Study On The DNA Electrochemical Biosensor Based On A DNA Tetrahedral Probe For The Detection Of Avian Influenza A(H7N9) Virus

In March2013, A novel reassortant avian influenza A(H7N9) virus,causing human respiratory infections firstly identified in China, has causedworldwide concern, and still cause sporadic infections now, making a severeimpact on economic and social development. The possibility that this viruscould obtain high transmissibility through gene mutation leading to an epidemiccannot be excluded in the future. Study on the adequate sensor detectiontechnology with a novel and unique probe for the on-site rapid identification ofA(H7N9) virus infected carriers, superior to the traditional diagnostic methods,which are labor-intensive laboratory procedures and time-consuming withexpensive, is crucial to prevent and control this disease.The DNA electrochemical biosensor, identifying nucleic acid sequence ofthe pathogenic microorganisms specificity, can detect and diagnose thepathogens at the gene level. Because of the advantages of rapid, sensitivity,cost-saving detection, promising portable and automatically, with the potentialof multiple detection of the pathogens in real time, The DNA electrochemical biosensor become the most promising application in the detection of thepathogens compared to other sensor detectors.The DNA tetrahedral structure is a three dimensional DNA nanostructuremolecule with a tetrahedral shape by self-assembly using four ssDNA sequencesas building blocks according to the Watson-Crick base pairs. DNA tetrahedralProbe (TEP) is a DNA tetrahedral structure with three thiol groups at the threevertices for anchoring its on the electrode surfaces by Au-S bonds, and with apendant probe DNA at the fourth vertex to recognize the target sequencespecifically. The advantages of the structure effect, surface effect and nanoscaleinterior pore space, promoting the biomolecular recognition between probe andtarget sequences, and enhancing the electron diffusion and transfer in electrodeface effectively, make the DNA electrochemical biosensor based on DNAtetrahedral probe (TEP biosensor) has a great application potential. Somebiosensors have been developed using the DNA tetrahedral probes to detectsynthetic ssDNA samples or other biomolecules with encouraging outcomes.However, to date, this method has not been applied to detect the clinical samplesof infectious pathogens. In order to rapidly detect the actual pathogens simply,sensitively and accurately, some challenges should be faced, such aspreprocessing the pathogen samples quickly, reducing the dependence on thePCR technique, or decreaseing the interference produced by background.A new DNA electrochemical biosensor based on the DNA tetrahedralprobe was developed to detect the avian influenza A(H7N9) virus at the genetic level with a simple, rapid, sensitive and specific tenet, for preventing andcontrolling the outbreak caused by this virus. The main work was concluded asfollows:1. Four ssDNA sequences were designed to self-assembly a DNAtetrahedral nanostructure by thermal denaturation, appended with a pendantssDNA probe to hybridize with a specific hemagglutinin (HA) nucleic acidfragment of avian influenza A(H7N9) virus, recommended by World HealthOrganization (WHO). The results, analyzed by polyacrylamide gelelectrophoresis (PAGE) with TotalLab software, demonstrated that the TEP wassynthesized successfully with yield as much as96%.2. The TEP was immobilized onto a gold electrode surface by Au-S bonds.The target sequence hybridized with a biotinylated-ssDNA detection probe wasthen captured to form a Sandwich model, and avidin-horseradish peroxides wereintroduced to produce an amperometric signal through the interaction with3,3,5,5tetramethylbenzidine (TMB) substrate. The manufacturing process ofthe TEP biosensor was characterized by cyclic voltammetry (CV) andelectrochemical impedance spectroscopy (EIS).3. In the detection of synthetic target DNA, the amperometric responsevalue of the TEP biosensor versus the logarithmic value of the targetconcentration presented two different linear relationships in differentconcentration ranges of synthetic target sequences and this phenomenon wasanalyzed by electrochemical version michaelis-menten equation. The TEP biosensor performed an excellent selectivity with the0.4pM of detection limitto synthetic target sequences. The TEP biosensor can discriminate the targetsequences from single-base, two-base, three-base mismatches or random controlDNA sequences, displaying a high selectivity to synthetic target DNA.4. The amperometric response signal of the TEP biosensor also became anexcellent linear relationship versus the logarithmic value of the target ssDNAsequences copied from clinical samples containing avian influenza A(H7N9)virus by asymmetric PCR with the detection limit of0.1pM. The biosensorcould specifically distinguish the avian influenza A(H7N9) virus from otherinfluenza A control viruses (avian influenza A(H1N1) virus and avian influenzaA(H1N1) virus). This demonstrated the significant selectivity and specificity ofthe TEP biosensor for the detection of the HA gene from influenza A(H7N9)virus.5. The TEP biosensor could distinguish the ssDNA products of only onecycle’s asymmetric PCR copied from clinical samples containing avianinfluenza A(H7N9) virus, with specificity for detection of the ssDNAasymmetric PCR products of the HA gene in three cycles, displaying a excellentsensitivity and specificity for the detection of asymmetric PCR ssDNA productsof the avian influenza A(H7N9) HA gene within limited cycles.