Construction And Application Of Electrochemical Biosensor Based On DNA Signal Amplification Technology

Electrochemical biosensor is fixed on the electrode with biological sensitive substances such as enzyme,antigen/antibody,deoxyribonucleic acid,polypeptide and so on as sensitive elements.The electrode is a transducer that can convert the physical and chemical signals generated during the specific recognition between biosensors and target molecules into electrochemical signals related to the concentration of target molecules.In recent years,electrochemical biosensors have been widely used in biomedicine,environmental monitoring,military,food and drug analysis because of their high sensitivity,good selectivity,low cost,fast analysis speed and small volume.Aptamer is a single strand of DNA screened in vitro,which can specifically identify the target,and has been widely used in DNA detection due to its advantages of high specificity,in vitro synthesis,great stability and easy modification.In order to develop high sensitivity sensors,many bio-amplification techniques have been applied to electrochemical analysis and detection,including target cyclic amplification,enzyme catalytic amplification,avidin-biotin amplification,DNA amplification and so on.Since the occurrence of many diseases are related to gene mutations,DNA detection can predict the risk of disease,which is extremely important for the clinical diagnosis of genetic diseases and the selection of appropriate drugs to improve the therapeutic effect.In this paper,based on Nucleic acid aptamer and DNA signal amplification technology,electrochemical biosensor was constructed for the detection of tumor-related genes,and the following work was carried out:1.An electrochemical biosensor based on toehold-mediated strand displacement reaction(SDR)and G-tetraplex/hemin was constructed for HPV16 gene detection.A hairpin capturing probe CP with 3’-end modified sulfhydryl(-SH)group was designed and mounted autonomously on a gold electrode(GE)via an Au-S bond,while 6-mercapto1-hexanol(MCH)was used to seal the vacant position on the electrode surface that was not covered by CP.The hairpin structure of CP opens in the presence of the target HPV16 gene.Compared with the HPV16 gene,HDNA and CP have stronger binding ability,and the target will be released in the hybridization process of HDNA and CP and used to start another cycle.After completion of the cycle,a large number of double-stranded bodies containing G-tetraplex were formed on the electrode surface.In the presence of K~+,hemins are stacked into the G-tetraplex region and form G-tetraplex/hemin complexes.Through its electron transfer to achieve the current response,so as to achieve the detection of the target.The sensor has high sensitivity,simple method and good stability for HPV16 gene detection.The detection limit is 0.03 p M and the detection range is 0.1 p M-5 n M.2.An electrochemical biosensor based on Exo III enzyme-assisted target cycling and hybridization chain reaction(HCR)amplification was constructed for Oral Cancer Overexpressed 1(ORAOV1)gene detection.First,the designed 5’-end modified sulfhydryl hairpin DNA(hp DNA)was self-assembled on the surface of the GE by Au-S binding.According to the Watson-Crick base pairing rule,self-assembled hp DNA forms a hairpin structure with a stem region and a ring region.Then,MCH was modified on the surface of hp DNA/GE to obtain MCH/hp DNA/GE,which completely blocked the active sites that were not occupied and effectively inhibited the non-specific adsorption effect.After the addition of ORAOV1,it specifically hybridized with a specific 3’-terminal sequence of hp DNA,resulting in the formation of DNA double strands and the opening of the hp DNA hairpin structure.Meanwhile,the Exo III enzyme binds to another hp DNA by degrading the ORAOV1 gene released by the double-stranded DNA,triggering the Exo III-assisted target cycle.HCR reactions can occur by hybridization with hairpin probes H1 and H2 using DNA fragments remaining on the electrode surface as primers,resulting in self-assembly into long double-stranded DNA with incisions.The electrochemical redox probe methylene blue(MB)can specifically bind to guanine in DNA molecules.When MB binds to long double-stranded DNA,the electrochemical signal of MB will be enhanced,thus realizing the detection of ORAOV1 gene.The sensor has a linear detection range of 0.5 p M-100 n M and a detection limit of 0.21 p M.In addition,the sensor has strong anti-interference ability and good stability,and can be used to detect ORAOV1 gene in artificial saliva samples.3.A ratio metric electrochemical biosensor was designed for the detection of p53 gene based on N.Bst NB I nuclease assisted target cycle and rolling circle amplification(RCA)signal amplification strategy.Firstly,the ferrocene(Fc)hairpin capture probe(Fc-HP-SH)labeled at the 3?-end was self-assembled on the surface of the gold electrode via Au-S bond,and the non-specific binding site on the electrode was closed with MCH.At this time,Fc was closer to the surface of the gold electrode and a strong electrochemical signal was generated.When both the p53 gene and N.Bst NB I nuclease were present,the p53 gene was specifically hybridized with the loop sequence of FC-HP-SH to unfold the hairpin structure,and then N.Bst NB I nuclease cleaved the DNA double strand to release the p53 gene for target recovery.The single stranded oligonucleotides remaining on the electrode surface were RCA extended by T4 DNA ligase,deoxyribonucleotides(d NTPs)and phi29 DNA polymerase,which increased the aggregation of electroactive probe MB on long polynucleotides.This change results in a change in the peak differential pulse voltammetry currents of Fc at+0.32 V(vs.Ag/Ag Cl)and MB at-0.25 V(vs.Ag/Ag Cl),and the ratio signal of the two peak currents is significantly enhanced.Under the optimal experimental conditions,the biosensor can detect the concentration of p53gene in the range of 1 f M-10 p M and the detection limit of 0.23 f M.The biosensor is highly sensitive to p53 gene in mutants and common proteins,and has been successfully applied to human serum samples.