Construction Of Highly Sensitive Fluorescence Biosensors Based On Proximity Hybridization-Mediated Isothermal Nucleic Acid Amplification And Their Application For Protein Detection

Proteins are important bioactive molecules,and they are involved in almost all normal physiological processes.In addition,abnormal expression of proteins often associates with many important diseases such as cancer,AIDS and diabetes.Therefore,highly sensitive and selective determination of disease-related protein biomarkers is of immense significance in the early diagnosis of diseases.Currently,immunoassay based on the highly specific recognition between antibody and antigen is the dominant analytical technology for clinical protein biomarker detection.However,conventional immunoassays such as radioimmunoassay,enzyme-linked immunosorbent assay,che-miluminescence immunoassay suffer from drawbacks such as complex experimental procedures and low sensitivity,and they have been unable to meet the demand of the detection of ultralow-abundance protein biomarkers in biological samples.In recent years,nanotechnology and nucleic acid signal amplification technology has become the two important methods to improve the sensitivity of analysis because of its powerful signal enhancement function,and plays an important role in the high sensitive biological detection.Especially in the field of protein analysis,the introduction of nanotechnology and nucleic acid signal amplification technology can improve the detection sensitivity of traditional protein analysis methods and achieve the determination of low abundance protein markers.However,most of the nanotechnology based and nucleic acid signal amplification techniques still require complex procedures such as separation,washing,or expensive instruments.On the other hand,with more and more disease-related protein markers to find and protein markers of clinical application of gradually thorough,put forward new requirements for the detection and analysis method of protein markers.Therefore,the research and development of new methods and techniques for protein analysis,which are generally applicable,easy to operate,fast and sensitive,are still the research focus in the field of biological analysis.In this paper,cancer related protein markers as the target,using specific molecular recognition of nucleic acid aptamers and antibodies with Proximity hybridization assay and nucleic acid signal amplification technology,a new method of fluorescence biosensor development with high sensitivity and high selectivity,and a function in determination of protein content in complex biological markers.Specific research contents are as follows:1.By coupling target induced self-assembly with DNAzyme-mediated signal amplification,we have developed a new fluorescent aptamer sensor for highly sensitive detection of carcinoembryonic antigen(CEA).This system mainly consists of trigger DNA,assistant DNA and gold nanoparticle-functionalized fluorescent substrate of hairpin DNAzyme.In the absence of CEA,the trigger DNA and assistant DNA are unable to bind with CEA to form whole DNAzyme sequence,and can not be catalytic cleavage substrate under the action of Zn²⁺,resulting in a relatively low background of the fluorescence value.However,upon introducing CEA,the trigger DNA and assistant DNA bind with CEA at the same time,and whole DNAzyme sequence is formed by proximity hybridization,which activates DNAzyme.The activated DNAzyme can catalytic cleavage substrate linked to gold nanoparticle under the action of Zn²⁺,resulting in the release of substrate fragments labeled fluorophores from gold nanoparticle surface,thus achieving signal amplification.Under the optimal conditions,a good linear response between the CEA concentration and and the fluorescence intensity were acquired in the range from of 6 pg/mL to 60 ng/mL.The detection limit was 3.4 pg/mL.The assay has been successfully applied to the detection of CEA in human serum,and the results are consistent with the results of tranditional ELISA.2,A new fluorescence aptasening method based on target-triggered proximity hybridization and isothermal exponential amplification reaction(EXPAR)for ultrasensitive and rapid detection of human vascular endothelial growth factor-165(VEGF₁65)has been developed.In this system,the two aptamer subunits of VEGF₁65 aptamer sequence are embeded in trigger DNA and assistant DNA(which with molecular beacon fragment and a nicking enzyme recognition site),and the trigger DNA is designed to be only six base to complementary to the assistant DNA.In the absence of VEGF₁65,the trigger DNA is unable to bind with assistant DNA because of the short complementary sequence,and cannot trigger EXPAR.In this case,the fluorescence intensity is relative low.While VEGF₁65 is introduced,because of the combination between VEGF₁65 and two aptamer subunits of the trigger DNA and assistant DNA,the trigger DNA and assistant DNA close to each other and complementary hybridization.In the presence of dNTPs and polymerase,the trigger DNA and assistant DNA can be used as trigger and template respectively to trigger polymerization,the stem-loop structure of molecular beacon is opened and to form double-stranded DNA with complete recognition site of the nicking enzyme,lead to increasing of fluorescence intensity.The forming double-stranded DNA is digested by nicking enzyme,resulting in the formation of a new polymerization site to trigger polymerization,then a new double-stranded DNA with complete recognition site is formed.At the same time,the cleavaged single-stranded DNA is displaced.The new forming double-stranded DNA can trigger another cleavage reaction,and to form cyclic polymerization-cleavage-displacement,resulting in the release of many single-stranded DNA.The released single-stranded DNA can hybridize with molecular beacon of assistant DNA to open stem-loop structure.The opened molecular beacon can bind with trigger DNA to trigge polymerization,after which a new double-stranded DNA with complete nicking enzyme recognition site is synthesized and single-stranded DNA is displaced simultaneously.While the displaced single-stranded DNA can hybridize with molecular beacon to initiate a new cycle polymerization and to form cycle single-stranded DNA,many double-stranded DNA are formed.The formed double-stranded DNA can also initiate the circular polymerization-cleavage-displacement reaction to achieve generation and circular of DNA.At last a large amount stem-loop structures of molecular beacon fragment are destroyed,which lead to a a substantial increase in the fluorescence value.This assay achieve the signal amplification detection of VEGF.Under the optimal conditions,the detection of VEGF₁65 was reached 1.25 fmol/L.Moreover,this method also exhibits high specificity,and has been successfully applied to the detection of VEGF 165 in human serum.3.Combine superior fluorescence quenching ability of gold nanoparticle with restriction endonuclease signal amplification based on target-mediated DNA proximity hybridization,we have developed a new multiplex fluorescence immunosensor for the detection of variety proteins simultaneously.This system mainly consists of three pairs antibody-conjugated DNA?three-color molecular beacon and restriction endonuclease BfuCI.In the absence of target protein,three pairs antibody-conjugated DNA cannot bind with each other among them and cannot trigger restriction endonuclease signal amplification,too.In this case,the fluorescence of molecular beacon is quenched by gold nanopartical.While upon introducing target protein into the system,the antibody of each pairs antibody-conjugated DNA bind to the corresponding target protein,then the two DNA hybridize to form proximity complex due to close to each other.This proximity complex hybridizes with the corresponding DNA hairpin at the gold nanopartical surface to form double-stranded DNA with whole recognition site of BfuCI.Molecular beacon at the gold nanopartical surface is cleavage in the prescence of restriction endonuclease BfuCI,fluorescent dye and proximity complex are released simultaneously and fluorescence recovery.The released proximity complex can also bind with another molecular beacon sequence at gold nanopartical surface to trigger a new circular cleavage,achieving signal amplification and amplified analysis of target proteins.We have verified the feasibility of this assay strategy by using prostate-specific antigen(PSA),carcinoembryonic antigen(CEA)and alpha-fetoprotein(AFP)as model analytes.Under the optimized experimental conditions,the detection limits of PSA,CEA and AFP were 1.42 pg/mL,0.56 pg/mL and 2.2 pg/mL,respectively.In addition,the assay also showed good specificity and no cross reaction.This method has been successfully applied to the determination of PSA,CEA and AFP in human serum simultaneously,and the results are consistent with the results of tranditional ELISA.