Study On Immunochromatographic Test Strip Based On Nanobody For Aflatoxin B₁ Detection

Aflatoxin B₁ is a group of secondary fungal metabolites produced by Aspergillus flavus and Aspergillus parasiticus.It is widely distributed,highly toxic and extremely harmful and was recognized as I carcinogen by the World Health Organization,seriously endangers human and animal safety.Therefore,the development of an efficient,sensitive and on-site detection method is of great significance for ensuring human health.Immunochromatographic test strips（ICTS）,as a cheap,accurate,and easy-operated method,is one of the most promising point-of-care diagnostic analytical and has been used broadly as a prospective detection technique in different fields,for example,food safety,clinical diagnosis,and environmental monitoring.However,traditional ICTS rely on monoclonal antibody（m Ab）as recognition elements,which limits the development and application of ICTS due to the complex preparation process,inability to mass-produce and high cost of m Ab.Nanobody（Nb）is derived from heavy-chain-only antibodies that occur naturally in the serum of camelids,which is the smallest genetic engineering antibody and can be expressed in various expression systems to form fusion protein with high yield and stability.In recent years,it has been widely used in the field of food safety detection.In this study,nanobodies were used as recognition elements,and gold nanoflowers（Au NFs）with different particle sizes and magnetic beads（MB）were used as signal labels.Based on the high specificity and stability of Nb and the good optical properties,large specific surface area and superparamagnetic properties of the nanomaterials.The efficient and sensitive ICTS were constructed to realize the rapid and real-time detection of AFB1 in food.The specific research contents are as follows:1.In this study,the Nb-G8 with desired specificity and high sensitivity against AFB₁was fused with the anti-DIG antibody for construction recombinant plasmid p ET25b-G8-DIG transformed into E.coli BL21（DE3）for expression,nanobody G8-DIG was obtained with molecular weight of about 30 KDa and evaluated protein activity using ic-ELISA.The G8-DIG was used as recognition elements,which could specifically recognize both AFB₁ and DIG-BSA.DIG-BSA can replace anti-His-m Ab for traditional ICTS based on nanobody,improving the stability and saving costs.Au NFs with a particle size of 110 nm were prepared,which were conjugated with G8-DIG by electrostatic adsorption as signal probes.A nanobody immunochromatographic test strip based on the Au NFs was constructed for the detection of AFB₁.The p H of Au NFs coupling with G8-DIG,the amount of G8-DIG,the detection time and temperature,the amount of Au NFs@G8-DIG and the concentration of AFB₁ and DIG-BSA were optimized.The standard curve was established under the optimal experimental conditions,the linear range(IC₂₀～IC₈₀)was 1.02 ng/m L～27.86 ng/m L,the limits of detection（LOD）was 0.1 ng/m L and the recoveries were 91%～100%.In addition,the nanobody-based ICTS have been successfully applied to the analysis of corn（quality control sample）providing a method for the detection of AFB₁ in actual samples.2.In order to improve the optical characteristics of signal labels and further enhance the detection sensitivity of ICTS,this study constructed a double probe signal amplification immunochromatographic test strip for AFB₁ detection based on the nanobody and biotin-Streptavidin system.Frist,Nanobody G8-Bio was obtained by chemical method,and the optimal molar ratio of Nb-G8 and biotin was 1:20.Secondly,Two sizes of Au NFs were prepared.The 65 nm Au NFs were conjugated with G8-Bio as signal probes,the 110 nm Au NFs were conjugated with SA as signal amplification probes,which were sprayed on the conjugated pad and amplification pad respectively.The high affinity between biotin and streptavidin was used for signal amplification and compared with ICTS without signal amplification.The results showed that under the optimal experimental conditions,the detection range of biotinized nanobody ICTS without signal amplification was 0.76ng/m L～85.26 ng/m L,and the LOD was 0.12 ng/m L.While the detection range of dual-probe signal amplification ICTS was 0.2 ng/m L～119.4 ng/m L,LOD was 0.03 ng/m L that the sensitivity was 4 times higher than that of the ICTS without signal amplification and with wider detection range.The recoveries of spiked corn samples were 100%～102%.3.In order to further improve the stability of the signal probe and realize the highly sensitive detection of AFB₁ in the actual samples with dark color.In this study,biotinylated nanobody G8 was conjugated with the magnetic beads-SA（MB-SA）as a signal probe using streptavidin and biotin interaction.The MB-SA@G8-Bio showed remarkable binding,separation and enrichment capacities toward AFB₁ even under complex sample matrices.Under the optimal detection conditions,the IC₁₀ and IC₅₀ of the method were 0.063ng/m L and4.62 ng/m L respectively.This ICTS were applied to detect AFB₁ in dark soy sauce,and its IC₁₀ was 0.069ng/m L,which was basically the same as the standard test,This method provides a reference for the detection of AFB₁ in dark actual samples.