Construction Of Immunomagnetic Beads SERS Sensor And Its Application In Detection Of Porcine Epidemic Diarrhoea Virus

Porcine epidemic diarrhea?PED?has the characteristics of rapid outbreak,rapid transmission and high mortality,which has a serious impact on the national pig industry.Therefore,the rapid and sensitive detection of its pathogenic virus-Porcine Epidemic Diarrhea Virus?PEDV?is of great significance for the prevention and control of PED.Surface Enhanced Raman Scattering?SERS?has the advantages of non-destructive,high sensitivity and fast detection,therefore has been widely used in many fields.However,the rapid identification and enrichment of targets in complex samples is still a challenge in the SERS application field.Immunomagnetic beads?IMBs?have good biocompatibility,which can be freely diffused in solution and easily separated by external magnets,achieving specific recognition and rapid separation of the target.This paper combined SERS technology with IMBs.By introducing biotin-streptavidin mode and rolling circle amplification mode,two SERS immunosensors were designed for rapid and sensitive detection of PEDV.The detailed contents and main results are described as follows:1. Construction of dandelion-type amplified SERS immunosensor based on silica-coated gold nanostar and magnetic separation and its application in PEDV detectionConstructed SERS-IMBs biosensors.The Raman signal molecule 4-mercaptobenzoic acid?4-MBA?was assembled to the surface of gold nanostars?Au NSs?through chemical bonds,then the Au NSs which attached to 4-MBA were wrapped with a silica shell and modified with biotin?Biotin?on the surface,constituted a biotinylated SERS probe with Si O₂@4-MBA@Au NSs structure.The monoclonal antibody was attached to the surface of magnetic beads through the covalent crosslinking of carboxyl groups and amino groups to form immunomagnetic beads?IMBs?and served as virus capture probes.The PEDV were bound to the immunomagnetic beads and the biotinylated polyclonal antibody,then used streptavidin?SA?as a bridging molecule to bind multiple SERS probes to form an immunodetection pattern which similar to dandelion structure centered on magnetic beads.The method for PDEV had a linear range from 5.3×10⁴copise/u L to 53 copise/u L.By the detection limit was 31.21 copies/u L and the recovery range from 94.34%to 115.13%.2. Construction of SERS immunosensor with magnetic separated and rolling circle amplification and its application in PEDVUsed rolling circle amplification?RCA?as a signal amplification method and combined SERS with immunomagnetic beads to construct a SERS-RCA-IMBs biosensor:the magnetic beads surface covalently modified monoclonal antibody as a capture probe to bind the PEDV,then boud to the biotinylated polyclonal antibody through immune recognition.Next used streptavidin as a bridging molecule to connect a single immunosandwich structure to multiple biotinylated primer DNA.Used circular DNA as a template,T4 DNA ligase,d NTP and phi29 polymerase were used to realize the RCA reaction,so that the primer DNA was extended to form a single-stranded DNA with multiple repeat fragments.The Au@4-MBA@Ag NPs SERS probe was constructed by using the signal-molecule embedded gold-core silver-shell nanoparticles as the signal probe,then modifyed the DNA complementary to the primer DNA repeat fragment on its surface.The amplified primer DNA was combined with a large number of SERS probes through the repeating structure to realize the amplification of the SERS signal.The linear range of PEDV detection by this method was 2.65×10⁴copise/u L?27 copies/u L,the detection limit was 7.48 copies/u L and the recovery range from 92.75%to 104.91%.