Ratiometric fluorescence immunoassay of SARS-CoV-2 nucleocapsid protein via Si-FITC nanoprobe-based inner filter effect

查看全文 作　　者：Guobin [1]Mao;Yang [3]Yang;Shijie [1,2]Cao;Silu [1]Ye;Yifang [1]Li;Wei [1]Zhao;Hongwei [2]An;Yingxia [3]Liu;Junbiao [1]Dai;Yingxin [1]Ma 机构地区：[1]CAS Key Laboratory of Quantitative Engineering Biology,Guangdong Provincial Key Laboratory of Synthetic Genomics and Shenzhen Key Laboratory of Synthetic Genomics,Shenzhen Institute of Synthetic Biology,Shenzhen Institutes of Advanced Technology,Chinese Academy of Sciences,Shenzhen 518055,China;[2]Guangxi University of Chinese Medicine,Nanning 530001,China;[3]Shenzhen Key Laboratory of Pathogen and Immunity,National Clinical Research Center for infectious disease,State Key Discipline of Infectious Disease,Shenzhen Third People’s Hospital,Second Hospital Affiliated to Southern University of Science and Technology,Shenzhen 518112,China 出　　处：《Nano Research》索引2023年第16卷第4期,共8页 基　　金：supported by the National Key Research and Development Program of China(No.2021YFA0910900);the National Natural Science Foundation(No.22104147);Youth Innovation Promotion Association CAS(No.2021359);the Natural Science Foundation of Guangdong(Nos.2018B030306046 and 2020A1515111130);Guangdong Provincial Key Laboratory of Synthetic Genomics(No.2019B030301006);Shenzhen Science and Technology Program(No.KQTD20180413181837372);Shenzhen Outstanding Talents Training Fund. 摘　　要：The global pandemic caused by severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)virus has necessitated rapid,easy-to-use,and accurate diagnostic methods to monitor the virus infection.Herein,a ratiometric fluorescence enzyme-linked immunosorbent assay(ELISA)was developed using Si-fluorescein isothiocyanate nanoparticles(FITC NPs)for detecting SARSCoV-2 nucleocapsid(N)protein.Si-FITC NPs were prepared by a one-pot hydrothermal method using 3-aminopropyl triethoxysilane(APTES)-FITC as the Si source.This method did not need post-modification and avoided the reduction in quantum yield and stability.The p-nitrophenyl(pNP)produced by the alkaline phosphatase(ALP)-mediated hydrolysis of pnitrophenyl phosphate(pNPP)could quench Si fluorescence in Si-FITC NPs via the inner filter effect.In ELISA,an immunocomplex was formed by the recognition of capture antibody/N protein/reporter antibody.ALP-linked secondary antibody bound to the reporter antibody and induced pNPP hydrolysis to specifically quench Si fluorescence in Si-FITC NPs.The change in fluorescence intensity ratio could be used for detecting N protein,with a wide linearity range(0.01-10.0 and 50-300 ng/mL)and low detection limit(0.002 ng/mL).The concentration of spiked SARS-CoV-2 N protein could be determined accurately in human serum.Moreover,this proposed method can accurately distinguish coronavirus disease 2019(COVID-19)and non-COVID-19 patient samples.Therefore,this simple,sensitive,and accurate method can be applied for the early diagnosis of SARS-CoV-2 virus infection. 关 键 词：Si-fluorescein isothiocyanate(FITC)nanoparticles ratiometric fluorescent probe severe acute respiratory syndrome coronavirus 2(SARS-CoV-2) inner filter effect enzyme-linked immunosorbent assay(ELISA)