detection limit is low to 43 fmol.(2)An enzyme-free amplification strategy for protein detection based on terminal protection of small-molecule-linked DNA combining with catalytic hairpin assembly(CHA).In this experiment,we also use the SA as the protein model.SA specifically binds to the small molecule of biotin attached to the DNA probe to form terminal protection.A hairpin DNA-AgNCs is designed as a fluorescent probe.The hairpin structure of DNA-AgNCs is opened due to the its hybridization to the protected DNA probe,Then,it hybridizes with a G-rich hairpin probe.Which narrows the distance between dark DNA-AgNCs and G-rich sequence,and stimulates the dark DNA-AgNCs to emit strong fluorescence.At the same time,the DNA probe protected by SA is replaced and combines with the new DNA-AgNCs probe to stimulate a new round of reaction,forming catalytic hairpin assembly amplification.The fluorescence intensity increases with the increase of SA.This method is simple and rapid,and no enzyme is involved in the amplification process,which can save the cost of detection.The using of AgNCs,a novel nanomaterial,develops a new idea for protein detection.