Surface Enhanced Raman Detection Of Aflatoxin B1 Based On Nano-composite Materials

Aflatoxin B1（AFB1）,relative to other mycotoxins such as zearalenone（ZEN）,is the most toxic natural harmful secondary metabolite,which is classified as Group I carcinogen.It can seriously threaten human health through direct or indirect means.The indirect mean is to induce the contamination of agricultural products such as peanuts through humid and heat conditions,which have low probability of damage in conventional food processing such as heat treatment and pasteurization.Moreover,it is still challenging to quickly monitor trace toxin with the existing technology.If it is consumed by the human for a long time,it leads to hepatocellular carcinoma in severe cases.Hence,constructing a novel detection method has important application value.As a spectroscopy technique that provides molecular structural information,surface-enhanced Raman scattering（SERS）refers to the enhanced surface electromagnetic field generated when excited light irradiates metal nanomaterials,which amplifies the weak Raman signal of molecules by several orders of magnitude Based on the advantages of high sensitivity,and resistance to fluorescence background interference,SERS has become a research hotspot in trace detection of food safety.However,unstable SERS signal easily reduces the sensitivity.Therefore,this work focuses on improving the quantitative signal of detection,combining the signal amplification advantages of nanomaterials and enzymatic reactions,using aptamers with good selectivity for the target,and constructing a simple and efficient SERS sensing method for rapid monitoring of low-concentration AFB1 in peanuts.Specifically,the work consists of:1.Based on the advantages of separation and purification for magnetic nanoflowers as SERS substrates,a label-based SERS sensor with stable SERS single probe was constructed for AFB1 detection.The Raman signaling molecule 4-mercaptobenzoic acid（4-MBA）was embedded in core-shell gold-silver nanostars（Au-4MBA@Ag NSs）,and the AFB1 aptamer was modified on Ag shell as highly sensitive signaling probes.Au-Ag alloy and the tip effect of the anisotropic nanoparticles caused a significant enhancement of the SERS signal.Meanwhile,ferric oxide@molybdenum disulfide nanoflowers（Fe₃O₄@Mo S₂ NFs）serve as magnetic substrates.The two parts are assembled through the non-covalent interaction between Mo S₂ nanosheets and nucleic acid strands,and the 4-MBA Raman signal is enriched by short-time magnetic separation.In the detection system,the specific binding of AFB1 to the aptamer induces part of the probe to detach from the substrate,resulting in a linear decrease of the Raman signal(I_4-MBA).The results showed that the sensing method exhibited a linear correlation（correlation coefficient R²=0.986）over the AFB1 concentration range of 0.1 ng/m L to 1000ng/m L,the detection limit was 58.9 pg/m L,and the recovery of spiked peanut was 97.9%to99.4%.2.Based on graphene oxide（GO）with intrinsic Raman signal as a corrected internal standard（IS）and amplifier for 4-MBA signal,a novel ratiometric SERS sensor for reliable detection of AFB1 was constructed,to improve the sensitivity decreased due to the uncontrollable fluctuation of a single signal in the previous chapter.4-MBA was embedded in core-shell gold-silver nanospheres（Au-4MBA@Ag NPs）,and the AFB1 aptamer was coupled to the Ag shell as a SERS signal probe.The GO film as protective layer was modified on gold nanoparticles/indium tin oxide glass（GO/Au NPs/ITO）to prepare stable internal standard substrates,and its good portability is beneficial for on-site detection.The AFB1 aptamer adsorbed on GO throughπ-πstacking,and the synergistic effect of the assembly promoted the further amplification of the 4-MBA signal.With the increase of AFB1 concentration,the high affinity of the aptamer to the target allows the probe to be gradually released,causing a reduction in the signal intensity of 4-MBA(I_4-MBA),while the signal intensity of GO(I_GO)remains relatively stable.The results showed that the sensing method based on the ratio value(I_4-MBA/I_GO)exhibited a good negative linear correlation in the AFB1 concentration range of0.0001 ng/m L to 100 ng/m L,the signal normalization improved the R² to 0.992,and the detection limit was 0.1 pg/m L,and the recovery of spiked peanut was 91.6%to 103.9%.3.Based on the catalase-like catalytic reaction of cuprous oxide nanomaterials,an output-signal SERS sensor was constructed for reliable detection of AFB1.The advantage is to change the indirect detection method based on SERS probes in the previous two chapters into a method for enzymatic reaction to generate products with Raman signal and further amplify the signal.Partial complementarity of nucleic acid sequences triggers the self-assembly of materials to form a novel and controllable nanocomposite.In this work,AFB1 aptamers and complementary chains were modified on hollow gold-silver alloy nanospheres（Au-Ag NPs）with SERS enhancement effects and cuprous oxide@gold nanocubes（Cu₂O@Au NCs）with catalytic effects,respectively,and self-assembled to form"satellite"nanocomposites.The aptamer binds to AFB1 more preferentially to regulate the self-assembly of nanocomposites.Next,in the3,3’,5,5’-tetramethylbenzidine/hydrogen peroxide（TMB/H₂O₂）reaction system,the nanocomposite acts as a nanozyme to catalyze and generate its oxidation product ox TMB（intrinsic Raman signal）.It diffused around the assembly,significantly amplifying the signal intensity.The results showed that in the AFB1 concentration range of 0.001 ng/m L to 100ng/m L,the sensing method based on the output signal value(I_{ox TMB})exhibited a good negative correlation（R²=0.991）,the detection limit was 0.7 pg/m L and the recovery of spiked peanut was 94.7%to 101.4%.