Extraction And Determination Of Aflatoxin B1 Based On Aptamer And Magnetic Nanoparticles

Pixian soybean paste has a long history and popularity,and enjoys the reputation of“Soul of Sichuan Cuisine”.With the popularization and promotion of Sichuan cuisine,the production and sales of Pixian soybean paste are also growing rapidly,and are deeply received by consumers at home and abroad.However,the Pixian soybean paste is still using a crude natural fermentation method,which is easy to produce aflatoxin and cause serious food safety hazards.As a fermented food,the composition of Pixian soybean paste matrix is very complicated,which brings a great challenge for the specific extraction and detection of aflatoxin.Therefore,in order to solve these problems,a new composite nano-material was prepared for the extraction of aflatoxin B1 from Pixian County.And on this basis,a method for detecting aflatoxin B1 with high sensitivity,good accuracy and strong specificity was developmented.The content and results are as follows:A magnetic solid phase extraction method based on AFB₁ aptamer and Fe₃O₄ was developmented.Firstly,the Fe3O4 were synthesized by solvothermal route.The TEOS and APTES was used to provide silica and amino groups on the surface of Fe3O4 The crystal form of Fe3O4 was characterized by X-ray diffraction?XRD?,the group of Fe3O4 was characterized by Fourier transform infrared spectroscopy?FT-IR?,and the surface morphology of Fe3O4 was characterized by transmission electron microscopy?TEM?.The AFB1 aptamer was assembled to the surface of the magnetic beads by glutaraldehyde reaction and avidin-biotin reaction to form a composite material?Fe3O4@SiO2-apt?,which was used to extract AFB₁ from the Pixian soybean paste.After optimization of the factors affecting the extraction efficiency,the optimal conditions were as follows:the amount of aptamer magnetic beads was 5 mg,the extraction time was 30 min,and the elution time was 5 min.Under the optimal conditions,the extraction recovery of AFB₁ by magnetic solid phase extraction was80.19%¹13.92%.In addition,after being reused twice,the extraction efficiency of AFB1 was maintained above 80%.This indicates that the material has good reusabilityAn aptamer-based hybridization chain reaction detection method was developmented by magnetic solid phase extraction as sample pretreatment.Firstly,a single-stranded DNA complementary to AFB₁?cDNA?and two hairpin probes?HP1 and HP2?were designed,and the terminal of HP1 was modified with FAM.cDNA as a trigger chain initiates continuous hybridization between HP1 and HP2 to assemble a nicked polymeric double-stranded DNA,the fluorescence produced by the fluorescent molecules on the double strand would not quenched by graphene oxide.In the presence of AFB₁,the aptamer binds to AFB₁to produce an enhanced fluorescence induced by HP1 and HP2 hybridization initiated by cDNA.After optimizing the relevant conditions,the optimal experimental conditions were as follows:the concentration of cDNA,HP1,HP2 and graphene oxide was 50 nmoL/L,60nmoL/L,60 nmoL/L and 50?g/mL,respectively.And the incubation time was 60 min.In the best case,this method has a linearity about 2⁶0 ng/mL?R²=0.971?.The recoveries ranged from 78.91%to 91.83%,and the relative standard deviation was 3.24%⁶.05%.The 12 kinds of Pixian soybean paste which come from different manufacturers were detected by this method.It was found that the average pollution level of AFB₁ was 3.42?g/kg in 10 samples and the highest pollution level was 4.50?g/kg,all of them are lower than the national standard limit of 5?g/kg.A method for detecting aflatoxin B1 in Pixian soybean paste by magnetic solid phase extraction-ultra performance liquid chromatography-triple quadrupole mass spectrometry?UPLC-MS/MS?was established.Electrospray ionization?ESI?,positive ion scanning multiple reaction monitoring?MRM?mode,and external standard method were used for quantification.After optimizing the mass spectrometry conditions and liquid phase conditions,a good linearity in the range of 0.39⁵0 ng/mL?R²=0.997?was obtained,the limit of detection is 0.08 ng/mL,and the limit of quantitation is 0.25 ng/mL.The recoveries ranged from 85.78%to 93.36%.It was compared with the hybridization chain reaction,and the correlation between the two methods was 0.9608,which indicated that the two methods have good accuracy and reliability,and can be well used for detection AFB₁ in actual samples.