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

Electrochemical biosensor is a kind of analytical tool which combines biometric identification technology and electrochemical detection technology.This sensor,which not only has the authenticity and sensitivity of biological detection,but also has the comprehensive advantages of simple electrochemical detection,miniaturization of instrument,and fast detection speed,are deeply developed and applied in the fields of medical and health,food safety,environmental monitoring,industry and agriculture.In recent years,a variety of materials combined with electrochemical sensing technology have been used to develop sensing platforms with superior detection performance,providing new ideas for solving practical problems.Among many materials,DNA has attracted much attention for its excellent recognition performance,diverse signal amplification techniques and extendable nanostructure,as well as its good stability,high biocompatibility,rapid reaction,and sensitive detetion.Therefore,based on DNA-assisted recognition,nucleic acid signal amplification and DNA nanostructure,the following works have been carried out in this paper:1.An aptamer electrochemical biosensor for kaname（Kana）detection was constructed based on Rec Jf enzyme-assisted target cycling and hybridization chain reaction（HCR）signal amplification strategy.Double-stranded DNA（ds DNA）was composed of hybridization of kanamycin aptamer（APT）and complementary DNA modified with sulfhydryl groups（SH-CDNA）,which was fixed on the surface of gold electrode by Au-S bond.In the presence of the mixture of kanamycin and Rec Jf enzyme,APT dissociates from ds DNA and binds to the ligand kanamycin,which is then hydrolyzed by Rec Jf e nzyme gradually to release kaname,thus triggering a cycle of more aptamers and ligands binding and release.The remaining SH-CDNA on the electrode surface initiated a hybridization chain reaction between hairpin DNA1（H1-Fc）and hairpin DNA2（H2-Fc）modified with ferrocene（Fc）,resulting in the formation of long double-stranded DNA nanostructures on the electrode surface.The logarithm of kaname concentration is proportional to the differential pulse voltammetry（DPV）signal of Fc with a detection limit of 7.8 p M at solution concentrations of 0.01～10 n M.The recoveries of kanamycin in actual samples ranged from 92.2%to 108.4%with relative standard deviation（RSD）of5.3%to 9.5%.The sensor has good selectivity and high sensitivity for kanamycin detectio n.The equipment used is simple and suitable for field detection.According to the specificity of the aptamer,the sensor provides a reference for the design of other detection targets,and has a great application prospect for food safety supervision.2.An ultrasensitive electrochemical biosensor for HPV16 oncogene was explored.Hairpin DNA-1,which can specifically bind with HPV16 oncogene,was fixed on the surface of gold electrode.Two hairpin DNAs underwent catalytic hairpin assembling with hairpin DNA-1 to construct Y-shaped DNA nanostructure,liberating HPV16 oncogene for target recycling.The 3’terminus of Y-shaped DNA nanostructure was prolonged under the catalysis of terminal deoxynucleotidyl transferase.Methylene blue（MB）was adsorbed onto DNA nanostructure to generate characteristic differential pulse voltammetry signal.This signal was increased with the concentration of HPV16 oncogene,and the detection limit of HPV16 oncogene was as low as0.19 f M.3.A label-free and ultrasensitive electrochemical biosensor for oral cancer overexpressed 1（ORAOV1）gene was constructed via exonuclease III-assisted target recycling and dual enzymes-assisted signal amplification strategy.Capture DNA with sulfhydryl group at 3’terminus was firstly modified onto the clean surface of gold electrode via strong Au-S bond.Assisted DNA hybridized with basal DNA to form hybrid DNA in advance.ORAOV1 gene could hybrid continuously with such hybrid DNA from the other terminus to construct intact double-stranded DNA.Exonuclease III digested basal DNA in such intact double-stranded DNA specifically and released both ORAOV1 gene and assisted DNA into solution.ORAOV1 gene induced another intact double-stranded DNA digestion for target recycling,while assisted DNA hybridized with the capture DNA to form double-stranded DNA on electrode surface.Unhybridized capture DNA was hydrolyzed by Rec Jf exonuclease to reduce the background electrochemical signal.The 3’terminus of double-stranded DNA was prolongated to be guanine-rich oligonucleotides under the catalysis of terminal deoxynucleotidyl transferase.In the presence of K⁺ion,hemin adsorbed onto oligonucleotides to construct multiple G-quadruplex/hemin complex with large steric hindrance effect to efficiently avoid the c harge transfer of[Fe（CN）₆]^3-/4-probe toward electrode surface.The electrochemical impedance value was increased significantly after the addition of ORAOV1 gene.The electrochemical impedance value was linearly related to logarithmic concentration of ORAOV1 gene in the range from 0.05f M to 20 p M,and the detection limit was low to 0.019 f M.Such biosensor was used to detect ORAOV1 gene in human saliva with satisfactory results.