Construction Of Electrochemical Biosensor For Aflatoxin M1 In Dairy Products Based On Covalent Organic Frameworks TpBD

Aflatoxin M1(AFM1)is a product of hydroxylated metabolism after animals ingeste aflatoxin B1 contaminated feed.Its toxicity and carcinogenicity are second only to aflatoxin B1.AFM1 mainly exists in milk and dairy products.The presence of AFM1 in raw milk causes serious harm to the whole dairy industry chain.Covalent organic frameworks are crystalline porous organic material.As a new porous nanomaterial,covalent organic frameworks have excellent physical and chemical properties,such as adjustable pore size,large specific surface area,good thermal and chemical stability,and have a good application prospect in separation,gas storage,catalysis,photoelectric sensing and other fields.However,there are few reports in the field of electrochemical sensing.In this paper,Tp BD(a kind of covalent organic frameworks)was used as the electrode modification material,AFM1 was used as the research target,combined with enzyme-linked immunosorbent assay and magnetic separation technology to construct the AFM1 electrochemical biosensor.The main research contents and results are as follows:(1)An electrochemical enzyme-linked immunoassay sensor for the detection of AFM1 in dairy products was constructed based on the covalent organic frameworks Tp BD modified glassy carbon electrode(GCE)and the rolling circle amplification technique.By constructing a primer-Au NPs-aptamer/AFM1/AFM1-antibody sandwich structure in a microplate and performing a rolling circle amplification reaction,the generated DNAzyme as catalyst to catalyze p-aminophenol to produce 3-aminophenazine.Tp BD/GCE was prepared by the drop coating method.Based on the ?-? adsorption and pore size matching effect of 3-aminophenolazine(10.4 ?)on Tp BD(23 ?),3-aminophenazine was enriched on the surface of the electrode and differential pulse voltammetry method was used for the determination of 3-aminophenazine.AFM1 incubation time,primer/aptamer ratio,rolling circle amplification reaction time,T4 DNA ligase concentration,phi29 DNA polymerase concentration,p-aminophenol oxidation time,hemin concentration,and H2O2 concentration were optimized.Under the optimized experimental conditions,an electrochemical-enzyme-linked immunoassay was constructed for the determination of AFM1 in dairy products.The linear range of this method was 0.5~80 ng/m L,and the detection limit was 0.15 ng/m L(signal-to-noise ratio S/N=3).On this basis,it was successfully applied to the determination of AFM1 in dairy products(milk powder,skimmed milk,milk beverage).The recoveris were 93.37%~104.01%(n=3),and the RSDs were less than 3.91%.(2)An electrochemical biosensor based on in-situ growth of covalent organic frameworks Tp BD on the electrode surface and magnetic separation technology was constructed to detect AFM1 in dairy products.The p-aminobenzoic acid was modified on the electrode surface by cyclic voltammetry,and then ethylenediamine was modified on the electrode surface by the condensation reaction between the amino group and the carboxyl group.Then Tp BD was covalently bonded on the surface of the glassy carbon electrode by magnetic stirring at room temperature to prepare a Tp BD-GCE modified electrode.AFM1 aptamer and ferrocene-modified signal probe were modified on the surface of magnetic nanoparticles.The signal probe and AFM1 aptamer were partially complementary.When there was AFM1,AFM1 interacted with the aptamer to dissociate the signal probe.The signal probe was adsorbed on the surface of Tp BD-GCE by ?-? interaction,and the ferrocene in the signal probe was determined by differential pulse voltammetry,thus an electrochemical biosensor for detecting AFM1 in dairy products was constructed.The process of electrode surface modification of Tp BD was characterized by electrochemical method,Raman spectroscopy,and scanning electron microscope.The linear range of this method was 0.5~100 ng/m L.The signal of the sensor kept at 76% of the original signal after 15 days storage at room temperature.AFM1 in milk samples(milk powder,skimmed milk,milk beverages)was determined by standars addition method.The recoveries were 80.74%~111.22%(n=3),and the RSDs were less than 3.32%.