Study On Rapid Detection Method Of Brucellosis Based On Graphene Oxide Silver Nanoparticles Modified DTP-LPFG Sensor

Brucellosis,short for Brucellosis,is one of the most prevalent and harmful zoonoses in the world,threatening the health of people and more than 60 species of animals.About half a million cases are reported globally each year.In recent years,with the rapid development of domestic economy and animal husbandry,as well as the more frequent and wider trading range of cattle and sheep,brucellosis has become one of the most rapidly rising infectious diseases in China.At present,China’s national standard serological diagnostic techniques include: Tiger Red plate agglutination test(RBT),test tube agglutination test(SAT)and complement binding test(CFT).Among them,RBT has high sensitivity and is often used in the preliminary screening method,but its specificity is low.SAT has high specificity and is often used for diagnosis,but its sensitivity is low,and it is easily affected by human factors,and it takes a long time.The specificity of CFT is higher than that of SAT,and it is a reliable method for diagnosis.However,it is complicated,time-consuming and labor-consuming,so it is not easy to be widely used.World Organization for Animal Health(OIE)designated international trade standard tests include RBT,CFT,indirect enzyme linked immunosorbent assay(IELISA),competitive enzyme linked immunosorbent assay(CELISA),fluorescence polarization assay(FPA),etc.IELISA is a high-throughput rapid detection method,its sensitivity is higher than RBT,and simple and easy to operate,the disadvantage is that the specificity is low,can not exclude the cross reaction,so it is often used in the primary screening method;The specificity of CELISA is higher than that of IELISA,which can effectively eliminate cross reaction,and can distinguish field virus infection and vaccine immunity to a certain extent.FPA is still in the evaluation stage in China.Therefore,it is of great significance to explore a quick,simple and sensitive detection technology for the prevention and control of brucellosis.Fiber Bragg Bragg Grating biosensors are widely used in biochemical detection because they are pollution-free,fast and real-time,portable,small in size and low in cost.However,in the field of fiber Bragg grating sensing,the greatest challenge is the lack of sensitivity for the application of biological small molecules and low concentration analytes.To solve this problem,researchers at home and abroad improve the sensing performance of fiber biosensors by using cladding etching,side casting and fiber taper.But these methods destroy the integrity of fiber structure.The Dispersion Turning Point long-period Fiber Grating(DTP-LPFG)can provide high sensitivity response to the external environment without damaging the Fiber structure.DTP-LPFG is coated with a high refractive index nanofilm and the cladding mode is located in the mode conversion region,which can further improve the sensitivity of DTP-LPFG to the environmental refractive index.Meanwhile,the range of DTP-LPFG refractive index sensing can be extended to solve the limitation that ordinary LPFG is sensitive only when the environmental refractive index is slightly lower than the cladding refractive index.In the early stage,our laboratory has successfully applied DTP-LPFG biosensor to detect avian influenza virus(AIV),and achieved relatively ideal research results,and published high-level research papers.In this paper,the DTP-LPFG biosensor developed earlier was further improved to improve its sensitivity,stability and specificity,and applied to the detection of brucellosis antibody.In this study,Brucella specific outer membrane protein BP26 was prepared by Escherichia coli expression system.The recombinant BP26 protein was immunized BALB/c mice,and the monoclonal antibody against BP26 was prepared by PEG1500 cell fusion technology,providing an effective reagent for the detection of Brucella.The DTP-LPFG biosensor detection platform was built,and the surface of DTP-LPFG was hydroxylated.Then,APTES was used to silanize the fiber surface.Continue to use EGO-AgNPs coating on the optical fiber surface;The carboxyl group on the surface of graphene oxide(EGO)was activated by EDC/NHS activator.Then,the recombinant Bp26 antigen was immobilized on the sensor surface by an amide reaction.By monitoring the resonance wavelength shift of EGO-AGNPs modified DTP-LPFG in antibody samples containing different concentrations,the antibody samples of brucellitis were detected in vitro.Finally,the detection results of the sensor were evaluated,including sensitivity,specificity and clinical performance.From the analysis of the detection results,it was concluded that the DTP-LPFG sensor antibody detection platform for brucellosis was established in this study,which could be used for the detection of clinical samples,and provided a new method for the diagnosis of brucellosis.At present,brucellosis vaccine is not widely used as a planned immunization vaccine.So the detection of serological antibody of brucellosis is of great significance in the diagnosis of brucellosis.In this paper,"EGO-AGNPs" was used to fix the highly specific Brucella-BP26 on the surface of DTP-LPFG,and the specific binding of Brucella antibody could greatly improve the sensitivity,stability and specificity of the immunosensor.The research contents of this paper are as follows:1.Construction of recombinant plasmid according to the gene sequence of BP26(accession number: U45996)published on the website of Gen Bank.The recombinant plasmid p ET30a(+)-BP26 was constructed by chemically synthesizing the whole BP26 gene and linking it with the vector p ET30a(+).Recombinant plasmid transformation to competent escherichia coli cells BL21(DE3)to express,respectively from the induction time,temperature and induced the inducer IPTG concentration select BP26 recombinant protein best induced condition,and a large number of expression,using ihs nickel-the tag column purification BP26 recombinant protein,purified BP26 recombinant protein by sds-page gel electrophoresis purity identification,with quinoline formic acid(BCA)concentration in the identification of kits,Western blot to identify the specificity.2.The above preparation BP26 recombinant protein as immunogen,using the method of routine immunization after four immune BALB/c mice to determine the serum titer,to fulfill