Construction Of An Allosteric Electrochemical Biosensor Based On Aatalytic Hairpin Assembly And Its Application In Strawberry Mottle Virus Detection

Strawberry mottle virus(SMo V)is a common virus in strawberries.The diseases caused by SMo V often occur in virus-free production and field cultivation,leading to a severe decline in strawberry yield and quality.The detection of SMo V has a prevention and monitoring effect on strawberry virus-free production and cultivation,which helps increase the economic value of strawberries.Traditional SMo V detection methods mainly include biological detection and electron microscope observation,which have a long detection cycle and poor accuracy.In recent years,RT-q PCR technology,the “gold standard” of molecular detection,has been widely used to detect strawberry virus with highly sensitivity.However,it still relies on large instruments,technical personnel and high cost.Electrochemical biosensors have the advantages of fast response,high sensitivity and specificity,which can meet the needs of rapid detection,combined with nucleic acid amplification strategy to realize signal amplification as well as ultra-sensitive detection.Catalyzed hairpin assembly(CHA),as a non-enzymatic nucleic acid amplification technique,has the characteristics of simple reaction conditions,exponential amplification and strong compatibility.It is an effective strategy to convert nucleic acid signals to electrochemical signals.In summary,this thesis constructs an allosteric electrochemical biosensor based on CHA.Through the programmed CHA design,the AP site is allosteric in the single and double chain structure,which achieve the efficient series effect of signal recognition and amplification,and realize the accurate and sensitive detection of SMo V.The main contents and results are as follows:1.The CHA-driven AP site allosteric sensing strategy was designed and verified.Since the APE1 enzyme is a double-stranded AP site-specific endonuclease,this section designed a single-stranded H1 hairpin structure embedded with the AP site.The nucleic acid targets were used to trigger CHA signal amplification to complete the allosteric of the AP site from single strand to double strand and realize the signal output,which is mediated by cleavage of AP site by APE1 enzyme.The results of polyacrylamide gel electrophoresis showed that CHA amplification was triggered when the target was present,and the allosteric products could be specifically recognized and cleaved by APE1 enzyme,which proved that the APE1 recognition series system caused by CHA allosterism was feasible.2.An AP site allosteric electrochemical biosensor based on CHA was constructed.Through the interface modification of the molecular hairpin,the target was detected by the biosensing strategy in the previous section.Firstly,the interface assembly was verified by Cyclic Voltammetry and Electrochemical Impedance Spectroscopy,indicating that the electrochemical sensing interface was successfully constructed.Differential Pulse Voltammetry(DPV)detection of the targets showed that the detection signal-to-noise ratio could reach more than three times.Under the optimal experimental conditions,the detection performance of the electrochemical biosensor was analyzed.The sensitive detection of SMo V can be realized in the range of 1 f M～10 n M,and the limit of detection(LOD)is0.6 f M,with good specificity and reproducibility.Moreover,It can be stably stored for 21 days at 4℃.3.The detection of SMo V in practical samples was realized by allosteric electrochemical biosensor.The total RNA extracted from strawberry leaves infected by strawberry mottle virus was used to synthesize c DNA.The detection results of c DNA at different concentrations showed that the LOD of the electrochemical biosensor was as low as 1.6 f M,which was higher than the traditional detection methods.This method can realize the accurate detection of SMo V and provide technical support for rapidly detecting of strawberry virus diseases.