| As we all know,nucleic acid is the material basis for species to maintainevolution and reproduction for generations.Traditional nucleic acid detection technology requires the use of enzymes and sophisticated temperature control instruments,which increase the detection cost.DNA electrochemical biosensor has been widely regarded as the most promising method for rapid detection of biomarkers due to its characteristics of high specificity,non-target amplification process and miniaturization.At present,the rapid detection method has attracted wide attention and research interest.In this study,the following research is carried out based on the electrochemical biosensor nucleic acid detection technology.Ratio signal detection has been widely used in the construction ofelectrochemical biosensors because of its good stability and anti-interference ability.However,how to construct a highly sensitive electrochemical biosensor through ratiometric detection is still a challenge.In this study,a ratio electrochemical nucleic acid detection technology based on the proximity-dependent surface hybridization structure and hairpin capture probe structure was developed in order to reduce the detection background.This method took advantages of the high specificity of the proximity-dependent hybridization and hairpin capture probe structure.When the target was absent,the designed hairpin capture probe could reduce the possibility of hybridization between the detection probe and hairpin capture probe,which in turn improved the detection specificity.Moreover,the ratio detection structure could eliminate the signal fluctuation error caused by experimental conditions and improve the detection stability.This biosensor had a good linear relationship between the nucleic acid target concentrations range of 1.0×10-12M to 1.0×10-16M.The detection limit can reach 6.4×10-17M.In addition,the biosensor had good selectivity and could identify the target of single base mismatch,which will have great application potential in nucleic acid detection and medical clinical diagnosis.In the preparation of traditional electrochemical biosensor,the modification ofcapture probe takes the longest time,and it usually takes 12 hours to overnight.Thus the detection efficiency of electrochemical biosensor can be improved by reducing the probe modification time.In this study,the ferrocene is added to the 5'end of the target DNA chain.Therefore,the hybridization can be characterized by detecting ferrocene signal.Firstly,the DNA probes were modified by chemical embedding method of pyrrole electropolymerization,as a result the capture probe was embedded in the network structure of polypyrrole by the external electric field.The results showed that the probe modified by this method were arranged irregularly on the electrode surface;therefore,an improvement was made.The 3'end of the capture probe was modified with pyrrole monomer,and the probe was modified by electropolymerization of pyrrole covalent bond.The probe formed on the surface of gold electrode was relatively regular.However,these two methods cannot detect ferrocene signal after target hybridization.According to the research,it is speculated that the pyrrole layer formed on the gold electrode surface is too thick or the capture probe is completely embedded in the polypyrrole layer,which hinders the electron transfer of ferrocene on the gold electrode surface or prevents the subsequent target hybridization process.Finally,the 3'end of the DNA capture probe was modified sulfhydryl and fixed on the surface of gold electrode by electrodeposition,and this process only took 5 minutes.This method of modifying capture probe meets the requirement of this study for detecting ferrocene electrochemical signals,and greatly shortens the time for electrochemical biosensors to detect targets.At the same time,it provides a new research idea for the mass production and high efficiency detection of target. |
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