Study Of The Detection Of AFB₁ And Ampicillin Based On Functional Nucleic Acids

In recent years,the problem of contamination of mycotoxins in food and the residues of veterinary drugs in food has been a hot issue of food safety that people pay close attention to.In order to solve the problem of food safety,new strategies have been developed by many researchers for the detection of food hazards.Among them,the biosensor based on aptamer(Apt)technology is a fast,sensitive,efficient and portable detection method,which can overcome the limitations of traditional methods(such as liquid chromatography,gas chromatography),including complex sample pretreatment,expensive instruments,long detection time and the need for professional technicians to operate the instrument.In this study,aptamers were used as recognition elements,the modified Enzyme Linked Immunosorbent Assay(ELISA)based on magnetic nanoparticles(MNPs)was developed for highly sensitive colorimetric detection of aflatoxin B₁(AFB₁).At the same time,a fluorescent sensing strategy based on hybrid chain reaction(HCR)technology was used to detect ampicillin(Amp).The main results are as follows:(1)Study on the detection of AFB₁ on the basis of aptamers and modified ELISA based on MNPsImprove and innovate on the basis of traditional ELISA.First,avidin-MNPs is used to be coupled with avidin-modified AFB₁ aptamer(MNPs@Apt).Through the adsorption of MNPs on HRP,MNPs@Apt@HRP detection probe is prepared.A combination of aptamer and antibody is used as a recognition element to jointly recognize the target.When the target exists,a sandwich structure is formed.By observing the 3,3',5,5'-tetramethylbenzidine(TMB)and H₂O₂ color reaction catalyzed by horseradish peroxidase(HRP),the visual detection of AFB₁was realized.The detection can also be completed by detecting the absorbance value.Under the best optimization conditions,in the linear range of 1×10^-8-1×10^-14 g/m L,the UV absorbance increases with the increase of AFB₁ concentration.The detection of AFB₁contamination in milk,peanuts,soy sauce,and vinegar has been completed,and it is basically consistent with the test results of commercial kits(the coincidence rate is 80.52%).(2)Ampicillin detection based on aptamer and HCR signal amplification technologyBased on HCR signal amplification technology,a sensor method for fluorescence detection of ampicillin was developed.First,through the DNA base complementary pairing,the carrier strand,aptamer and HCR priming strand are hybridized together to complete the preparation of the probe.The aptamer-HCR initiation chain was obtained by the ligation reaction of T4 DNA ligase(T4)and the degradation reaction of Exonuclease III(EXO III).The binding of aptamer-HCR was captured on a 96-polystyrene microwell plate by using the binding action of biotin-avidin.After the capture was completed,the HCR signal was amplified.In the presence of the target,the detection of ampicillin was achieved by observing the fluorescence intensity of SYBR Green I at 520 nm.In the linear range of 1×10^-10-1×10^-18g/m L,the fluorescence intensity decreases with the increase of the ampicillin concentration,and the detection sensitivity reaches 1×10^-18g/m L,which can meet the needs of rapid detection of trace ampicillin in food.Finally,the detection of ampicillin residues in milk is completed,and the Relative Standard Deviation(RSD)does not exceed 10%.