RESUMEN
A lateral flow assay (LFA) strip based on dual 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB)-encoded satellite Fe3O4@Au (Mag@Au) SERS tags with nanogap is reported for ultrasensitive and simultaneous diagnosis of two SARS-CoV-2 functional proteins. Composed of Fe3O4 core, satellite gold shell with nanogaps, and double-layer DTNB, the Mag@Au nanoparticles with an average size of 238 nm were designed as multifunctional tags to efficiently enrich the target SARS-CoV-2 protein from complex samples, significantly enhancing the SERS signal of the LFA strip and provide quantitative SERS detection of analyte on test lines. The developed dual DTNB-encoded satellite Mag@Au-based LFA allowed simultaneous quantification of spike (S) protein and nucleocapsid (NP) protein with detection limits of 23 pg mL-1 and 2 pg mL-1, respectively, lower than commercial ELISA kits and reported SERS-LFA detection system-based Au NPs and Fe3O4@3 nm Au MNPs. This magnetic SERS-LFA also showed high performance of multi-variant strain detection and further distinguished clinical samples of Omicron variant infection, demonstrating the potential of in situ detection of respiratory virus diseases.
Asunto(s)
COVID-19 , Nanopartículas del Metal , Humanos , COVID-19/diagnóstico , Ácido Ditionitrobenzoico , Oro , SARS-CoV-2RESUMEN
SARS-CoV-2 is the culprit causing Coronavirus Disease 2019 (COVID-19). For the study of SARS-CoV-2 infection in a BSL-2 laboratory, a SARS-CoV-2 pseudovirus particle (SARS2pp) production and infection system was constructed by using a lentiviral vector bearing dual-reporter genes eGFP and firefly luciferase (Luc2) for easy observation and analysis. Comparison of SARS2pp different production conditions revealed that the pseudovirus titer could be greatly improved by: 1) removing the last 19 amino acids of the spike protein and replacing the signal peptide with the mouse Igk signal sequence; 2) expressing the spike protein using CMV promoter other than CAG (a hybrid promoter consisting of a CMV enhancer, beta-actin promoter, splice donor, and a beta-globin splice acceptor); 3) screening better optimized spike protein sequences for SARS2pp production; and 4) adding 1 % BSA in the SARS2pp production medium. For infection, this SARS2pp system showed a good linear relationship between MOI 2-0.0002 and then was successfully used to evaluate SARS-CoV-2 infection inhibitors including recombinant human ACE2 proteins and SARS-CoV-2 neutralizing antibodies. The kidney, liver and small intestine-derived cell lines were also found to show different susceptibility to SARSpp and SARS2pp. Given its robustness and good performance, it is believed that this pseudovirus particle production and infection system will greatly promote future research for SARS-CoV-2 entry mechanisms and inhibitors and can be easily applied to study new emerging SARS-CoV-2 variants.
Asunto(s)
Pruebas de Neutralización/métodos , SARS-CoV-2/fisiología , Internalización del Virus , Enzima Convertidora de Angiotensina 2/farmacología , Animales , Anticuerpos Neutralizantes/farmacología , Antivirales/farmacología , Línea Celular , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Lentivirus/genética , Luciferasas de Luciérnaga/genética , Luciferasas de Luciérnaga/metabolismo , Proteínas Recombinantes/farmacología , SARS-CoV-2/efectos de los fármacos , SARS-CoV-2/genética , SARS-CoV-2/inmunología , Glicoproteína de la Espiga del Coronavirus/genética , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virión , Internalización del Virus/efectos de los fármacosRESUMEN
A surface-enhanced Raman scattering (SERS) immunochromatographic assay (ICA) has been developed for rapid, ultrasensitive, and quantitative detection of rotavirus in feces using double Raman molecule-labeled Au-core Ag-shell nanoparticles. The Raman signals are generated by 5,5'-dithiobis-(2-nitrobenzoic acid) and the intensity of the characteristic peak at 1334-1 cm was detected as the analytical signal. The Raman signals were enhanced by the SERS-enhanced effect of both Au and Ag, the large amount of Raman molecules, and the hot-spot effect in the narrow gap between the Au core and Ag shell. The SERS ICA can quantitatively detect rotavirus in a concentration range of 8- 40,000 pg/mL, with detection limits of 80 pg/mL and 8 pg/mL based on naked eye observation and SERS signal detection, respectively. No cross-reaction was observed from other common pathogens. The standard deviation of the intra- and inter-batch repetitive tests is less than 10%, and the coincidence between SERS ICA and RT-qPCR as well as commercial colloidal gold ICA is 100%. The results indicated that this SERS ICA is able to quantitatively detect rotavirus in feces in 20 min with high sensitivity, selectivity, reproducibility, and accuracy and might be a promising method for the early detection of rotavirus in clinical analysis.
