Build And Application Of Fluorescent Biosensor Based On Nucleic Acid Isothermal Cycle Amplification

At present,the study of fluorescent biosensors has become a very important and concerning development direction in the field of bioanalytical chemistry.It has the advantages of simple operation,sensitive analysis and low cost in the analysis and detection of a variety of disease markers,and the application prospect is very broad.However,because the content of the target in the sample to be tested is often at a low level in the actual analysis and detection,it is of great significance to establish a simple,sensitive and practical detection method with the help of some signal amplification strategies.In this paper,we design some new fluorescent biosensors by combining with chain replacement,a variety of enzymes and DNAzyme assisted nucleic acid isothermal cycle signal amplification technology,and use fluorescent markers to realize the highly sensitive detection of large proteins or small molecular nucleic acids.The research content includes the following three aspects:（1）In this work,based on the powerful cycle amplification cascades of proximity hybridization-induced hybridization chain reaction and catalyzed hairpin assembly,we engineered a nonenzymatic and ultrasensitive method which combined the Mg²⁺-DNAzyme recycling signal amplification for the analysis of the human prostate specific antigen.Herein,we adopted PSA-conjugates as triggers in the self-assembly process of two hairpin DNAs（H1,H2）into the products of the CHA which could activate the HCR to induce repeated hybridization.And both ends of each adjacent sequence of the HCR products could form a unit of Mg²⁺-DNAzyme which in presence of cofactor Mg²⁺could recognize and cyclically cleave the hairpin probes in the solution and thus generate observably enhanced fluorescent signal.Benefit from the nucleic acid circuit amplification strategy,PSA of concentration low to 0.73 pg/m L was detected in this system.This homogeneous sensing method in solution avoids the use of the sophisticated equipment and complex operation,as well as addition of artificial enzyme,thus greatly reducing the constraints and complexity of experimental conditions.Moreover,considering most protein biomarkers in serum don’t have their corresponding aptamers,this sensing method provide a general sensing approach for homogeneous sensitive detection of.（2）We developed an enzyme-assisted multiple signal amplification reaction for target nucleic acid detection.In this strategy,the target nucleic acid as triggers,and in the presence of two specific primers,DNA polymerase and DNA nicking endonuclease act in concert to promote the repetitive cycle DNA replication process,leading to an exponential increase in nucleic acid sequence.Both ends of each sequence of the amplification products and the specific primer could form a unit of Mg²⁺-DNAzyme which in the presence of cofactor Mg²⁺could recognize and cyclically cleave the hairpin probes in the solution and thus realize real-time fluorescence detection of HIV-1 DNA.Under optimal conditions,the detection limit of this method is 10^-17 M.The simple in design,isothermal conditions and easy fluorescence measurement of this method exhibits superior sensitivity and specificity,and thus holds great potential to be a promising assay for quantitative detection of DNA or RNA in theoretical and clinical studies.（3）We propose a non-enzymatic catalyzed self-assembly nucleic acid signal amplification method mediated by three-dimensional DNA nanostructures,and construct a novel DNA hexahedral nanofluorescent probe that can rapidly and sensitively detect tumor-related mi RNA in living cells.Traditional CHA is performed by random free diffusion of DNA hairpins,which is very inefficient and dynamic.However,the CHA reaction kinetics is accelerated by self-assembly of functional branch chains to the five vertices of the DNA hexahedron,forming a structurally stable DNA nanostructure.And can move freely across cell membranes and react quickly in living cells.This method combines nucleic acid isothermal amplification technology with DNA nanostructure technology.The new hexahedral nanoprobe has good stability and biocompatibility,can respond to the target quickly,sensitively identify cancer cells,and distinguish them from normal cells.It has been widely used in the detection of cancer biomarkers and has shown a more obvious application prospect in diagnosis and treatment of cancer.