| Avian influenza viruses(AIV)have the characteristics of short incubation period,strong infectivity,and rapid transmission.They have high clinical morbidity and mortality,and may cause major epidemics,it poses a huge threat to public health and the poultry farming industry.Threatened.Therefore,the development of a rapid,highly sensitive,specific,in-situ,non-destructive,and real-time biosensor detection method is essential to control the early spread of avian influenza virus(AIV).Surface-enhanced Raman scattering(SERS)has excellent resolution,ultra-high sensitivity,the ability to amplify signals,and can quickly and non-destructively detect samples.It can also provide molecular structure vibration fingerprint information so that SERS is widely used in many fields.This research was carried out on the basis of screening avian influenza virus SERS enhancement substrate.By fixing the avian influenza virus,the SERS detection method after the virus fixation was established,and the immunomagnetic beads with good magnetic response were introduced.Finally,the SERS specific detection method for the avian influenza virus was developed.The new method provides a new technical means for the detection of avian influenza virus.The research content and results of this study are as follows:1.Screening of SERS enhancers and establishment of avian influenza virus SERS detection methodsGold nanostars(Au NSs),gold nanocages(Au NCs),and gold nanoshells(Au NShs)were used as SERS enhancement substrates for SERS detection of H3N2subtype avian influenza virus,and the best enhancer was determined to be Au NSs.In the range of 500-2000 cm-1,the H3N2 subtype avian influenza virus has strong and symmetrical Raman peaks at 1052 cm-1,1456 cm-1,and 1662 cm-1.The conditions were optimized for the SERS method for detecting avian influenza virus based on Au NSs.The optimal conditions for obtaining the strongest virus Raman signal are:virus preparation buffer is 10 m M PBS,the incubation time of Au NSs and virus is 15min,and Au NSs and virus are incubated in a volume of 1:1.The method constructed in this study can complete the rapid detection of the virus within 35 minutes,and the peak shape and peak intensity of the 10 batches of samples prepared at the same time are uniform and stable.The Raman signal can still be detected when the original virus solution is diluted to 108 times.2.Research on the establishment of SERS detection method for avian influenza virus after chemical fixation based on Au NSsDue to the harm of virus and microorganisms,based on Au NSs as a basis for biological safety considerations,we compared the Raman spectrum peaks of H3N2subtype avian influenza virus after a variety of common chemical fixatives.After PCA analysis,the most suitable fixative for SERS detection was selected as formaldehyde.Next,the Raman spectra of formaldehyde on three different subtypes of avian influenza viruses,H1N1,H3N2,and H5N1,were studied.PCA analysis was used to identify and distinguish the three different subtypes of avian influenza viruses based on Au NSs.The method constructed in this study can complete the rapid detection of the fixed virus within 35 minutes.At the same time,the SERS detection method of avian influenza virus after chemical fixation based on Au NSs is evaluated.The SERS spectra of the three strains have good reproducibility.Raman signal can be detected when the virus solution is diluted to 108 times.3.Research on the establishment of SERS immunomagnetic beads for detection of avian influenza virus based on Ag NPsWe established IMBM-HA@Ag NPs"primary antibody sandwich"and IMBM-HAP@Ag NPs"double antibody sandwich"two structures in situ reduction to detect H5N1 subtype avian influenza virus.Using immunomagnetic beads separation techniques(IMBS),the H5N1 subtype avian influenza virus HA monoclonal antibody is coupled with streptavidin magnetic beads to prepare immunomagnetic beads.IMBMcaptures antigens to form IMBM-HA,and binds HA polyclonal antibody forms a"double-antibody sandwich"complex IMBM-HAP.And modified Ag NPs on its surface,and coated on the surface of IMBM-HA and IMBM-HAP to form IMBM-HA@Ag NPs and IMBM-HAP@Ag NPs.By comparing the Raman spectra of the two structures,it is found that the Raman intensity of the latter at 1053 cm-1 is nearly 7 times that of the former.The structure of the latter was optimized.0.2 mg of streptavidin magnetic beads coupled with 6.8?g monoclonal antibody can make the200?L H5N1 avian influenza virus enrichment efficiency of 5.99×103 copies/?L reach 53.75%,coupled with 20?g of polyclonal antibody at the same time,can increase the Raman intensity of the sample at 1053 cm-1 by 2 times;when the Ag NO3and Na BH4 concentrations are both 10 m M and incubated for 10 min and 5 min,respectively,it is at 1053 cm-1 The Raman intensity is up to 1232 a.u.Compared with traditional Raman detection,the method constructed in this study has strong specificity,good reproducibility,and higher sensitivity by introducing immunoma-gnetic beads.The dilution factor of the virus solution is up to 109 times,and the detection can be completed within 1 h.Rapid virus detection.This test is based on the micromagnetic particle capture system combined with SERS technology.The established SERS immunoassay method for detecting avian influenza virus can achieve in-situ,non-destructive,high-efficiency,specific,high-sensitivity,rapid,and low-concentration detection level,which solves SERS The problem of its own specificity provides a new detection method for avian influenza virus.As a new analysis method,SESR technology is expected to become a low-cost,high-efficiency,sensitive and in-situ detection of pathogenic microorganisms. |
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