A Novel Assay For Electrochemical Sensing Of EBV-related DNA Based On Silver Nanocomposites And Enzyme-assisted Target Recycling

Epstein-Barr virus(EBV)is a linear double-stranded DNA(dsDNA)virus that mainly latently infects B lymphocytes,and the population is generally susceptible.Diseases associated with EBV infection include infectious mononucleosis,Burkitt's lymphoma,Hodgkin's lymphoma,and nasopharyngeal cancer.Although serological testing is a common method for clinical diagnosis of EBV infection,it has low detection rate and high false positive results.Nucleic acid analysis owns the merits of high sensitivity and specificity.Clinical detection of viral nucleic acid can reflect viral infection and viral replication in time.Currently,fluorescence quantitative polymerase chain reaction(FQ-PCR)is the most used method for EBV nucleic acid detection.However,FQ-PCR still requires complex labeling,high-precision thermal cycling,and strict experimental conditions,limiting its popularization and application in primary-level medical units.Herein,based on the high conductivity and strong redox capacity of the silver nanocomposites and the signal amplification effect of the enzyme-assisted target cycle reaction,a simple and isothermal method for electrochemical sensor detection was designed for label-free and ultrasensitive detection of EBV related DNA.In this work,the conjugates of PAMAM dendrimers-encapsulated silver nanocomposites(AgDNCs),DNA-encapsulated silver nanoclusters(DNA/AgNCs)and probe DNA(pDNA)were used as dual template enhanced signal tags(pDNA-AgDNCs@DNA/AgNCs conjugates).The signal tags without labeling bio-enzymes could produce electrochemical signals directly with the robust electro-oxidation ability of silver.Based on the noncanonical properties of lambda exonuclease(? exo),a noncanonical ? exo-assisted target recycling(LNTR)system was designed without the requirements of phosphorylated DNA substrates.The LNTR reaction could be triggered specifically by target DNA,recycling target DNA and liberating plentiful single-stranded DNA(ssDNA)products.The ssDNA products were attached to the electrode surface and then bound with signal tags.The electrochemical signal enhanced significantly owing to the powerful electroconductivity of the silver nanocomposites and the cyclic amplification property of LNTR.The developed electrochemical biosensor could realize the label-free and ultrasensitive detection of target DNA in a linear range from 1fM to1 nM with a limit of detection of 0.38 fM.In addition,the method with excellent specificity was successfully applied to assay target DNA in complex bio-matrix.The established electrochemical biosensing strategy does not require complex labeling and thermal cycling processes.This method is simple and efficient,providing strong technical support for early diagnosis of EBV infection,and is expected to provide potential application tools for the prediction and screening of tumors and other major diseases,pathogen detection and biomedical research.