Study On Simultaneous Detection Of Ochratoxin A And Aflatoxin B₁ Based On Fluorescence Sensor

With the improvement of the quality of life,food safety has attracted more and more attention,especially the problems of mycotoxin contamination.Among them,ochratoxin A（OTA）and Aflatoxin B₁（AFB₁）are widely distributed in agricultural products and are very harmful to human health.More seriously,studies have shown that OTA and AFB₁ often coexist in agricultural products,and their toxicity is enhanced due to the synergistic and additive effects of toxins.Therefore,the establishment of a method that can detect OTA and AFB₁ at the same time is of great significance to food safety control,and it has obvious advantages in detection cost and time compared with single toxin detection method.Based on the advantages of good stability and high specificity of the aptamer,this project uses it as a specific recognition probe and combines a fluorescence analysis method with simple operation and fast signal response.With the help of DNA nanostructure and signal sensing strategies such as fluorescence resonance energy transfer（FRET）,a fluorescent aptasensor with simple operation,good specificity and high sensitivity has been constructed,which can be used to simultaneously detect OTA and AFB₁.The specific research content is as follows:1、Based on the FRET and aptamer-specific recognition,a simple double-labeled DNA strand was designed to be used as a signal probe,where the double-labeled DNA strand is complemented by the long chain C1,short chain C2 and C3.The two ends of C1 are labeled with Cy3 and Cy5,C2 and C3 are labeled with BHQ2,respectively.OTA aptamer and AFB₁aptamer can respectively replace C2 and C3 by chain replacement,which will cause the fluorescence intensity of Cy3 and Cy5 to change.Based on this,a simple and fast fluorescent aptasensor is constructed for simultaneous detection of OTA and AFB₁.The optimal reaction time for the toxin is 30 min.The standard curve of the sensor was established under optimal conditions.The linear ranges of OTA and AFB₁ were 0.01-100 ng/m L,0.1-100 ng/m L,and the detection limits were 0.007 ng/m L,0.03 ng/m L,respectively.The spiked corn samples and spiked wine samples were tested,and the recoveries obtained were in the range of 96%-104%and 92%-109%,respectively.In the specific study,even in the presence of interfering toxins20 times higher than the concentration of OTA and AFB₁,the prepared sensor still showed good selectivity.The method is simple to operate,and the whole process is less than 2 hours from the preparation of the sensor to the completion of the detection.2、DNA double-cross molecular structure that can detect Cy3 and Cy5 at the same time is designed as a signal probe;based on this,a fluorescent aptasensor that can simultaneously detect OTA and AFB₁ is prepared.The DNA double-cross molecule is composed of Cy3labeled OTA aptamer,Cy5 labeled AFB₁ aptamer,central circular chain C2,two long arm chains C1 and C3 labeled BHQ2 after denaturation and annealing.Among them,C1,C2 and C3 can form the backbone of the DNA double-cross molecule,and the aptamer of OTA and AFB₁ can be partially complementary to the two ends of the structure.The experimental parameters were optimized,and the optimal concentration of DNA double-crossing molecules was 200 n M,and the optimal p H of the buffer was 8.0.Under the optimal conditions,the linear range of OTA is 0.01-50 ng/m L and AFB₁ is 0.05-100 ng/m L,the detection limits are 0.008ng/m L and 0.04 ng/m L,respectively.For the spiked corn samples and spiked wine samples,the recovery rates obtained were in the range of 96%-101%and 92%-106%,respectively.In the specific study,even in the presence of interfering toxins 10 times the concentration of OTA and AFB₁,the prepared sensor still showed good selectivity.3、Using the three-dimensional structure of the DNA tetrahedron and the dynamic conformational transformation of the hairpin structure on the edges,a fluorescence aptasensor based on the FRET mechanism was designed for simultaneous detection of OTA and AFB₁.DNA tetrahedron is self-assembled by S1,S2,S3 and S4 after denaturation annealing.Among them,both end of S3 and S4 are labeled with Cy3 and BHQ2,Cy5 and BHQ2,respectively.OTA aptamer and AFB₁ aptamer can respectively mediate the opening and closing of the hairpin structure on the edge of the tetrahedron,and then regulate the spatial distance of Cy3,Cy5 and BHQ2 respectively,resulting in changes in the corresponding fluorescence intensity.The reaction time of the sensor was optimized,and the best reaction time was 30 min.The 100n M DNA tetrahedron concentration was optimized to obtain the best concentration.Under optimal conditions,the linear ranges of the aptasensor for OTA and AFB₁ detection are 0.01-100 ng/m L,0.05-100 ng/m L,respectively;the detection limits are 0.005 ng/m L and 0.01ng/m L,respectively.The recoveries of spiked corn meal and spiked wine samples were 95%-106%and 95%-101%,respectively.In the specific study,even in the presence of interfering toxins 20 times higher than the concentration of OTA and AFB₁,the prepared sensor still showed good selectivity.