Immunoassays Based On Carbonized Polymer Dots And Prussian Blue Nanoparticles For Zearalenone Detection

Currently,the world is facing a crisis in food security caused by climate change and regional conflicts.In recent years,our country’s main grain-producing areas have been frequently affected by natural disasters,which have impacted the quantity and quality of grain production.The long-term high temperature and humidity environment have accelerated the growth and reproduction of fungi,leading to widespread contamination of crops.Fusarium is a common factor causing fungal pollution.Zearalenone（ZEN）is the secondary metabolite produced by Fusarium species.It can cause pollution to various crops.Long-term consumption of grains or grain products contaminated with ZEN can pose health risks to both humans and animals.Traditional detection techniques for ZEN mainly rely on instrumental analysis methods such as thin-layer chromatography and high-performance liquid chromatography.Although these analytical methods have high sensitivity,the cost of detection equipment is high,and there are many pretreatment steps,which require skilled operators.Enzyme-linked immunosorbent assay（ELISA）is an immunological detection technique that utilizes antigen-antibody reactions.It has the advantages of high detection sensitivity,easy operation and low cost.Nanoparticles have unique physical and chemical properties,good biocompatibility,and strong stability.When applied in ELISA detection,they can serve as signal carriers or reporter molecules,enhancing the signal intensity and improving detection sensitivity.Carbonized polymer dots（CPDs）are nanoparticles with many advantages,such as simple synthesis method,excellent fluorescence performance,good biocompatibility and strong photostability.Applying CPDs in immunological detection not only reduces detection costs but also achieves high sensitivity.Prussian blue nanoparticles（PB NPs）are blue-colored nanoparticles,and the preparation method is simple,cheap and fast.Combining the excellent performance of PB NPs with immunological detection technology can shorten the detection time and improve the visualization of detection.This study combines CPDs and PB NPs with ELISA or lateral flow immunoassay technology to establish four immunological methods for detecting ZEN.The main research contents are as follows:1.Establishment of an indirect competitive ELISA（ic-ELISA）Horseradish peroxidase（HRP）was used to catalyze colorless substrates 3,3’,5,5’-tetramethylbenzidine（TMB）and hydrogen peroxide（H₂O₂）into colored products.The reaction was terminated by adding acid.ZEN could be quantitatively detected by measuring the change in absorbance of ultraviolet-visible absorption spectrum（UV-vis）at 450 nm.After optimizing the parameters of the experiment,the standard curve equation was y=84.844x-80.943,R²=0.990.According to the standard curve,the sensitivity(IC₅₀)was 34.32 pg/m L,and the linear detection range was 14.78 pg/m L to79.69 pg/m L.There was no cross reaction with the other five mycotoxins.The recovery rate of ZEN in corn flour samples ranged from 89.65%to 93.76%,the relative standard deviation（RSD）ranged from 5.11%to 9.29%.The recovery rate of commercial ELISA kits ranged from 91.65%to 96.40%and the RSD ranged from 4.80%to 8.69%.They all met the requirements of the Association of Official Analytical Chemists（AOAC）for sample addition and recovery experiment.The established ic-ELISA method had the characteristics of high sensitivity and specificity,and was suitable for the detection of a large number of samples.2.Establishment of a colorimetric and fluorescence dual-signal method based on the generation of ox TMB by HRP catalysis for quenching CPD fluorescence.Using citric acid and urea as precursors,CPDs with fluorescence characteristics were synthesized by microwave method.On the basis of ic-ELISA,the colorless TMB and H₂O₂ substrates were catalyzed by HRP to produce blue and oxidized TMB（ox TMB）.After adding CPDs,ox TMB reduced the fluorescence of CPDs through inner filter effect.Finally,the absorbance of ox TMB at 650 nm and the fluorescence intensity of CPDs at 430 nm were detected by a multifunctional enzyme-labeled instrument,so that ZEN could be quantitatively detected by colorimetric and fluorescence signals.After optimizing the parameters of the experiment,the standard curve equation of colorimetry was y=-0.630x+0.502,R²=0.983.The standard curve equation of fluorescence method was y=1550.153x+8946.036,R²=0.980.According to the standard curve of colorimetry,the limit of detection（LOD）was 0.048 ng/m L,and the linear range was 0.05 ng/m L to 0.5 ng/m L.According