Application Research On Rapid Detection Of Mycotoxins In Cereals Based On Lanthanide-doped UCNPs Immunochromatographic Method

Aflatoxin B1（AFB1）is the most toxic and carcinogenic mycotoxin,which is listed as a Class I carcinogen by the World Health Organization Cancer Institute.It can induce severe liver damage in humans and animals,and even lead to liver cancer and death.Zearalenone（ZEN）is an estrogenic,widespread mycotoxin that exhibits strong reproductive toxicity in humans and animals.AFB1 and ZEN mainly exist in crops and their products,and their mixed contamination can lead to significant cooperative and synergistic effects,which greatly increase their toxicity.Immunochromatography assay（ICA）has been widely used in the on-site quantitative detection of single or multiple mycotoxins due to its advantages of rapid detection,low production cost,simple operation,and intuitive results.In this study,two improved up-conversion nanoparticles（UCNPs）were prepared by doping lanthanide rare earth ions and used as markers to construct immunochromatographic strips for ZEN detection.By comparing the detective sensitivity of the two UCNPs-ICAs,Lu-UCNPs were selected as the marker to construct the dual-channel test strips to achieve the simultaneous detection of AFB1 and ZEN in grain samples.The main results are as follows:1.Rapid detection of ZEN using La³⁺doped UCNPs-ICAIn this study,La³⁺doped UCNPs at five different ratios were synthesized using the thermal decomposition method,and their ion doping ratio was optimized.The results showed that the UCNPs doped with 20%La³⁺exhibited optimal morphology,structure,and fluorescence properties,which were recorded as La-UCNPs.Polyacrylic acid（PAA）was used to modify the surface of La-UCNPs with carboxyl groups and then coupled with ZEN-m Ab to prepare immunoprobes.Subsequently,a new method for the determination of ZEN in cereals by fluorescence ICA was established.The dosage of PAA,the amounts of antibody labeling,the detection time,and the concentrations of fluorescent immunoprobe were optimized to be2.0 m L,20μg,10 min,and 8 mg/m L,respectively.Under the optimized conditions,the limit of detection（LOD）and linear correlation coefficient（R²）of La-UCNPs-ICA for ZEN standard solution was 0.25 ng/m L and 0.9944,with the coefficient of variation（CV）less than8.67%.Besides,the specificity test showed that the prepared test strip was highly specific for ZEN.The LOD of ZEN in wheat and maize by this method was 0.5μg/kg.The recoveries of spiking test were 87.65%～99.26%and 84.12%～95.12%,respectively,and the corresponding CVs were 1.289%～7.143%and 4.033%～9.013%.Compared with colloidal gold strips and high-performance liquid chromatography（HPLC）,La-UCNPs-ICA had better sensitivity and matrix interference resistance,and the three methods showed high consistency in the ZEN detection of natural grain samples.2.Rapid detection of ZEN using Lu³⁺doped UCNPs-ICAA series of Lu³⁺doped UCNPs were synthesized using the thermal decomposition method and were characterized via scanning electron microscopy,X-ray diffraction,energy dispersive spectroscopy,Fourier transform infrared spectroscopy,and fluorescence spectrometer.The results found that UCNPs doped with 30%Lu³⁺showed the best particle properties and fluorescence properties,which were recorded as Lu-UCNPs and used as markers for the development of subsequent test strips.After the detection conditions of the test strips were optimized,the optimal antibody labeling amount,T-line antigen concentration,fluorescent probe concentration,and sample dilution were determined to be 10μg,0.8 mg/m L,10 mg/m L,and PBST diluents.Under optimal detection conditions,the LOD of Lu-UCNPs-ICA for ZEN standard solution was 0.1 ng/m L,with a good linear relationship（R²=0.9946）in the range of0～100 ng/m L.Moreover,there was no cross-reaction with other mycotoxins.The LODs of ZEN in maize and wheat using this method were all 0.16μg/kg,and the recovery rate of spiking test were 82.11～100.76%and 79.64～98.51%respectively.Additionally,the detection results of Lu-UCNPs-ICA were consistent with those of colloidal gold strips and HPLC in natural grain samples.Therefore,this novel Lu-UCNPs-ICA can be applied to quantitatively monitor the trace residue of ZEN in grain samples.3.Application of improved UCNPs-based immunochromatographic competitive assay for simultaneous detection of AFB1 and ZENComparing the fluorescence performance and the detection sensitivity between the constructed two ICA of La-UCNPs and Lu-UCNPs,the Lu-UCNPs with better performance were select to determine as the markers for the test strips,which were recorded as IUCNPs（Improved UCNPs）.A single-channel detection strip for AFB1 detection was constructed based on IUCNPs,and the optimized antibody labeling amount and fluorescence probe concentration were 20μg and 8 mg/m L,respectively.Additionally,the LOD of ZEN standard solution was 0.1 ng/m L.Under the optimized single-channel detection conditions,a novel dual-detection test strip was established based on IUCNPs,which can reach the simultaneous detection of AFB1 and ZEN in 10 min.The LODs of AFB1 and ZEN were 0.025 and 0.1ng/m L respectively,with the R²of calibration curves being 0.9931 and 0.9982.After being stored at room temperature in a dark and dry environment for 2 months,the average relative strengths of the T1/C and T2/C values in the test strips were 98.24%and 99.65%,respectively.Furthermore,the CVs of intra-assay and inter-assay tests were all less than 10%.The LODs of AFB1 and ZEN in maize samples using this method were 0.025μg/kg and 0.25μg/kg,respectively,indicating that it had excellent sensitivity and matrix tolerance.15 naturally contaminated maize samples were detected with the test strips,and the detection results were consistent with the HPLC analysis,confirming the reliability and practicability of the method.In summary,the two improved UCNPs prepared in this study showed excellent morphological structure and fluorescence properties,which would have broad application prospects in the field of immunochromatographic markers.The single immunochromatographic technology proposed based on two UCNPs could achieve quantitative detection of ZEN in cereals and exhibited better detection performance than traditional UCNPs-ICA and CG-ICA.At the same time,the dual detection test strips constructed based on Lu-UCNPs provided a great reference value for the simultaneous detection of mycotoxins in cereals.