Gold Nanoflower And Anti-idiotypic Nanobody In Immunoassay Application For The Detection Of Mycotoxins

Mycotoxins are a chemically diverse group of fungal secondary metabolites,which found worldwide in agricultural products and food.Several of these mycotoxins have been associated with human and animal diseases,which can lead to cancer or even killed,especially aflatoxin A1(AFB1)and ochratoxin A(OTA)have more strong toxicity.Therefore,developing a rapid,sensitive and specific detection method is particularly important.Among the numerous detection methods,immunoassay is the common method due to it specificity,rapidity,simplicity and cost effective for high throughput screening.But with the increment of world import and export,people's demand for safe food is increasing constantly,resulting in the immunoassay cannot meeting their needs.Therefore,the development of immunoassay with high sensitivity is imperative.In this thesis,AFB1 and OTA as two model mycotoxins,and aims to develop immunoassay with high sensitivity and specificity,sensitivity were improve from two aspects:detection signal and immune recognition element.First,an ultrasensitive and quantitative immunochromatographic test strips(ICTS)was develop using gold nanoflowers with high optical and stability as new labeled probes,which provides new ideas and technical direction for food safty.At the same time,anti-OTA monoclonal antibody was used as the target for biopanning from a naive alpaca nanobody phage display library,and an anti-idiotypic nanobody was isolated for OTA.An environmentally friendly immunoassay for OTA was performed based on the anti-idiotypic nanobody(AId-Nb)to substitute the synthetic antigen,which provides new technical perform.The main contents are as follow:1)High-quality gold nanoparticles(AuNPs)were synthesized in the presence of hydroquinone through a seed-mediated growth approach.A series of factors on the shape and size of AuNPs have been studied at various volume of seeds,HAuCl4 and hydroquinone,and the gold nanoparticles were characterized by color chang,UV-Vis spectrophotometer,TEM and Zeta potential.Results show that:the size of AuNPs strongly depended on the volume of seeds,the size of AuNPs becomes larger,and favorable for the formation of AuNFs with the increasing of volume of seeds(?500?L).The volum of HAuCl4 and hydroquinone are main factors that control significantly the shape of AuNPs,increasing the volume of hydroquinone(300?L?V?400?L)and reducing the volume of HAuCl4(?150?L)are beneficial for formation of AuNFs.Moreover,AuNFs has better stability and optical properties than the same size of AuNSs,which means it might have a better promising application prospect.2)A new ultrasensitive and quantitative immunochromatographic test strips(ICTS)was established using AuNFs as a signal amplification probe for AFB1 in rice.A portable optical strip reader was used to record the optical densities of test and control lines of the strip.Under the optimal conditions,the AuNF based ICTS system accurately detected AFB1 linearly and dynamically over the range of 0.5-25 pg/mL with a half maximal inhibitory concentration at 4.17 pg/mL.The inhibitory concentration was achieved 10 times lower than that of the traditional AuNS based ICTS systems(41.25 pg/mL).The limit of detection for AFB1 in rice extract was achieved at 0.32 pg/mL.In summary,AuNFs are a novel probe that exhibited excellent sensitivity in the ICTS system and could be used for ultrasensitive detection of other analytes in food safety monitoring,and even medical diagnostics.Low CVs revealed that the new AuNF-ICTS system is suitable for AFB iquantitative analysis.AuNF-ICTS was further compared with a commercially available ELISA kitor 50 AFB1 spiked rice samples,shows that the two methods exhibited consistent results with a highly significant correlation(R2=0.93).AuNFs are a novel probe that exhibited excellent sensitivity in the ICTS system and could be used for ultrasensitive detection of other analytes in food safety monitoring,and even medical diagnostics.3)The phage displayed anti-AId-Nb was selected from a naive nanobody library.After four cycles of panning,a phage displayed AId-Nb was isolated using a competition-binding biopanning strategy.The half-inhibition concentration(IC50)of the phage-ELISA was 300 pg/mL,which was 8.83-fold better than that of a conventional ELISA.Furthermore,a environment friendly quantitative Q-IPCR assay was also developed for the detection of ochratoxin A in agri-products based on phage displayed OTA-AId-Nbs.The limit of detection(LOD)of the assay is 4.17 pg mL-1,which exhibits a 9-fold improvement over the phage-ELISA.The developed method was successfully validated using OTA contaminated agri-products.Furthermore,novel and environmentally friendly Q-IPCR might have potential applications in a general method for the immunoassay of various toxic small molecules.4)The OTA-AId-Nbs was expression and purified from the expression vector.A competitive immunoassay for OTA was performed using the nanobody as the coating antigen.The sensitivity of ELISA was improved 10 times than the synthetic antigen based ELIS A under the optimal conditions.In addition,the specificity of the assay was tested,but negligible cross reactivity was observed with AFB1,FB1,ZEN,DON.At the same time,AuNFs as a signal amplification probe,a sensitive immunochromatographic test strips(ICTS)was develop for OTA using AId-Nbs to replace the synthetic antigen,the cutoff value is 4 ng/mL.Furthermore,novel and environmentally friendly ELISA and ICTS might have potential applications in a general method for the immunoassay of various toxic small molecules.