to the standard curve of fluorescence method,the LOD was 0.421 ng/m L,and the linear range was 0.05 ng/m L to 1.5 ng/m L.The recovery rate of ZEN in corn flour samples by colorimetry was between 93.42%and 101.67%,and the RSD was between 7.44%and 8.36%.The recovery rate of fluorescence method was between 90%and 100.25%,and the RSD was between 2.58%and 6.68%.The recovery rate of commercial ELISA kit was between 90.32%and 101.31%,and the RSD was between 5.28%and 8.71%.They all met the requirements of AOAC for sample addition and recovery experiment.The established HRP-triggered dual-signal detection method could mutually verify the results during detection,and had the characteristics of high sensitivity and good accuracy.3.Establishment of a multicolor colorimetric and electrochemical dual-signal detection of ZEN based on ALP-triggered in situ generation of PB NPsBased on ic-ELISA,the formation of PB NPs was triggered by ALP.The blue PB NPs in the solution were mixed with orange-yellow L-ascorbic acid-2-phosphate（AA2P）-Fe³⁺complexes to present visible multicolor changes.The absorbance at 700nm was measured by UV-vis to detect the generation of PB NPs.In addition,the consumption of K₃[Fe（CN）₆],another substance in the solution,could be monitored by electrochemical method.Thereby,it achieved quantitative detection of ZEN through multi-color colorimetric signals and current signals.After optimizing the parameters of the experiment,the standard curve equation of colorimetry was y=2.628 log C+1.845,R²=0.987.The standard curve of electrochemical method was y=358.169 log C+361.065,R²=0.993.According to the standard curve of colorimetry,the LOD of colorimetry was 0.04 ng/m L,and the linear detection range was 0.2 ng/m L to 0.8 ng/m L.According to the standard curve of electrochemical method,the LOD of electrochemical method was 0.08 ng/m L and the linear detection range was 0.125ng/m L to 0.5 ng/m L.The recovery rate of ZEN in corn flour samples determined by colorimetry was between 102.9%and 118.1%,and the RSD was between 2.6%and5.7%.The recovery rate of electrochemical method was between 81.7%and 114.8%,and the RSD was between 4.3%and 6.2%.The recovery rate of commercial ELISA kit was between 81.9%and 93.6%,and the RSD was between 2.8%and 5.1%.They all met the requirements of AOAC for sample addition and recovery experiment.Overall,this method had the advantages of good specificity,simple operation and intuitive judgment with naked eyes.4.Establishment of a lateral flow immunoassay strip based on PB NPs and HRP catalyzed DAB substratesThe color of PB NPs itself was used as the color signal,and PB NPs and HRP were used to jointly catalyze 3,3’-Diaminobenzidine（DAB）substrates,thereby achieving signal enhancement.After optimizing the experimental parameters,the visual limits of detection（v LOD）of this method before adding DAB substrate was 0.63 ng/m L,and the v LOD after adding DAB substrate was 1.25 ng/m L.At the same time,the color of T-line was converted into gray value by using Fiji image recognition software,and the standard curve equation of ZEN concentration and color gray value was y=-44.023x+39.625,R²=0.976.According to the standard curve,the LOD of this method was0.04 ng/m L,and its linear detection range was 0.08 ng/m L to 0.63 ng/m L.ZEN in corn flour samples was determined by this method,the recovery rate was between 88.89%and 102.59%,and the RSD was between 4.08%and 7.71%.The recovery rate of commercial ELISA kit was between 84.76%and 106.09%,and the RSD was between8.20%and 9.76%.The developed lateral flow immunoassay strip had an integrated structure,and there was no need to pre-mix the sample with the probe before use.After adding the sample for 8 minutes,it could be directly interpreted by the naked eye.In summary,this study combined CPDs,PB NPs,and immunological detection techniques to establish various methods for detecting ZEN.Among them,the ic-ELISA method didn’t require large instruments and could be used for high-throughput detection.The colorimetric and fluorescence dual-signal detection method broadened the detection range of ic-ELISA.The results of the multi-color colorimetric and electrochemical dual-signal detection methods could be preliminarily judged by the naked eye before instrument detection.The lateral flow immunoassay strip had an integrated structure,simple operation,short detection time,and the detection results could be read directly by the naked eye or quantitatively analyzed using image recognition software.Moreover,after replacing the corresponding antigen or antibody,these methods can also be used for the detection of other small molecules.