Sensitive Detection Of Cell Membrane Proteins Based On Hybridization Chain Reaction Fluorescence Biosensor

Biofilms are composed of carbohydrates,lipids and proteins.As a kind of proteins,membrane protein is related to the occurrence of many diseases.After normal cells become cancerous,membrane protein will be abnormally expressed.In addition,membrane proteins,as potential cancer markers are also the major targets of anticancer drugs.Therefore,the development of highly sensitive quantitative analysis methods of membrane proteins is particularly important for disease diagnosis and drug development.Here,we developed a fluorescent biosensor based on aptamers and signal amplification technology without enzyme assistance for rapid and highly sensitive detection of cell membrane proteins.Tyrosine protein kinase 7(PTK7)membrane protein of acute human lymphocytic leukemia(CCRF-CEM)and tyrosine protein kinase Met membrane protein of lung cancer cell(A549)were used as target proteins.The specific research contents of the thesis are as follows:1.A fluorescent biosensor based on aptamers and signal amplification technology was designed for the rapid and sensitive detection of cell membrane proteins.The detection system included two classes of DNA: The aptamer sgc8 of membrane protein PTK7 and the hairpins H1 and H2 of HCR.H1 was modified with cholesterol at the 3’end and Cy3 at the 5’ end,H2 was tagged used cholesterol at the 3’ end and Cy5 at the T base of the hairpin loop.In addition,the trigger chain(I)that triggered HCR was connected to sgc8,so trigger chain was anchored on membrane protein via the recognition between sgc8 aptamer and PTK7,HCR reaction was initiated by I anchored on cell surface,H1 and H2 were alternately opened,forming a long chain polymer like helical,resulting in the close proximity of Cy3 and Cy5 and further occuence of fluorescence resonance energy transfer(FRET).Repeat this process to achieve signal amplification,so that the FRET fluorescence signal finally detected is significantly enhanced,and the amplified detection of membrane protein PTK7 is realized.This method improved the sensitivity of detection,speeded up the reaction speed,provided faster reaction kinetics.This method can be used to detect other types of membrane proteins by simply replacing the aptamers,which is expected to provide a new platform for the detection of membrane proteins in trace amounts and when the expression level of membrane proteins is relatively low in the early stage of cancer.2.A fluorescent biosensor based on nucleic acid aptamer enzyme-free signal amplification technique for the detection of cell membrane protein dimerization was investigated.The detection system uses two kinds of DNA sequences and recombinant hepatocyte growth factor(HGF),two kinds of DNA sequences are aptamer of membrane protein Met and HCR hairpin probe H7 and H8.H7 was modified with cholesterol at the 3’ end;H8 was modified Cy5 at the 5’ end and dispersed in solution.The trigger chain(I)was divided into two parts,which are respectively connected to the aptamer of Met protein.cmet i1 and cmet i2 can be paired with eight complementary bases,cmet i1 and cmet i2 could be stable in solution.When cmet i1 and cmet i2 are targeted to the cell membrane and added to HGF,Met protein dimerization is induced,cmet i1 and cmet i2 get close to each other,base complementary pairing occurs,i1 and i2 are adjacent,forming a complete trigger chain,which triggers H7,H8 and HCR reactions,and finally the enhanced fluorescence of Cy5 on the cell membrane is detected.This method is expected to improve the sensitivity of cell membrane protein dimerization detection by using aptamers and HCR signal amplification.