the requirements of fusion sterile separation after spleen cells,and cell fusion with myeloma cells using PEG1500,to establish an indirect ELISA screening method,qing antibody screening holes of cell fusion,will be merged to twice the positive hybridoma clones,get stable can secrete antibody monoclonal hybridoma cell lines.After expanded culture,mouse ascites monoclonal antibody was prepared,antibody subtypes were detected by Sigma kit,and ascites was purified by Protein A column.Finally,biological characteristics of the purified antibodies were identified,including concentration determination by BCA method,purity identification by SDS-PAGE,titer determination by ELISA,and specificity identification by Western-blot.3.By designing and changing the period length of LPFG to make its phase matching curve near the dispersion inflection point,the ordinary LPFG can be changed into LPFG with dispersion inflection point.The gate area was soaked with 5% dilute NHO3,and then cleaned with deionized water(RO water)and anhydrous ethanol.By combining the EGO-AgNPs with electrostatic bonding way,using Na OH hydroxylation was carried out on the fiber surface exposed fiber surface carboxyl groups,using silane coupling agent(APTES)throμgh covalent bond combination of silicon alkylation was carried out on the fiber surface,after the graphene oxide coated silver nanoparticles(EGO-AgNPs)composites modified in fiber surface,with 1-ethyl-(3-dimethyl amino propyl)carbonyl two imine(EDC)and N-hydroxy succinimide and activated surface carboxyl(NHS).BP26 antigen throμgh covalent bond combination of fixed in the fiber surface,with 5% skimmed milk powder to closed of optical fiber,then BP26 single resistance of different concentration detection sensitivity,with bovine serum albumin(BSA)and bovine serum as the avian influenza virus(AIV)healthy control,is used to detect the specificity,finally to clinical serum samples for testing.After the detection,the sensitivity,specificity and clinical characteristics of the sensor were evaluated.Results:1.The recombinant plasmid by Nde Ⅰ and Xhol Ⅰ double enzyme after appraisal,at 698 bp has obvious stripe,conforms to the sequencing results expected results show that recombinant plasmid build is successful.The recombinant plasmid p ET30a(+)-BP26 was successfully transformed into E.coli competent cell BL21(DE3),and the recombinant genetic engineering bacterium p ET30a(+)-BP26 /BL21 was constructed.Select BP26 recombinant protein best induced conditions: temperature 30 ℃,induction time 8 h,1mmol/L IPTG concentration,using the best induction conditions of a large number of protein expression purpose,and use the Ni column affinity chromatography purification,purification of BP26 recombinant protein reached the electrophoresis purity pure,BCA method measured concentration of 1.430 mg/m L,Western blot appraisal result shows significant at 27.8 Kd protein imprinting,consistent with the expected purpose stripe.2.The serum antibody titer of mice was detected after four immunizations,and cell fusion could be carried out when the titer reached more than 1:10000.Splenic cells of mice were collected and fused with myeloma cells.Five 96-well plates were divided into 480 Wells.Cell status was observed and counted under a microscope 7 days after fusion.The number of successful fusion cells was 403,the fusion rate was 83.9%,and the number of positive cells was 372,the positive rate was 77.5%.Four monoclonal hybridioma cell lines(B12,C12,C1,and A12)that could secrete antibodies were obtained after two times of subclonal and ELISA screening.The results of monoclonal antibody subtype identification showed that B12 and C1 were Ig G2 a,A12 was Ig G2 b,and C12 was Ig G1.Two cell lines B12 and A12 were selected for the preparation of ascites antibody after expanded culture,and the ascites antibody was purified by Proteina column.SDS-PAGE identified that the purity of Ig G antibody reached over 90%,and found that there were bands at 25 KD and55KD,respectively,without heterozygophylly,which was consistent with the light chain and heavy chain and size of Ig G antibody,indicating that the purification effect was good.After purification,the titers of B12 and A12 reached more than 1:128000,and the highest antibody concentration could reach 1.5367mg/m L by BCA method.Western-blot results showed that the prepared monoclonal antibody was specific to Bp26 antigen.3.A DTP-LPFG detection platform was established and a brucellosis antibody detection method was established.After hydroxylation,silanization,modification of EGO-AgNPs composite,activation,modification of brucelliasis antigen and blocking with skimmed milk powder,DTP-LPFG was detected,and the relative changes of bimodal spacing from bare fiber to coated EGO-AgNPs composite and to closed resonance were4.995 nm and 9.02 nm.The changes of each step relative to bare fiber were 1.3725 nm,4.995 nm,12.06 nm,13.6575 nm,13.905 nm.The lower limit of the biosensor for the detection of BP26 antibody is 100pg/m L,and the detection range is 100pg/m L-10 μg/m L.The specificity of the sensor was verified by detecting BSA,AIV virus and healthy bovine serum.The results showed that BSA and healthy bovine serum did not react specifically with the BP26 antigen on the sensor,indicating that the specificity was good.The results showed that the resonant wavelengths of several clinical samples changed obviously,and the sensors gradually approached saturation,which indicated that the sensors had good clinical performance and could be used for clinical detection.Conclusion:In this study,the genetically engineered strain p ET30a(+)-BP26 /BL21(DE3)was successfully constructed,and the Brucella specific BP26 protein was expressed efficiently in the Escherichia coli expression system.After long-term immunization,cell fusion and subcloning,4 cell lines were obtained which could secrete BP26 monoclonal antibodies stably.Using the BP26 antigen and antibody developed,a rapid detection method of brucelliae antibody based on EGO-AgNPs modified DTP-LPFG biosensor was established.The sensitivity,specificity and detection of the method in clinical samples were evaluated.The results showed that this method has the advantages of high sensitivity,specificity and no labeling,and can be used for the detection of brucellosis antibodies in clinical samples,which provides a new detection technology and means for the diagnosis of brucellosis.