ABSTRACT
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 SARS-CoV-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 p-nitrophenyl 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. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra); condition optimization of ALP response (fluorescence intensity ratio change); mechanism investigation of ALP response (fluorescence lifetime decay curves and UV-vis absorption spectra); detection of N protein using commercial ELISA Kit; analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection; and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.
ABSTRACT
Coronavirus disease 2019 (COVID-19) highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission. Here, we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles (NPs) for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N protein). The synthesis of Si-FITC NPs did not need post-modification, which addressed the issue of quantum yield reduction during the coupling reaction. Si-FITC NPs showed two distinct peaks, Si fluorescence at λ em = 385 nm and FITC fluorescence at λ em = 490 nm. In the presence of KMnO4, Si fluorescence was decreased and FITC fluorescence was enhanced. Briefly, in the presence of N protein, catalase (CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates. Subsequently, hydrogen peroxide (H2O2) and Si-FITC NPs/KMnO4 were injected into the microplate together. The decomposition of H2O2 by CAT resulted in remaining of KMnO4, which changed the fluorescence intensity ratio of Si-FITC NPs. The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL, and the detection limit was 0.003 ng/mL, which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL. The N protein concentration can be accurately determined in human serum. Furthermore, the COVID-19 and non-COVID-19 patients were distinguishable by this method. Therefore, the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity. Electronic Supplementary Material: Supplementary material (characterization of Si-FITC NPs (FTIR, HRXPS); stability investigation of Si-FITC NPs (photostability, pH stability, anti-interference ability); stability investigation of free FITC (pH value, KMnO4); quenching mechanism of KMnO4 (UV-vis absorption spectra, fluorescence lifetime decay curves); reaction condition optimization of biotin-CAT with H2O2 (pH value, temperature, time); detection of N protein using commercial ELISA Kit; selectivity investigation of assays for SARS-CoV-2 N protein detection; determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-5005-z.
ABSTRACT
Coronavirus disease 2019 (COVID-19) highlights the importance of rapid and reliable diagnostic assays for the management of virus transmission. Here, we developed a one-pot hydrothermal method to prepare Si-FITC nanoparticles (NPs) for the fluorescent immunoassay of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleocapsid protein (N protein). The synthesis of Si-FITC NPs did not need post-modification, which addressed the issue of quantum yield reduction during the coupling reaction. Si-FITC NPs showed two distinct peaks, Si fluorescence at λem = 385 nm and FITC fluorescence at λem = 490 nm. In the presence of KMnO4, Si fluorescence was decreased and FITC fluorescence was enhanced. Briefly, in the presence of N protein, catalase (CAT)-linked secondary antibody/reporter antibody/N protein/capture antibody immunocomplexes were formed on microplates. Subsequently, hydrogen peroxide (H2O2) and Si-FITC NPs/KMnO4 were injected into the microplate together. The decomposition of H2O2 by CAT resulted in remaining of KMnO4, which changed the fluorescence intensity ratio of Si-FITC NPs. The fluorescence intensity ratio correlated significantly with the N protein concentration ranging from 0.02 to 50.00 ng/mL, and the detection limit was 0.003 ng/mL, which was more sensitive than the commercial ELISA kit with a detection limit of 0.057 ng/mL. The N protein concentration can be accurately determined in human serum. Furthermore, the COVID-19 and non-COVID-19 patients were distinguishable by this method. Therefore, the ratiometric fluorescent immunoassay can be used for SARS-CoV-2 infection diagnosis with a high sensitivity and selectivity. Electronic Supplementary Material Supplementary material (characterization of Si-FITC NPs (FTIR, HRXPS);stability investigation of Si-FITC NPs (photostability, pH stability, anti-interference ability);stability investigation of free FITC (pH value, KMnO4);quenching mechanism of KMnO4 (UV-vis absorption spectra, fluorescence lifetime decay curves);reaction condition optimization of biotin-CAT with H2O2 (pH value, temperature, time);detection of N protein using commercial ELISA Kit;selectivity investigation of assays for SARS-CoV-2 N protein detection;determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-5005-z.
ABSTRACT
The coronavirus disease-2019 (COVID-19) pandemic caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has seriously affected public health around the world. In-depth studies on the pathogenic mechanisms of SARS-CoV-2 is urgently necessary for pandemic prevention. However, most laboratory studies on SARS-CoV-2 have to be carried out in bio-safety level 3 (BSL-3) laboratories, greatly restricting the progress of relevant experiments. In this study, we used a bacterial artificial chromosome (BAC) method to assemble a SARS-CoV-2 replication and transcription system in Vero E6 cells without virion envelope formation, thus avoiding the risk of coronavirus exposure. Furthermore, an improved real-time quantitative reverse transcription PCR (RT-qPCR) approach was used to distinguish the replication of full-length replicon RNAs and transcription of subgenomic RNAs (sgRNAs). Using the SARS-CoV-2 replicon, we demonstrated that the nucleocapsid (N) protein of SARS-CoV-2 facilitates the transcription of sgRNAs in the discontinuous synthesis process. Moreover, two high-frequency mutants of N protein, R203K and S194L, can obviously enhance the transcription level of the replicon, hinting that these mutations likely allow SARS-CoV-2 to spread and reproduce more quickly. In addition, remdesivir and chloroquine, two well-known drugs demonstrated to be effective against coronavirus in previous studies, also inhibited the transcription of our replicon, indicating the potential applications of this system in antiviral drug discovery. Overall, we developed a bio-safe and valuable replicon system of SARS-CoV-2 that is useful to study the mechanisms of viral RNA synthesis and has potential in novel antiviral drug screening.
ABSTRACT
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 SARS-CoV-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 p-nitrophenyl 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. Electronic Supplementary Material Supplementary material (characterization of Si-FITC NPs (FTIR spectrum, XRD spectra, and synchronous fluorescence spectra);condition optimization of ALP response (fluorescence intensity ratio change);mechanism investigation of ALP response (fluorescence lifetime decay curves and UV—vis absorption spectra);detection of N protein using commercial ELISA Kit;analytical performance of assays for ALP detection or SARS-CoV-2 N protein detection;and determination results of SARS-CoV-2 N protein in human serum) is available in the online version of this article at 10.1007/s12274-022-4740-5.
ABSTRACT
The continuing global spread of Coronavirus Disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) infection, has led to an unprecedented global health crisis. Effective and affordable methods are needed to diagnose SARS-CoV-2 infection. In this work, a ratiometric fluorescence probe, Si-Mn:ZnSe nanoparticles, was constructed through the electrostatic interaction between Si dots and Mn:ZnSe QDs, and the fluorescence of Mn:ZnSe QDs has a specifical response to H2O2. An immunocomplex was formed by the recognition of capture antibody/spike (S) protein/spike neutralizing antibody/biotinylated second antibody/streptavidin/biotinylated catalase (CAT). In the presence of S protein, CAT effectively catalyzed the decomposition of H2O2 in the system, and the fluorescence of Mn:ZnSe QDs was not specifically quenched. Based on this principle, a ratiometric immunoassay of SARS-CoV-2 S protein was established. The sensitivity of the proposed ELISA method was comparable to that of the commercial kit. In addition, this method can effectively distinguish the pseudo-SARS-CoV-2 virus and other pseudovirus. Therefore, this method provided a reliable and potential direction for diagnosing SARS-CoV-2 infection.