Microchannel Resistance Immunosensor Based On Magnetic Nanomaterials For The Detection Of Aflatoxin B₁ In Grains

Grains are an important part of sustaining human life activities and are also one of the foods most susceptible to mycotoxins contamination.Aflatoxin,a mycotoxin produced by Aspergillus,is the strongest carcinogen discovered so far.Among them,aflatoxin B₁（AFB₁）is a class I carcinogen,which is extremely toxic and may cause liver damage at low levels,even threaten people’s life.Therefore,even if AFB₁ exists in extremely small amounts in grain samples,its toxicity cannot be ignored.In addition,grains contain a lot of starch and fat,which undoubtedly increases the difficulty of AFB₁extraction,enrichment and accurate detection.However,the existing methods have the problems that the pretreatment steps are cumbersome,it is difficult to fully extract the target substance,and the detection method for trace substances is not sensitive enough.Therefore,it is urgent to establish an efficient sample pretreatment method and a high-sensitivity detection method for the analysis of trace AFB₁ in grains.In this study,Fe₃O₄was surface-bonded and surface-coated based on surface modification technology to obtain magnetic nanomaterials with good dispersion,uniformity and magnetic responsiveness,which were used as a magnetic separation and enrichment material,and then combined with the microchannel resistance immunosensor for the rapid and accurate detection of AFB₁ in grains.The main research contents are as follows:1.Microwave-assisted hydrothermal synthesis of Fe₃O₄ magnetic nanoparticlesFe₃O₄ magnetic nanoparticles with uniform particle size and strong magnetic responsiveness were successfully prepared by microwave-assisted hydrothermal method.The effects of the molar ratio of Fe Cl₃ 6H₂O to Na Ac 3H₂O,synthesis temperature and synthesis time on the particle size uniformity,dispersibility and magnetic properties of Fe₃O₄ magnetic nanoparticles were investigated.The Fe₃O₄ magnetic nanoparticles were characterized by scanning electron microscopy（SEM）,dynamic light scattering analysis（DLS）,fourier transform infrared spectroscopy（FT-IR）,X-ray diffraction（XRD）and vibrating sample magnetometer（VSM）.The experimental results showed that the optimal synthesis conditions were Fe Cl₃·6H₂O and Na Ac·3H₂O molar ratio 1:2.5,heating temperature 220℃,heating time 60 min.The as-synthesized Fe₃O₄ magnetic nanoparticles were spherical with regular shape and the average particle size was about300 nm.FT-IR、XRD and VSM results showed that Fe₃O₄ magnetic nanoparticles exhibited stretching vibration peaks around 586 cm^-1 and 3400 cm^-1,corresponding to the absorption peaks of Fe-O and-OH,and the X-ray diffraction peaks at 30.1°（220）,35.5°（311）,43.2°（400）,53.4°（422）,57.1°（511）and 62.5°（440）are consistent with the JCPDS NO.75e0449 standard,and the saturation magnetization at room temperature was65.13 emu/g.The above characterization results showed that Fe₃O₄ nanoparticles with uniform particle size,good dispersion and good magnetic properties were successfully synthesized,and the synthesis time can be shortened from 12-24 h in the classical hydrothermal synthesis method to 1 h.2.Detection of aflatoxin B₁ in grains by microchannel resistance immunosensor based on Mg/Zn-MOF74@Fe₃O₄ magnetic solid phase extraction materialThe as-synthesized Fe₃O₄ magnetic nanoparticles were subjected to bonding surface modification by solvothermal method,and an organic framework material（Mg/Zn-MOF74@Fe₃O₄）mixed with two metals,Mg and Zn,was assembled on the surface,and the material was used as a magnetic solid phase extraction carrier.Since Zn²⁺in this material can generate stable chemical bonding with theβ-dicarbonyl group in the structure of AFB₁,the efficient adsorption of AFB₁ in grains can be achieved.After the AFB₁ adsorbed on the material was eluted,it would compete with polystyrene microspheres coupled with AFB₁ complete antigen（PS-BSA-AFB₁）and Fe₃O₄ coupled with AFB₁ antibody（Fe₃O₄-AFB₁-Ab）,The unreacted PS-BSA-AFB₁ after magnetic separation was detected by a microchannel resistive immunosensor based on the Coulter counting principle,which can convert the PS-BSA-AFB₁ concentration signal related to the AFB₁ concentration in the sample into an electrical signal,so as to achieve accurate and sensitive quantification of AFB₁ in grain samples.The research results showed that the synthesized Mg/Zn-MOF74@Fe₃O₄ magnetic nanomaterials are regular hexagonal clusters with an average particle size of 380-480 nm,the adsorption capacity for AFB₁ is about 8.921 mg/g,the recovery rate is between 79.38%and 95.84%in complex samples,and it had a wonderful stability and reusability.The constructed microchannel resistive immunosensor based on Mg/Zn-MOF74@Fe₃O₄ magnetic solid-phase extraction material had a linear range of 10 pg/m L～20 ng/m L for the detection of AFB₁,and the detection limit was 4.75 pg/m L.The recovery rate was between 78.13%and 116.60%,and the coefficient of variation was under 15.83%.In the wheat blind sample test,the method has good consistency with high performance liquid chromatography,and the quantification can be completed within 25-30 min.3.Detection of aflatoxin B₁ in grains based on antibody-SiO₂@Fe₃O₄immunomagnetic enrichment microchannel resistive immunosensorFe₃O₄ magnetic nanoparticles were coated with SiO₂ by the improved stober method,and an antibody was coupled on the surface of the material as an immunomagnetic separation carrier Antibody（Ab）-SiO₂@Fe₃O₄ to capture AFB₁ from the extract.The carrier was directly immunoreacted with the extract and PS-BSA-AFB₁.After magnetic separation,the microchannel resistance immunosensor can be used for signal readout.The concentration signal is converted into a quantified electrical signal for quantitative analysis of the target substance.The experimental results showed that the SiO₂@Fe₃O₄ magnetic nanomaterials are uniformly dispersed spherical,with an average size of about 280 nm,and the thickness of the coating layer was between 20 and 30 nm.The adsorption capacity for AFB₁ was about 68.283 mg/g,and the recoveries in the samples were between 81.34%and 89.96%.The microchannel resistance immunosensor was used for quantitative analysis under optimal conditions,and the results showed that AFB₁ had a good linear relationship in the range of 10 pg/m L～50 ng/m L,the detection limit was 4.12 pg/m L,and the recovery rate was 79.68%～110.67%,the coefficient of variation was under 12.62%,and the detection results of the actual samples were in good agreement with high performance liquid chromatography method.In contrast,the material has a larger adsorption capacity for AFB₁,does not require an elution process,and is more convenient to operate.The quantification can be completed within 20 minutes,and the combination with the microchannel resistance immunosensor can realize a relatively smooth and complete detection,thereby simplifying the operation procedure,improving the detection efficiency,and realizing the high-sensitivity,accurate and convenient detection of AFB₁.