Multi-microorganisms Carriers-based High-sensitivity Immunochromatographic Assay For Zearalenone Detection

Zearalenone（ZEN）is an estrogenic fungal toxin produced by fusarium,which can enter the food chain by contaminating grains and feedstuffs,causing serious harm to human and animal health,and has become an area of emphasis in food safety research.Consequently,it is of important practical significance to develop ZEN monitoring techniques to fulfill the demands of rapid,sensitive,and accurate detection methods in the field of global food security.Immunochromatographic assay（ICA）is a rapid immunoassay method combining the development of immunoassay,chromatography,and nanomaterials technology,which has the advantages of simple operation,low cost and does not rely on large equipment,and is widely used in mycotoxin detection.However,the traditional gold nanoparticles-based ICAs（Au NPs-ICAs）usually suffer from poor sensitivity resulting from the small size and poor stability of Au NPs.Compared to the Au NPs,microorganisms（MOs）has the advantages of large surface-to-volume ratio and abundant functional groups,can be used as efficient templates for the production of multifunctional carriers,which is expected to develop a new MOs-based ICA to overcome the limitations of traditional Au NPs and improve the detection performance of the ICAs.Therefore,this article combined with the many advantages of MOs,four MOs carriers with excellent properties were designed and their synthesis mechanisms were explored,and a series of novel MOs-ICAs were constructed for high-sensitivity detection of ZEN in corn and millet,and the related detection performance and practical application potential were evaluated respectively.The results of the study are as follows:1.Immunochromatographic test for ZEN detection based on gold nanoparticles-functionalized microorganism with improved sensitivityMicroorganism loaded gold nanoparticles composites（Yeast@Au NPs and LAB@Au NPs）were synthesized by reacting Yeast/Lactobacillus（LAB）with HAu Cl₄ via biosorption and subsequent bioreduction.By exploring the synthesis conditions（p H and reaction time）and main characteristics（morphology,diameter,and functional groups）of microorganisms@Au NPs（MO@Au NPs）,it is proved that the synthesized MO@Au NPs had large size（about 2μm）,strong adsorption capacity and the low amount of labeled monoclonal antibodies（m Abs）,significantly reducing the detection limit of ICA.After optimization,the linear detection of ZEN using Yeast@Au NPs-ICA and LAB@Au NPs-ICA ranged from 0.375to 12 ng/m L and 0.18 to 12 ng/m L,and the limits of detection（LOD）were 0.078 and 0.118ng/m L,which were nearly 14-fold lower than traditional Au NPs-ICA.Meanwhile,the proposed strategy also possessed high specificity for ZEN against other mycotoxins,which could detect the lowest levels of ZEN in maize and millet about 1.5μg/kg,and the average recoveries were 81.55–127.70%.The above results indicate that the method has good stability and accuracy.2.Dual-signal immunochromatographic test of ZEN sensing based on SABPDs-directed modified antibodiesAccording to the characteristic that protein A on the surface of Staphylococcus aureus（SA）can specifically bind to fragment crystallizable（Fc）region of monoclonal antibodies（m Abs）,in this work,a SA-biosynthesized polymer dots（SABPDs）were intelligently fabricated via Schiff base reaction and self-assembly strategy using SA and hydroquinone（HQ）as precursors.Then a dual-signal immunochromatography assay based on SABPDs-directed labeling was constructed（SABPDs-DICA）.Experiments demonstrated that SABPDs possessed uniform size coccus morphology,blue-green fluorescence and visible brown signal,and the ability to target the Fc region of m Abs.The affinity constant（k_a）and equilibrium dissociation constant（K_D）of SABPDs-labeled m Abs were calculated,which verified that the carrier could retain the inherent biological activity of m Abs.Therefore,the prepared SABPDs can not only effectively expose the antigen-binding site of the m Abs,but also endow the ICA with excellent colorimetric-fluorescence dual signals,which can further improve the sensitivity.Under the optimal conditions,the constructed test strip can detect ZEN as low as 0.036 ng/m L with excellent specificity and reproducibility.The lowest detectable ZEN levels of SABPDs-DICT in maize and millet were 0.72μg/kg with good recovery,which were consistent with liquid chromatography-mass spectrometry（LC-MS/MS）,and false positive and false negative rates were 0%.The above detection results were consistent with liquid chromatography-mass spectrometry（LC-MS/MS）,realizing the high sensitivity and accuracy detection of ZEN by this method.3.A SAD universal immunochromatographic assay for detection of ZEN with one-step biological stainingBased on the advantages of dyes with bright colors,low cost,and fast binding to microbial proteins,we used the biostaining self-assembly strategy to create SA-dye（SAD）carriers for ICA,choosing congo red（CR）and fluorescein isothiocyanate（FITC）were embedded in SA to make SACR and SAFITC,respectively,which provided high brightness and abundant specific binding sites for the directional immobilization of m Abs.The mechanism of SAD directional binding to m Abs was verified,and the calculation of ka showed that SAD had higher antibody biological activity(k_a was about 10⁹ M^-1),which provided a basis for further improving the sensitivity.Furthermore,SACR and SAFITC were applied to ICA to achieve colorimetric and fluorescent signal detection of ZEN,respectively.The results showed that the constructed SAD-ICAs had lower detection limit（0.013 ng/m L）,good linearity（R²>0.98）,reproducibility（RSD<8%）,selectivity and stability.SAD-ICAs could detect the lowest level of ZEN in corn and millet at 0.36μg/kg,with good recoveries in the range of 81.5-124.6%,consistent with LC-MS/MS.Moreover,SACR and SAFITC were mixed and applied to the ICA to achieve a dual-readout assay of ZEN.Thus,the microorganism self-synthesizing carrier-based immunoassays can be skillfully generalized to on-site and multimode detection of other food safety hazards by simply altering the different dyes and corresponding m Abs.4.Double-readout immunochromatography assay for detection of ZEN based on Ru（bpy）₃²⁺-doped SAQDs with enhanced signalIn order to further improve the detection perceformance,this study adopted a"space-time coupling"strategy to in situ synthesize SA-quantum dots（SAQDs）,then Ru（bpy）₃²⁺was incorporated into SAQDs to obtain SAQDs Ru as a novel carrier,a double-readout immunochromatography assay for detection ZEN based on Ru（bpy）₃²⁺-doped SAQDs（SAQDs Ru-DICA）with enhanced signal was established.The synthesis mechanism of SAQDs Ru was explored,and then characterization of SAQDs Ru-labeled m Ab（SAQDs Ru-m Ab）probe demonstrated that this probe had strong luminescence,excellent stability,and high affinity with ZEN,which could significantly improve the detection sensitivity.After optimization,SAQDs Ru-DICA indicated a limit of detection reaching down to 0.008 ng/m L（colorimetric mode）and 0.0058 ng/m L（fluorescent mode）,possessing favorable specificity and repeatability.Subsequently,by combining SAQDs Ru-DICA with a smartphone-based APP,a portable sensing system was developed for convenient detection of ZEN.In addition,SAQDs Ru-DICA could detect the lowest level of ZEN in corn and millet at 0.16μg/kg,the recoveries were 85.58–118.83%with RSD less than 5.62%.At the same time,the actual samples of natural contamination were also tested,and the results showed that they were consistent with detection results of LC-MS/MS,indicating that the method has broad application prospects in the field of food safety.