Study Of High Effective Seperation And Purification Of Sample Pretreatment Techniques Based On Aflatoxins Broad-spectrum Antibody

Veterinary drugs,pesticides,mycotoxins,and environment hamful contaminats are the huge threat for human health.The study of these compounds are always the hot issues in the field of analytical chemistry.However,due to the complexity of the sample matrix,these compounds are mostly trace or ultra-trace,the existing pre-treatment technology is mostly cumbersome operation,low efficiency,specificity,consumption of a large number of organic solvents,can not meet the actual testing requirements.The development of a simple,fast,effective and green enviroment friendly sample pretreatment method for the determination of compounds is especially necessary.In this paper,nanomaterials with high surface area,good adsorption performance and structural stability are combined with high affinity and high specific bio-identification materials.The new sample pretreatment technology is developed and applied to aflatoxin detection.Herein we designed a multifunctional nanoarchitecture by integrating magnetic property of Fe₃0₄,functional groups of CTS and selective monoclonal antibody（Fe₃O₄@CTS@AF_s-mAb）.The nanostructured Fe₃O₄@CTS were prepared with a simple one-step method and characterized in detail.The resultant composite Fe₃0₄@CTS was characterized using X-ray powder diffraction（XRD）,scanning electron microscope（SEM）,transmission electron microscopy（TEM）,fourier transform infrared spectroscopy（FTIR）,energy dispersive spectroscopy（EDS）,and thermogravimetric analysis（TGA）.The results indicated that the Fe₃0₄@CTS particles were well crystalized,core-shell structure,and successfully coated by the CTS layer.Further,a monoclonal antibody directed against six AFs was immobilized on the nanocomposite through the EDC/sulfo-NHS reaction,generating the Fe₃O₄@CTS@AFs-mAb adsorbent.Fe₃O₄@CTS nanoparticle have high adsorption capacities（23.5 mg/g）and high coupling rates（more than 90%）.The maximum aflatoxins-binding capacity was 337,351,306,113,462 and 389ng/mL for AFB₁,AFB₂,AFG₁,AFG₂,AFM₁ and AFM₂,respectively.A pretreatment method for the detection of six aflatoxins（AF_s）from corn,peanut,and olive oil samples based on Fe₃O₄@CTS@AF_s-mAb was developed.AFs were extracted from samples with methanol-water（60:40,v/v）solution and were purified by Fe₃O₄@CTS@AF_s-mAb.Six toxic AFs were enriched by the nanoarchitecture in less than 1 min from foodstuff samples.The efficiency of Fe₃O₄@CTS@AF_s-mAb in extracting AFs has been found to be more than 60 times higher than both conventional immunoaffinity chromatography and solid-phase extraction.After UPLC-MS/MS detection,the results showed that the LODs for six AFs ranged from 0.003-0.007 μ/kg.The recoveries ranged from 63%to 112%,with relative standard deviations less than 15.6%.The developed method is further validated by three kinds of reference materials.In this paper,we proposed a method for preparing modified reduced graphene oxide films that were easily conjugated to monoclonal antibodies（rGO@PBA@SA@B-AF_s-mAb）.A group of AFs could be efficiently and selectively captured by the rGO@PBA@SA@B-AFs-mAb films in rabbit serum samples.First,the GO films were fabricated by vacuum filtration method.And,the rGO films were reduced via thermal reduction in a vacuum.Pyrenylbutyric acid（PBA）was immobilized onto rGO film by π-π stacking,thus introducing more carboxyl groups,compared to original rGO film,for conjugating streptavidin（SA）by EDC/sulfo-NHS.Finally,biotinylated AFs-mAb was immobilized onto the SA-rGO films with coupling rates of 99.5%.The resulting rGO@PBA@SA@B-AFs-mAb films can successfully act as sensitive biointerfaces to recognize specific AFs.After the serum samples were digested,100μL was added dropwise to the surface of rGO@PBA@SA@B-AFs-mAb films.After purification,the samples were analyzed by UPLC-MS/MS.LODs for six AFs ranged from 0.05-0.17 μg/L.The average recoveries of AFs in spiked rabbit serum samples were from 55.1%to 75.3%with the relative standard deviations of less than 9.4%.This study described a reliable immunoaffinity chromatography separation technology for the the simultaneous purification of aflatoxins（AFB₁,AFB₂,AFG₁,AFG₂）in eight foodstuffs such as maize,rice,peanut,peanut oil,soybean oil,bear,soybean sauce,and soybean paste.The immunoaffinity chromatography of AFs（AFs-IAC）was prepared by coupling CNBr-activated Sepharose-4B with the AFs-mAb,with the coupling rates more than 90%.The home-made IAC capacity was in the range of 45-293 ng/mL gel for 4 AFs.Samples were extracted with methanol-water and the extracts were then purified on an AFs-IAC,which then washed with deionized water.The aim compounds were eluted by pure methanol before UPLC-MS/MS analysis.LODs for four AFs ranged from 0.003-0.005 μg/kg.The results showed that the recoveries ranged from 60.2%to 119.8%and the relative standard deviations were below 15.7%.