Construction And Application Of Electrochemical DNA Sensor Based On DNA Probe Fixation And Nucleic Acid Signal Amplification

Electrochemical DNA sensors are a biosensor which immobilize DNA probe on substrate surface to recognize or indirectly recognize target sequences,and convert sequences recognition signal into electrical signal by transducer.Electrochemical DNA sensors have been widely focused by many researchers because of their rapid response,high sensitivity and excellent selectivity.However,electrochemical DNA sensors still face the challenges of low fixing efficiency of DNA probe and poor analytical sensitivity.In this paper,different DNA probe immobilization techniques are used to improve the assembly efficiency and reaction ability of the probe,and reduce the non-specific adsorption of the probe on the electrode surface.In addition,various nucleic acid signal amplification techniques are combined to improve the analytical performance of the detection system.Specific work contents are as follows:1.An electrochemical DNA sensor is constructed for the specific and sensitive detection of human T-lymphotropic virus type I DNA（HTLV-I DNA）based onλ-Exo assisted target recovery and template-free DNA extension mediated by terminal deoxynucleotide transferase（Td T）.In the process of theλ-Exo assisted target recovery,Hp-DNA hybridizes with HTLV-I DNA to form double strands DNA（ds DNA）.The ds DNA with a blunt 5’terminal phosphorylated is then digested byλ-Exo,releasing the HTLV-I DNA into the recycling and repeating the"hybridization-digestion"process to produce rich s DNA.On the electrode interface,the Au-S covalent bond is formed between CP-DNA and gold electrode（GE）through the 3’-terminal modified sulfhydryl group.CP-DNA captures a large amount of s DNA exported during theλ-Exo assisted target recovery to form CP-DNA/s DNA complexes.When Td T and d NTPs are presented on the electrode interface,the 3’-OH terminal of s DNA is extended to form long single stranded DNA（ss DNA）.Methylene blue（MB）is used as electrochemical active substance,which can be combined with both ss DNA and ds DNA to achieve significantly enhanced electrochemical signal.When the two signal amplification strategies are combined,the detection limit is as low as 0.069 f M（S/N=3）.The detection platform has good analytical performance and provides a potential method for the sensitive and specific detection of HTLV-I DNA.2.The ultrasensitive electrochemical analysis platform of p53 gene is proposed based on molecular beacon（cDNA）-mediated circular strand displacement（CSD）and terminal deoxynucleotide transferase（Td T）-mediated template-free DNA extension signal amplification strategy.First,cDNA modified with a 5’sulfhydryl group is assembled on the gold electrode（GE）surface by forming a strong specific Au-S bond with GE.Non-specific adsorption sites on the surface of cDNA/GE are blocked by mercaptohexanol（MCH）.After the addition of p53 gene into the detection system,p53 gene can specifically recognize the nucleotide sequences in the circular part of cDNA structure,and release the hybridization region of primer DNA.Then primer DNA hybridizes with the 3’-terminal of the cDNA.With the help of d NTPs and phi29 DNA polymerase,primer DNA extends in the direction of cDNA and hybridizes with it to form double strands DNA（ds DNA）,triggering the cDNA-mediated CSD process,while releasing the p53 gene captured on the electrode surface into the circulation to continue hybridizing with the unopened cDNA.Td T catalyzes the template-free DNA extension reaction at the 3’-OH terminal of cDNA.The resulting single stranded DNA（ss DNA）contains abundant guanine bases and provides a large number of adsorption sites for the electroactive probe MB.A significant characteristic response signal of MB is detected on the electrode surface.Therefore,the proposed electrochemical method can be used for ultrasensitive and specific analysis of p53 gene in complex substrates.3.An electrochemical DNA sensor based on triblock polyadenine probe（TPP）integrates Exonuclease Ⅲ（Exo Ⅲ）-assisted target recycling and hybridization chain reaction（HCR）for the ultrasensitive and rapid detection of carcinoembryonic antigen（CEA）.In the solution of Exo Ⅲ-assisted CEA recycling,CEA is introduced into the mixture of H1,H2,and Exo Ⅲ,and hybridizes with aptamer sequences in H1,resulting in a transformation of the conformation of H1 from hairpin structure to CEA@H1 complex structure.Exposed regions in the H1 structure can hybridize with the prominent 3’-terminal of H2 to form a blunt 3’-terminal.Exo Ⅲ catalyzes the gradual removal of the 3’-terminal mononucleotides of H2,resulting in the release of the CEA@H1 complex and the eventual production of output DNA sequences（output DNA,o DNA）.The poly A fragment of TPP is fixed on the interface of the gold electrode（GE）,and two flanking capture probes（PAP）at both ends acts with o DNA by specific hybridization on the electrode interface.Part of the sequences in o DNA acts as a catalyst between H3 and H4 to trigger HCR,with the purpose of generating double strands DNA（ds DNA）polymer.The polymer has a large steric hindrance effect,which inhibits electron transfer from the[Fe（CN）₆]^3-/4-probe to the electrode interface,resulting in a large electrochemical impedance signal(R_et).The recoveries of 87.0%-108%are obtained in the actual samples.The relative standard deviation（RSD）ranges from 1.2%to 9.2%.The designed electrochemical sensing strategy has high sensitivity and specificity for CEA detection.