Study On The Detection Methods For Multiple Mycotoxins Based On Upconversion Nanoparticles And Aptamer Recognition

Food safety is connected with human life and health.Mycotoxin is an important small molecule pollutant endangering food safety,which has a strong toxic effect on humans and animals.Therefore,constructing accurate,sensitive,specific,and rapid detection methods for mycotoxin is of great significance to ensure food safety and human health.Upconversion nanoparticles(UCNPs)have the advantages of large anti-Stokes shift,long fluorescence life,good photochemical stability,low biotoxicity,resistance to photobleaching and light scintillation,and no autofluorescence interference,which exhibits great application potential in the biological detection of a complex matrix.In this thesis,808 nm-excited core-shell UCNPs were prepared by the high-temperature pyrolysis method and then used as fluorescence labels.By using mycotoxin aptamer as a specific recognition molecule and further combining the CRISPR technology,dual-signal detection,and paper-based analytical technology,respectively,three new biosensing methods with high sensitivity and specificity were constructed for mycotoxins.The main research contents include:1.A CRISPR-Cas12 a mediated fluorescence resonance energy transfer(FRET)sensing platform was constructed for deoxynivalenol(DON)based on the use of UCNPs and gold nanoparticles loaded MXene(MXene-Au)nanosheets as the energy donor-receptor pair and the aptamer as activation sequence.The binding of aptamer and DON inhibits the trans-cleavage ability of CRISPR-Cas12 a to the single-stranded DNA modified onto UCNPs,resulting in UCNPs being adsorbed to the surface of MXene-Au and the fluorescence is quenched.Polyacrylamide gel electrophoresis and SYBR Green I fluorescence experiments verified that the DON could effectively inhibit the activation of the trans-cleavage ability of CRISPRCas12 a.Under the optimum conditions,the relative fluorescence intensity shows a good linear relationship with the logarithm of DON concentration.The linear range is 1 ng/m L～500 ng/m L with the limit of detection(LOD)of 0.64 ng/m L.The recovery of DON in corn flour is96.2%～104.6%.This method integrates mycotoxin aptamer with CRISPR technology,which broadens the detection range of CRISPR-based sensors for non-nucleic acid small molecule food hazards.2.To improve the reliability and applicability of detection,a fluorescence-surfaceenhanced Raman scattering(SERS)dual-signal detection method was constructed for ochratoxin A(OTA)based on the conversion of aptamer conformation.The aptamer complementary sequence modified UCNPs and aptamer modified gold nanourchins(GNUs)were used as fluorescence labels and SERS enhancement substrates;the 6-carboxytetramethyl rhodamine(TAMRA)dye was utilized as both quencher and SERS reporter.The UCNPs and GNUs can form nanocomplexes through DNA hybridization.When the aptamer specifically binds with OTA to form a "stem-loop" structure,the TAMRA modified at the other end of the aptamer is far away from UCNPs and in the meantime is close to the surface of GNUs,which eventually leads to the recovery of upconversion fluorescence and the enhancement of SERS signal.Under the optimal conditions,a good linear relationship is observed between the relative fluorescence and SERS signals and the logarithm of OTA concentrations with linear ranges of0.01 ng/m L～100 ng/m L(LOD: 3.2 pg/m L)and 0.01 ng/m L～50 ng/m L(LOD: 8.6 pg/m L),respectively.The recoveries of OTA spiked in beer are 95.2%～103% and 92.4%～108% for fluorescence and SERS signals,respectively.The two detection signals of this method can be checked by each other,which is conducive to improving detection accuracy and reliability.3.To further improve the efficiency and convenience of detection,a dual-color paperbased sensor was constructed for simultaneous visual detection of zearalenone(ZEN)and OTA with the assistance of a smartphone.The dual-color UCNPs modified with aptamers and CuTCPP nanosheets are used as fluorescence donors and receptors,respectively.The ZEN and OTA located in the sample region of paper substrate migrate to two detection regions under the action of capillary force.After the specific binding of the mycotoxins with corresponding aptamers,the spatial distance between the dual-color UCNPs and Cu-TCPP nanosheets increased,and the green and blue fluorescence were recovered.Then the smartphone and color recognition software were used to collect fluorescent images and analyze RGB signals,respectively.Under the optimal conditions,the ΔG value of green fluorescent images and ΔB value of blue fluorescent images exhibit excellent linear relationships with the logarithmic ZEN and OTA concentrations,and the linear ranges were 0.5 ng/m L～100 ng/m L(LOD: 0.44 ng/m L)and 0.1 ng/m L～50 ng/m L(LOD: 0.098 ng/m L),respectively.The spiked recoveries in corn flour are 94.5%～103.7% and 92.2%～106.8% for ZEN and OTA,respectively.This sensor possesses the merits of low reagent and sample consumption,high sensitivity,desirable selectivity,low cross-reactivity,and on-site rapid detection.It provides a choice for rapid screening of multiple mycotoxins in complex food matrix at the same time.