Application Of Immuno-magnetic Nanoparticle In Rapid Detection For Food Safety

In recent years,immuno-magnetic nanoparticles(IMNPs),with its unique advantages,had been widely used in the food safety field for sample pretreatment before detection and being as indicator in immunoassay.In this paper,based on the unique magnetic and optical properties of magnetic nanoparticles(MNPs),a variety of new types of immunochromatographic assay with immune magnetic nanoparticles as probe were developed,mainly including two step immunochromatography,competitive immunochromatography for determination of macro-molecular targets and sandwich assay with enrichment from big volume sample.The methods above were applied in food-safety-related target detection.Compared with the conventional methods,all the methods we developed exhibited many advantages.In the first chapter of this study,MNPs,immunochromatographic assay,and their current applications in the field of food safety were reviewed.In the second chapter,poly-clonal antibodies against Salmonella were prepared by injecting Salmonella antigen to rabbits.The shortest antibody preparation period was only 25 d.The estimated Salmonella antibody production in one rabbit was more than 183.7 mg,and the biggest antibody production was up to 466.7 mg.The titer of antiserum from Q1 rabbit was as high as 12,800,000.All the five antibodies were capable to recognize representative Salmonella strains from A,B,C1,D,E1 group.It revealed that all the antibodies had strong potential advantages in both immunological detection and immune sample pretreatment.In the third chapter,the outer membrane protein extracts and other Salmonella immunogen mixture were used as injected immunogen for BALB/c mice.After a series of steps,44 strains of anti-Salmonella monoclonal cell line were prepared.Among the cell lines,there were 16 strains cells that could produce antibody with high specificity(no cross reaction with other Salmonella serotypes).Antibody produced by most of the rest of the 44 cell lines could combine typical Salmonella strains belonging to four different groups,except Salmonella choleraesuis.The high specific antibodies could be applied to rapid detection for some specific serotypes and even to Salmonella serotypes identification as there was no cross reactions.And some strains of antibodies could be applied together for rapid screening of Salmonella genus.In chapter 4,an immunochromatographic sandwich assay with effective sample enrichment was developed based on IMNPs.In the assay,IMNPs was used as the tool for samples enrichment processing and bio-markers of immunochromatography.By optimizing the pH in coupling procedure,blocking agents,the largest amount on NC membrane,the IMNPs was used as a probe.Compared with conventional way,probes after capture procedure can be directly tested in immunochromatography without any traditional elution steps,and the test results can be read by naked eye.For detection of Salmonella enteritidis,the sensitivity of the method was 1.95×105 CFU/mL.Compared with conventional IMNPs based immunochromatography(no enrichment)and colloidal gold immunochromatography,the sensitivity was enhanced 10 times and 1000 times respectively.In our research condition,the detection antibody amount used in detection was only 3 ?g,less than that of the traditional gold immunochromatography(4.8 ?g).Thus,MNPs,as a bio-marker,has potential economic and industrial value.In chapter 5,a competitive lateral flow assay for the rapid detection of Salmonella choleraesuis was developed.IMNPs were produced by covalently coupling anti-Salmonella choleraesuis antibody to MNPs.The IMNPs were used as visually detected probes in the subsequent assay.Compared with the traditional sandwich assay which is used for detecting macro-molecules,this new method was developed based on the competitive relationship between S.choleraesuis in sample and the outer membrane protein immobilized on the T line.Thus,only one antibody was necessary in the new assay,whereas a pair of rigorously selected antibodies were required in the sandwich assay.The sensitivity of the competitive assay for S.choleraesuis was 1.2×107 CFU/mL.In addition,no cross reactions were found in the 18 common non-Salmonella bacteria strains and in the 3 Salmonella strains of other serotypes.Thus,with satisfactory sensitivity and specificity,the assay can be applied for the rapid detection of pre-enriched culture that may contain S.choleraesuis.In chapter 6,a modified lateral flow immunoassay(two-step assay)was developed to detect trace aflatoxin M1(AFM1)in raw milk.In contrast to conventional LFIA,two kinds of IMNPs were used.IMNP with high antibody concentration was used to capture AFM1 in the test sample,whereas the other IMNP with low antibody concentration was used to elucidate the results of the test.The two-step assay exhibited an ideal sensitivity to screen trace AFM1 in milk samples without extra sample pretreatment.The cutoff value of the naked eye was 0.02 ?g/L and satisfied the European Union's maximum level of AFM1 in raw milk,heat-treated milk,and milk used to manufacture milk-based products and even in baby foods.With the same antibody,sensitivity was enhanced approximately 25 and 50 times when compared with conventional IMNP-based LFIA and gold-based LFIA,respectively.Corresponding results of 10 raw milk samples were obtained between this two-step assay and referenced enzyme-linked immunosorbent assay.