A Nanoenzyme Linked Immunochromatographic Sensor For Rapid And Quantitative Detection Of SARS-CoV-2 Nucleocapsid Protein In Human Blood

Background and purpose2019 coronavirus disease(COVID-19),a severe acute respiratory infectious disease that endangers human health and social development,is caused by the novel coronavirus(SARS-CoV-2),with strong infectivity,long latency,and general vulnerability of the population.Its clinical symptoms were mainly fever,dry cough,and weakness.If it was serious,it would be accompanied by acute respiratory distress syndrome.Virus nucleic acid testing(NAT)is the "gold standard " for the diagnosis of COVID-19,but it is time-consuming,demanding for sample preservation,testing equipment,laboratory requirements,professional operators,and prone to false negatives during the actual detection.Therefore,a rapid,easy,sensitive and reliable detection method is urgently needed to diagnose the infection of SARS-CoV-2 early and control the spread of the outbreak in a timely manner.In this study,by preparing monoclonal antibodies against the nucleocapsid protein(NP)of SARS-CoV-2 and platinum coated gold nanoparticles(Au@PtNPs),a smartphone based nanoenzyme linked immunochromatographic sensor(NLICS)was developed for rapid,sensitive,convenient and quantitative detection of SARS-CoV-2 NP antigen in human blood.MethodsNP specific monoclonal antibodies were screened and prepared by traditional monoclonal hybridoma cell preparation technology and indirect ELISA.The paired antibodies of NP were screened by double antibody sandwich ELISA.Au@PtNPs was prepared by reduction method,and the detection antibody in the paired antibody was labeled with Au@PtNPs,and then sprayed on the conjugate pad;The coated antibody in the paired antibody was fixed on the detection line(T line)of NC membrane with a scriber.The sample pad,conjugate pad,NC membrane and absorbent paper were assembled in a special U-shaped groove in order to form an immunochromatographic device of nanoenzyme linked immunochromatographic sensor,namely U-shaped ICA.The U-shaped ICA was assembled with reading device and photometer,and connected with smart phone through Bluetooth to development nanoenzyme linked immunosensor based on smart phone.The sensor was applied to the detection of 21 clinical coronavirus positive samples and the screening of plasma samples from 80 healthy blood donors,and compared with the commercial antigen detection kit to evaluate the detection performance of NLICS,so as to expect to apply it to the detection and screening of coronavirus antigen in large-scale population.Results1.Previously,five NP monoclonal antibodies with high titer and specificity were prepared,and a pair of paired antibodies with strong sensitivity and specificity were screened,including coated antibody 4G11 and detection antibody 5E4.2.With the detection of NP standards,the detection linear range of NLICS was 0.05-1.6ng/mL,and the limit of detection was 0.026ng/mL,which was better than the traditional immunochromatographic detection card(ICA)(the limit of detection was 0.1ng/mL).Intra assay and inter assay variations of NLICS were less than 10%and 15%,respectively.The sensor has high reliability,stability and repeatability,and the detection report can be obtained in only 25 minutes.3.In 21 cases of novel coronavirus pneumonia positive serum samples,16 cases were positive and 5 cases were negative for NLICS,with a sensitivity of 76.2%and accuracy of 95.1%.The commercial antigen ELISA Kit detected 10 positive cases and 11 negative cases,indicating a sensitivity of 47.6%and an accuracy of 89.1%.Of the blood samples tested by nlics and ELISA,80%were negative.The performance of NLICS was better than that of commercial ELISA kit.In the negative plasma samples of 80 healthy blood donors,the results of both methods were negative,which meant that the specific of NLICS and ELISA were 100%.ConclusionThe smartphone based nanoenzyme linked immunochromatographic sensor achieves rapid and quantitative detection of SARS-CoV-2 NP with high sensitivity,accuracy and stability.As a complement and innovation to the COVID-19 diagnostic method,NLICS is a point of care testing(POCT)with reliable,simple,sensitive and low-cost,and suitable for the screening of suspected SARS-CoV-2 infection and the dynamic monitoring of SARS-CoV-2 NP content in patients.Innovation1.Monoclonal antibodies against the nucleocapsid protein of novel coronavirus with high sensitivity and specificity were prepared and screened,which were 4G11 antibody strain and 5E4 antibody strain respectively.2.The developed NLICS combined with smart phones can specifically detect sars-cov-2 NP antigen in human blood,which takes a short time and can provide quantitative detection results.Its research results have been published on sensors and actuators:B.chemical(2021).