The Construction Of DNA Fluorescent Sensor For Aflatoxin B1 Detection

Aflatoxin B1（AFB1） is a typical mycotoxin,which is easily retained in agricultural products and poses a great threat to humans and animals when accumulated to a certain amount.Therefore,accurate and rapid determination of AFB1 is essential for environmental analysis and food safety.In this paper,three kinds of DNA fluorescent aptasensors were constructed for the detection of AFB1 by making use of the advantages of simple operation,low cost and good stability of the fluorescent sensor,combined with the high affinity and specificity of the aptamer,and introducing different sensing strategies such as magnetic beads,DNA nanostructures,Exo Ⅲ and catalytic hairpin self-assembly.The specific research content is as follows:1.A novel nitrogen-doped carbon-supported palladium（C-N-Pd）material was synthesized and used as an energy acceptor for a fluorescent aptamer sensor for the first time.A fluorescent sensor for AFB1 detection was constructed using C-N-Pd as a new quenching material and Exo Ⅲ as an auxiliary signal amplification.The fluorescence response value of the sensor with Exo Ⅲ was increased by 74.7%compared with that without the enzyme.The fluorescent sensor showed good selectivity for AFB1 with a limit of detection（LOD）as low as 9 pg/m L.AFB1 was detected in corn,peanut and wine samples with LOD of 15 pg/m L,13pg/m L and 18 pg/m L,respectively.In addition,the proposed method was compared with high performance liquid chromatography（HPLC）,and the t-value was 1.588,which was less than the theoretical t-value of 4.303.The result indicats that the two methods had good agreement at the 95%confidence interval.The method is not only simple to operate,but also only takes2 hours from the preparation of the sensor to the realization of detection.2.Previous studies have found that single signal may produce false positives under the influence of the environment.Moreover,the auxiliary signal amplification strategy based on Exo Ⅲ is easily affected by temperature and p H.In this experiment,a ratio-type sensor based on fluorescence resonance energy transfer（FRET）is designed,and the ratio of fluorescence intensity at two different wavelengths is used as the response signal,which can greatly weaken the negative impact of complex environment on fluorescence and achieve the effect of internal correction.At the same time,the catalytic hairpin self-assembly without enzyme amplification was used to amplify the signal,which was used for the specific detection of AFB1 in food.Compared with the single signal mode,the ratio signal mode based on F_Cy3/F_Cy5 had a wider linear range（0.1-100 ng/m L）and higher correlation coefficient（R²=0.991）for AFB1 detection.For the spiking experiments of peanut,soybean and wine samples,the average recovery rates of three standard curves,including F_Cy3,F_Cy5 and F_Cy3/F_Cy5,were used as the reference value,and the ratio signal had higher accuracy than the single signal.At the same time,the existence of dendritic scaffolds not only improved the stability of the sensor,but also improved the efficiency of FRET.The response signal difference of the dendritic DNA nanostructure was5.7 times greater than that of the non-dendritic DNA nanostructure throughout the detection.In addition,catalytic hairpin self-assembly,an enzyme-free amplification strategy,was introduced to achieve signal amplification through the cycling of aptamer complementary strand（c-DNA）.3.Previous studies have found that the ratiometric sensor has higher accuracy,and the self-synthesized DNA nanostructures can improve the efficiency of FRET.In this experiment,h-DNA with hairpin structure was introduced,and gold palladium nanoparticles（Au-Pd NPs）were used as fluorescence quenching substrate for the first time to independently synthesize carbon spots（CDs）,and SYBR Green I（SG I）dye was introduced.A ratiometric sensor with F_CDs/F_{SG I} as signal was constructed for the specific detection of AFB1 in food.The addition of h-DNA greatly improves the efficiency of its loading aptamer（Apt）.The quenching effect of Au-Pd NPs on CDs was 1.4 times that of Au NPs and 3.4 times that of Pd NPs,respectively,and the quenching ability was significantly improved.The self-synthesized carbon spot and label-free probe SG I used in this sensor had cost saving advantages compared with labeled probes.In the range of 1-100 ng/m L,F_CDs/F_{SG I} showed a good linear relationship with the logarithm of the concentration of AFB1,and the LOD was as low as 0.07 ng/m L.At the same time,AFB1 in peanut and wine samples was detected by spiking,and the recovery rate was between 91%and 115%,indicating that the sensor showed high application potential in actual sample detection.