A Soft Metal-polyphenol Capsule-based Electrochemical Immunoassay For Ultrasensitive Detection Of Epstein-barr (EB) Virus Antibody

Background and ObjectivesNasopharyngeal carcinoma（NPC）is a type of epithelial carcinoma originating in the mucosa of the nasopharynx and is particularly prevalent in East and Southeast Asia.The age-standardized incidence rate in China is reported to be 3 cases per 100,000population,with a 5-year overall survival rate of 61%.Epstein-Barr virus（EBV）has been proved to be the main pathogenic factor of NPC,so effective screening of EBV population plays an important role in early diagnosis of NPC.PCR analysis of plasma EBV DNA is considered to be an effective method for detecting EBV,but due to the lack of standardized detection methods,the results of the test vary significantly between laboratories.Recently,serological tests for anti-EBV antibodies have been developed as part of routine screening for NPCs.However,the traditional ELISA-based antibody detection methods have low sensitivity and specificity.Therefore,a highly sensitive,specific and stable anti-EBV antibody detection method is urgently needed.We propose an electrochemical immunoassay based on metal-polyphenol capsules（sMPC）for the ultra-sensitive detection of Epstein-Barr virus capsid antigen antibody（EBVCA-Ig A）,a biomarker of nasopharyngeal carcinoma.MethodsThe preparation of metal-polyphenol capsules（sMPC）is simple and the whole process can be completed in 30 minutes.The metal ions were mixed with tannic acid（TA）and immediately incubated with micron-sized calcium carbonate（Ca CO₃）microspheres.Due to the high affinity of polyphenols with metal ions,metal-polyphenol（MP）networks are formed and deposited on Ca CO₃ microspheres.Then adjust the p H of the solution to 5.0 to dissolve the template.The soft and hollow capsules were centrifugally collected and incubated with anti-human Ig A to form a metal probe for detection.When the sMPC form the classic sandwich structure on the electrode surface,HNO₃ is added to dissolve them,and the metal ions released can be used as signal molecules.Anodic stripping voltammetry（ASV）is one of the most widely used electrochemical techniques.Each sMPC contains a large number of metal ions,so the ASV can detect the amplified current signal,thus enabling the ultra-sensitive detection of tumor biomarkers.Results and conclusionThe results show that hollow and soft sMPC move freely on the electrode surface,so they have more opportunities to bind to the corresponding analyte.Compared with solid sMPC,the sMPC designed by our group has better analytical recognition efficiency.At the same time,the tiny SMPC contains a lot of metal ions,which help amplify the signal.On this basis,the sensitivity of the design strategy is greatly improved and allows detection of the sub-fetomolar concentration of the analyte.In the experiment,the ultra-low detection limit of EBVCA-Ig A antibody for EBV capsid antigen（EBVCA-Ig A）was 0.46 Fm,which could also be used for the analysis of actual samples.To our knowledge,this is the first sMPC-based electrochemical strategy report.This method has the advantages of convenient preparation,high sensitivity and good selectivity,and has a broad application prospect in the field diagnosis of virus-related diseases.In addition,the surface of sMPC can be easily modified by other recognition molecules such as DNA aptamers,peptides or antibodies,so this method can be easily extended to other analytes,offering great potential for detection of other biomarkers in clinical diagnosis.