Investigation Of Ochratoxin A And Aflatoxin B₁ Based On DNA Fluorescence Sensor

In China’s traditional diet culture,people’s main food is cereals,which are easily contaminated by mycotoxins.Among them,ochratoxin A（OTA）and aflatoxin B₁（AFB₁）are both highly toxic and common food contaminants that pose a serious threat to human and animal health.Therefore,it is of great practical importance to establish analytical methods that can rapidly and accurately detect OTA and AFB₁.Based on aptamers that can specifically identify the target,fluorescence resonance energy transfer（FRET）action,DNA nanostructure design,magnetic separation technology,we construct simple,sensitive,and specific DNA fluorescence sensors for OTA and AFB₁detection using fluorescence and paper-based analysis methods.The details of the study are as follows:1.Based on the specific recognition of aptamer and FRET interaction between FAM and BHQ1,a homogeneous fluorescence sensor for simple and rapid detection of OTA in food was constructed.The binding of OTA aptamer to its complementary DNA was simulated using NUPACK software.The competitive binding of OTA and complementary DNA to OTA aptamer was also investigated experimentally.Based on the software simulation and experimental results,the best complementary DNA was selected as c DNA1-BHQ1.The optimal binding method was selected as OTA aptamer and OTA were incubated for 30 min before adding c DNA1-BHQ1 for 30 min,the optimal incubation time was 60 min,the optimal c DNA to Apt dosage ratio was 1:1,and the p H value of the optimal reaction system was 7.4.Under the optimal detection conditions,the linear range of OTA detected by the sensor is 0.5-100 ng/m L,and the lowest detection limit is 0.13 ng/m L.The sensor was used to detect OTA in corn meal and wine samples with average recoveries of100.0%to 102.0%and 98.0%to 102.2%,respectively.The paper-based sensor preparation process is simple,portable and easy to miniaturize.In order to achieve convenient and rapid detection of OTA,a simple,low-cost and easy long-term storage paper-based fluorescence sensor was constructed for the detection of OTA in food based on the prepared homogeneous fluorescence sensor using paper as the sensor substrate.Under the optimal detection conditions,the linear range of OTA detected by the sensor is 10-2000 ng/m L,and the lowest detection limit is 1.2 ng/m L.The sensor can be successfully used to detect OTA in rice flour and Mung bean flour samples with average recoveries of 89.2%-111.0%and 89.0%-103.0%,respectively.2.A simple two-dimensional DNA nanostructure was designed and an X-shaped DNA fluorescence sensor was constructed to achieve efficient detection of AFB₁using a fast magnetic separation technique.The X-shaped DNA nanostructure consisted of DNA1,DNA2 and four strips of FAM-Apt.DNA1 and DNA2 form the backbone part of the X-shaped DNA nanostructure,with each of the four FAM-Apt strands partially complementing each other at both ends of the structure via base complementary pairing.DNA1 and DNA2 formed the backbone part of the X-shaped DNA nanostructure,with each of the four FAM-Apt strands partially complementing each other at both ends of the structure via base complementary pairing.Comparing the X-shaped DNA fluorescence sensor with the conventional shaped DNA double-stranded fluorescence sensor,we found that the X-shaped DNA fluorescence sensor can achieve signal amplification,save the amount of magnetic beads to reduce the cost of detection,reduce the LOD,and improve the detection range of AFB₁.By optimizing the experimental parameters,the optimal amount of magnetic beads（MBs）was determined as 1.5μL and the optimal incubation time of AFB₁was 40 min.Under optimal detection conditions,the linear extent of AFB₁detected by the sensor ranged from 0.05 to 100 ng/m L,with the lowest detection limit of 0.009 ng/m L.The average recovery rate for AFB₁was 92.6%to 107.3%,and for the black tea and corn meal samples was 92.1%to 104.4%.3.In this chapter,we designed a stable two-dimensional DNA nanostructure and constructed a tuning fork-shaped DNA（TF-DNA）fluorescence sensor for the simultaneous detection of OTA and AFB₁using a fast magnetic separation technique.DNA1 and DNA2formed the backbone part of the TF-DNA nanostructure,and FAM-Apt and ROX-Apt were partially complementary at the two ends of the structure,respectively.The experimental conditions were optimized,and the optimal amount of MBs was determined to be 3μL,the optimal DNA1 was DNA1-51,the optimal DNA2 was DNA2-45,and the optimal incubation time for the toxin was 60 min.Under the optimal detection conditions,the prepared TF-DNA fluorescence sensor detected OTA in the concentration range of 0.05-100 ng/m L with a detection limit of 0.015 ng/m L,and AFB₁in the concentration range of 0.1-100 ng/m L with a detection limit of 0.045 ng/m L.The sensor was highly specific for both OTA and AFB₁.The average recoveries of OTA and AFB₁were 94.1%-106.7%and 92.8%-110.6%for black tea and wheat flour samples spiked and tested,respectively.