Development And Application Of The Detection Methods For Mycotoxins And Heavy Metals Using Aptamer Recognition And Enzyme Catalysis-induced Chromogenic Reaction

Mycotoxins and heavy metals are important risk factors that endanger food safety.The pollution caused by mycotoxins and heavy metals is widely distributed and difficult to prevent and control,which contaminates crops,drinking water and food from farmland to dining table,and adversely affect human health and public health.Therefore,it is extremely essential to measure and monitor the pollution level.The purpose of this research is to develop fast detection methods towrads mycotoxins and heavy metals with advantages of short time-consuming,intuitive results and simple processes.This study focoused on four pollutants,Fumonisin B₁,zearalenone,cadmium and lead,nucleic acid aptamers with good specificity and high affinity were served as probes,and the visual detection based on enzymatic reactions was employed,taking the flexible advantages and signal enhancement capabilities of nanoenzymes and nanomaterials,combined with signal amplification systems,by the means of enzymatic reactions and colorimetry,a series of interrelated and progressive visual detection methods based on aptamers and enzymatic color reaction are developed,providing new ideas for enriching and improving food safety technology.Firstly,a detection assay based on aptamer-horseradish peroxidase?HRP?direct labeling for the detection of Fumonisin B₁ was developed.The biotin-labeled fumonisin B₁ aptamer was immobilized on the microtiter plate through the biotin-streptavidin reaction,and then HRP was labeled on the complementary chain of the aptamer by the biotin-streptavidin reaction again,the complementary chain competes with the target mycotoxin for binding to the aptamer.The aptamer preferentially captures the target mycotoxin.As the concentration of fumonisin B₁ increases,the color reaction signal decreases,the color of the solution changes from deep yellow to light yellow and colorless,achieving visual detection,and it can be further quantified by measuring the absorbance value of 450 nm.Under optimal conditions,good linearity was observed within the concentration range of 0.5 to 300 ng/mL,with a detection of limit of 0.30 ng/m L.The developed method has been used to detect corn and beer samples which reported have a high detection rate of fumonisin B₁,and the results are consistent with the enzyme-linked immunoassay method.Secondly,tyramine signal amplification technology is introduced to develop a detection system based on aptamers and tyramine-Au NPs signal amplification system for the detection of zearalenone.The biotinylated aptamer is immobilzed on the microplate,zearalenone and complementary chain compete for capturing the aptamer,and then the complementary probe connects the streptavidin-HRP complex.As a substrate of HRP,tyramine plays a signal amplification role,in the presence of hydrogen peroxide,a large amount of tyramine deposited around the reaction site of HRP.Through the biotin-streptavidin system,a large amount of catalase is combined,and then hydrogen peroxide and chloroauric acid are added to the system.Hydrogen peroxide can reduce chloroauric acid to Au NPs,and catalase preferentially catalyzes the decomposition of hydrogen peroxide.The concentration of hydrogen peroxide affects the reduction rate of chloroauric acid and generates agglomerated AuNPs with blue color or dispersed gold nanoparticles with red color,the concentration of zearalenone corresponds to the color of the solution,achieving the visual detection.Under optimal conditions,the detection range of this method is 0.2-200 ng/mL,and the detection limit is 0.11 ng/mL.This method is used to detect corn samples,and the results are consistent with the enzyme-linked immunoassay method.Thirdly,nanostructured peroxidase-like enzyme mimics are employed to initiate the enzymatic reaction,a detection system based on aptamers and AuNP-MoS₂ nanozyme for the detection of heavy metal cadmium was developed.The gold nanoparticles are loaded on the surface of MoS₂ nanosheets to form nanocomposites,and the Cd²⁺aptamer is immobilized on the mocroplate.The complementary chain of the aptamer is connected to AuNP-MoS₂nanocomposites through van der Waals force and coordination bond to form detection probes.The aptamer preferentially captures the heavy metal.As the concentration of Cd²⁺increases,the inensity of color reaction decreases,and the color of the solution tends to be yellow.After loaded by gold nanoparticles,the peroxidase-like catalytic ability of molybdenum disulfide is enhanced,which makes the signal enhancement.The discrimination of color changes and visualization effect exhibit more obvious.Under optimal conditions,the detection range of this method is 0.3-500 ng/m L,and the detection limit is 0.18 ng/m L.This method is used to detect white wine samples,and the results are consistent with national standard method graphite furnace atomic absorption spectrometry.Fourthly,the nanozyme composite composed of multiple nanozymes was explored;a detection assay based on aptamer and graphene/Fe₃O₄/AuNP nanozyme composite was developed for the detection of heavy metal lead.The lead aptamer is connected to the aminated magnetic bead to form a capture probe.Based on the graphene's peroxidase-like ability,graphene/Fe₃O₄/AuNP nanozyme is prepared.The Fe₃O₄ nanoparticles and gold nanoparticles which loaded on the surface of graphene also have certain peroxidase-like catalytic activity;the nanocomposites play a synergistic effect,and further promote the catalytic ability of graphene.The aptamers modified magnetic beads preferentially bind to the target.After the magnetic separation,the complementary oligonucleotide chain of the aptamer is adsorbed by graphene due to?-?conjugation,occupying the catalytic site of the nanozyme composite,which inhibits the activity of the nanozyme and affects the signal intensity of enzyme catalysis-induced chromogenic reaction,thereby achieves the visual detection.Under optimal conditions,the detection range of this method is 0.1 to 300 ng/mL,and the detection limit is as low as 0.057 ng/m L.This developed assay is used to detect tap water samples,and the results are consistent with the graphite furnace atomic absorption spectrometry.