Study On Rapid And Highly Sensitive Quantitative Detection Technology Of Respiratory Viruses Based On Magnetic SERS Immunochromatography

Objective:Respiratory viruses generally induced similar clinical symptoms in early infection.Early accurate pathogen diagnosis is of great significance for early treatment and epidemic prevention and control.Surface-enhanced Raman scattering-based lateral flow immunoassay（SERS LFA）combines the advantages of SERS（high sensitivity and multi-channel detection）and immunochromatography（fast and simple）.And SERS LFA greatly improves the detection sensitivity and quantitative detection ability of immunochromatography,which has been widely applied to the detection of nucleic acid,protein,bacteria,virus and et al.However,the complex composition of clinical respiratory samples and little volume of adding sample,may cause non-specific adsorption and false negative results.Herein,the high-performance and monodisperse magnetic SERS substrates were synthesized in this study.Antibodies that can specifically identify respiratory viruses were modified on the SERS substrates to prepare magnetic SERS immune tags.Magnetic SERS tags can specifically capture and enrich the target viruses from the complex sample.The formed tags-virus immunocomplex will be remained on the test line（T line）of the multi-channel strip by combining with the detection antibody pre-coated on the strip.Rapid and highly sensitive quantitative detection of respiratory viruses in the biological samples could be realized by collecting and analyzing the SERS signals on the T line.Methods:1.The Fe₃O₄@Au Magnetic nanoparticles（Mag NPs）were synthesized by polyethyleneimine（PEI）-mediated seed growth method.First,a layer of positively charged PEI molecules was adsorbed on the surface of Fe₃O₄ particles under sonicating.Then small diameter of colloidal Au NPs（negatively charged）were adsorbed by electrostatic interaction,and the formed Au seeds provided nucleation sites for the subsequent synthesis of Au shells.The morphologic characteristics and properties of Fe₃O₄@Au Mag NPs were characterized by HR-TEM,SEM,Zeta potential,and UV-vis hysteresis curves.And the SERS properties of the as-prepared magnetic SERS probes were evaluated,including the stability of p H and time,and the reproductivity.2.Double-layer Raman molecule（DTNB）-modified Fe₃O₄@Au Mag NPs was used as magnetic SERS substrates.The specific capture antibodies of influenza A（H1N1）virus,severe acute respiratory syndrome coronavirus 2（SARS-Co V-2）and respiratory syncytial virus（RSV）were conjugated on the SERS substrates to prepare magnetic SERS tags.The antibodies sprayed on the top-to-bottom NC membrane were goat anti-mouse Ig G,H1N1,SARS-Co V-2,and RSV sequentially by the Biodot XYZ3050 plotter,namely,C line,T₁,T₂ and T₃.Three parallel T lines were used to simultaneously and rapid diagnose three respiratory viruses on one strip.By analyzing the SERS signals on three T lines,the quantitative detection of H1N1,SARS-Co V-2,and RSV were achieved.3.Firstly,two kinds of Raman reporter molecules（DTNB and 4-MBA）were modified on Fe₃O₄@30Au Mag NPs.And the specific capture antibodies of SARS-Co V-2 and H1N1 were conjugated on Fe₃O₄@30Au/DTNB and Fe₃O₄@30Au/MBA,respectively,to obtain two kinds of magnetic SERS tags with different characteristic Raman shifts.The mixed detection antibodies of SARS-Co V-2 and H1N1 were sprayed on the T line of the NC membrane.While SARS-Co V-2 and H1N1 both existed in the sample,the two kinds of magnetic SERS tags will be combined on the T line at the same time.The SERS signals of the T line were then collected by the line spot Raman detector developed in this study,with～3 mm width of the line spot.Quantitative detection of SARS-Co V-2 and H1N1 viruses can be completed by analyzing the SERS intensities at characteristic shifts(1330 and 1584 cm^-1).Results:1.The spherical,homogeneous,and monodispersed Fe₃O₄@Au Mag NPs were synthesized successfully by PEI-mediated seed growth method.The as-prepared magnetic SERS probes based on Fe₃O₄@Au Mag NPs showed relatively stable SERS signal intensities in the different p H（p H 6-14）of water solutions.In addition,the stability of time of magnetic SERS probes stored in the ethanol were evaluated,and showed strong SERS signal within 90 days,1.8%～10%lower than the freshly prepared.2.Simultaneous,highly sensitive and quantitative detection of three kinds of respiratory viruses was realized based on multi-channel magnetic SERS LFA.The limit detection limits（LOD）of H1N1 inactive viruses,SARS-Co V-2 spike proteins and RSV antigen were calculated to be 85 copies/m L,8 pg/m L and 8 pg/m L,respectively,which are 100 times higher than that of colloidal Au LFA and 5～500 times higher than commercial ELISA.In addition,the detection properties of the proposed method for target viruses in throat swab samples were verified.3.The dual-channel LFA realized the simultaneous detection of two respiratory viruses on one T-line via two kinds of magnetic SERS tags.And the SERS signals of the whole T line were collected by the line spot Raman detector.The detection process of SARS-Co V-2 and H1N1 inactivated viruses can be finished within 26 min via the bi-channel strips,with LODs of 870 and 263 copies/m L for SARS-Co V-2 and H1N1,respectively.In addition,the repeatability and specificity of this method were also confirmed,exhibiting high sensitivity,strong specificity and strong stability in simulated clinical samples.Conclusions:The multi-channel magnetic SERS LFA established in this study can enrich and capture the target viruses from the complicated samples without pretreatment via the magnetic SERS tags,and achieved highly sensitive,quantitation and simultaneous detection of multiple respiratory viruses.In addition,a new portable line spot Raman detector was specifically developed for SERS signal collection on the immune chromatographic strips in this study,and can realize multiple detection by single laser source,greatly improving the detection efficiency.Notably,the line spot produced by the line spot Raman detector could completely cover the whole width of the strip,greatly shortening the signal collection time and reducing the measurement error.The magnetic SERS LFA established in this study showed great detection results in simulated throat swab samples,revealing its great potential in the early and rapid differential diagnosis of respiratory viruses,which could be used for large-scale screening of clinical samples.