Study On Analytical Methods Of Ochratoxin A Based On Nanomaterial-enabled Fluorescent Sensors

Ochratoxin A（OTA）is a toxic metabolite derived mainly from Aspergillus ochraceus and Penicillium verrucosum,which are widely presented as contaminants in a variety of products,including crops and their products.OTA is found to cause severe toxic effects,such as teratogenic,embryotoxic,genotoxic,neurotoxic,immunosuppressive,carcinogenic,and nephrotoxic to humans.Current analysis of OTA is conducted by various methods,although these methods have both high accuracy and sensitivity,they still have some shortcomings,such as time consuming,tedious sample pretreatment and expensive instruments.Therefore,it would be highly desirable to establish a rapid,convenient,low-cost,and sensitive method for OTA detection.Recently,fluorescent analysis methods have become hotspots in the detection field due to their advantages of high sensitivity,good selectivity,and adjustable fluorescence.Nanomaterials have unique optical and electrical properties,good biocompatibility,and better water solubility,which have become auxiliary methods for fluorescent sensors.Based on this,this project developed nanomaterials and aptamer-based fluorescent sensor for the determination of OTA in food and traditional Chinese medicine,which is expected to provide a novel tool for the rapid and sensitive detection of OTA.The main research works are as follows:1.Development of labled fluorescent aptasensor for detection of OTA based on Carbon nanoparticles（CNPs）oxide.FAM-modified aptamers were used as donors,and water-soluble CNPs oxides were used as acceptors.Utilizing folded structure formed by the aptamers and OTA could not combine with CNPs oxide,we constructed a fluorescent sensor based on CNPs oxide and labeled aptamer for OTA detection.The ultraviolet-visible spectrum（UV）was used to characterize CNPs oxides and their mixture with aptamers.The experimental conditions such as CNPs oxide concentration and mixing time were optimized.Under optimized conditions,the linear range of the fluorescent sensor for OTA detection was 10-80 ng mL^-1,and the detection limit（LOD）was 3.681ng mL^-1.The fluorescent sensor has good selectivity and has been used for OTA detection in wine samples with a recovery rate of 98.9%-106.11%.2.Development of label-free fluorescent aptasensor for OTA of detection based on porphyrin and CdTe Quantum Dots.In this study,water-soluble CdTe QDs were used as fluorescent probes,and（N-methyl-4-pyridy）porphyrin（TMPyP）was used as a quencher.A novel label-free fluorescent aptasensor utilizing the interaction between OTA-triggered antiparallel G-quadruplex and TMPyP for the rapid and sensitive determination of OTA was established.The Circular Dichroism（CD）spectrum was used to characterize the G-quadruplex formed by the aptamer and OTA,and the experimental conditions such as the concentration of TMPyP and the incubation time of the aptamer and OTA were optimized.Under optimized conditions,the LOD of the designed aptasensor was 0.16 ng mL^-1,with a linear range of 0.2 to 20 ng mL^-1 and good selectivity.Furthermore,this aptasensor was applied in Astragalus membranaceus samples with a recovery rate of 98.9-102.2%,indicating that the sensor can be used to detect OTA in actual samples.3.Development of“Turn off-on”fluorescent sensor for OTA detection based on ZnCdSe QDs and self-assembled zinc porphyrin.In this study,a self-assembled zinc porphyrin was used as a quencher,and a water-soluble ZnCdSe QDs was used as fluorescent probes.A“Turn off-on”fluorescent sensor was established for the detection of OTA.Using environmentally friendly dodecyl dimethyl betaine as“soft template”,zinc5,10,15,20-tetra（4-pyridyl）-21H-23H-porphine（ZnTPyP）was self-assembled into nanorods through a green process,which has significantly improved their water solubility and optical performance.Under the optimized experimental conditions,the newly developed fluorescent sensor showed a linear response in concentration range 0.5–80 ng mL^-1,with a LOD of 0.33 ng mL^-1,good stability,high selective,and short detection time.The excellent applicability of the system has been verifed in milk and coffee samples,with relative recoveries between 98.33%and 103.70%.The results obtained were further verified by Ultra-high performance liquid chromatography–tandem mass spectrometry（UHPLC–MS/MS）.