The Construction Of Fluorescence-magnetic Nanosensor Platform And Its Application In The Detection Of Mycotoxin In Grain

Mycotoxin pollution in grain seriously threatens human health and causes huge economic losses.Therefore,it is of great significance to establish an accurate,rapid and sensitive mycotoxin detection method for ensuring food quality and safety.Fluorescent nanosensors have the characteristics of high sensitivity and fast response,while magnetic nanosensors have low background interference and easy pretreatment.Combining fluorescence and magnetic sensing technologies to construct fluorescence-magnetic nanosensors can realize the complementary advantages of the two,which has a good application prospect in the field of food safety rapid detection.In this thesis,mycotoxins in grain were taken as detection objects,we construct magnetic relaxation/fluorescence dual-mode sensor,magnetic encoding fluorescence immunosensor and magnetron fluorescence visualization sensor for the sensitive and rapid detection of mycotoxins in grain,respectively.The main research content of the thesis includes three parts:（1）A magnetic relaxation/fluorescence dual-mode sensor based on Gd₂O₃:Eu³⁺nanoparticles were constructed for the detection of AFB₁ in wheat and corn.It is found that the synthesized Gd₂O₃:Eu³⁺nanoparticles can generate fluorescence and magnetic relaxation signals at the same time,and the signal intensity is dependent on its concentration.Therefore,Gd₂O₃:Eu³⁺conjugated to AFB₁ polyclonal antibody to prepare a signal probe,and magnetic beads（MB）were conjugated to AFB₁ monoclonal antibody to prepare a separation probe.A sandwich type immuno-optical-magnetic dual-mode sensor was constructed,which was successfully used for the rapid and highly sensitive detection of AFB₁ in wheat and corn.Under the optimal conditions,the linear ranges of magnetic relaxation and fluorescence signals for AFB₁ detection is 0.5～100μg/kg,and the detection limits are 0.142μg/kg and 0.049μg/kg,respectively.（2）A competitive immunosensor based on magnetic coded fluorescence nanoparticles was constructed for simultaneous detection of AFB₁,deoxynifuenol（DON）,ocher atoxin A（OTA）and zealenone（ZEN）in wheat.Firstly,two kinds of nanoparticles with different magnetic responses（S-MNPs and W-MNPs）were synthesized by regulating the amount ofγ-Fe₂O₃in the silica microspheres（MPS）.Based on the first study,fluorescent nanoparticles(Gd₂O₃:Eu³⁺and Gd₂O₃:Tb³⁺)with emission wavelengths of 609 and 541 nm were synthesized by doping luminescent rare earth ions(Eu³⁺or Tb³⁺).Combined with magnetic nanoparticles,a 2×2 array encoding four mycotoxins was constructed.During the assay,the mycotoxins are first separated according to the magnetic response difference,and then each mycotoxin is quantified by the different emitted fluorescence signals.The detection limits of AFB₁,DON,OTA and ZEN were0.032,0.141,0.097 and 0.376μg/kg,respectively.The recoveries of wheat samples ranged from81.6 to 120.0%.（3）A fluorescence visualization sensor based on G-quadruplex DNAzyme was constructed,combined with magnetic chip and portable fluorescence detection platform to achieve rapid,sensitive,and on-site detection of OTA in wheat.It was found that OTA aptamer can self-assemble with two segments of DNA and Hemin chloride to form G-quadruplex DNAzyme structure,which can catalyze the oxidation reaction of ADHP with H₂O₂,turning the solution red and producing red fluorescence.When the target is introduced,the G-quadruplex DNAzyme structure breaks down,leading to a decrease in catalytic activity.On this basis,magnetic DNAzyme（MB-DNAzyme）probe was prepared by coupling OTA aptamer with MB,and a magnetic chip integrating reaction,washing and detection was designed.The detection process was completed by shuttle of the probe on each slot of the chip,which greatly reduced the influence of the background signal and improved the detection efficiency and sensitivity.The sensor was successfully used for the detection of OTA in wheat samples with the detection limits of 0.011μg/kg under fluorescence mode and 0.008μg/kg under colorimetric mode.