Study On Detection Method Of Ampicillin In Milk And Tannic Acid In Feed Based On Up-conversion Fluorescence Technology

In recent years,the increasing demand for milk has promoted the rapid development of dairy farming.However,rapid and disorderly expansion has led to a lot of chaos,such as the overuse of antibiotics to treat infectious diseases in dairy cows,which may lead to mass transfer to milk and harm human health;The antioxidants added in the feed accumulate excessively in the animal body,damaging the animal’s health.Therefore,it is particularly important to accurately determine antibiotics in milk and antioxidants in feed.At present,most of the traditional detection methods require expensive equipment and professional personnel to operate,and cannot meet the needs of on-site detection.One of the effective ways to solve the above problems is to construct a biosensor method to realize the rapid,economical and real-time detection of the target object.Upconversion nanoparticles have been widely used to construct fluorescent biosensors for detection because of their unique anti-Tusks properties,strong stability and low background interference.Single-atom nanozymes are also popular research objects for the construction of colorimetric biosensors due to their excellent catalytic properties.Accordingly,this thesis intends to carry out the following research:（1）Construction of an aptamer biosensing based on fluorescence energy resonance transfer between upconverting nanoparticles and gold nanoparticles and its application to the detection of ampicillin in milk.Up-converting particles（UCNPs）are used as energy donors and gold nanoparticles（AuNPs）are used as energy acceptors.The detection mechanism is based on the quenching of the fluorescence of UCNPs by AuNPs and the specific binding of ampicillin to its aptamer,which changes the conformation of the aptamer.Therefore,AuNPs aggregated in different degrees in saline solution,and the resonance energy transfer between it and UCNPs also changed accordingly.Under the optimal conditions,as the concentration of ampicillin increased from 10 ng/m L to 150 ng/m L,the fluorescence intensity showed a linear upward trend,and the detection limit was 3.9 ng/m L.The aptasensor has good selectivity for ampicillin and is not interfered by other antibiotics,and has been successfully used for the detection of ampicillin in milk with high accuracy.（2）Preparation,characterization and mechanism exploration of FeCe/NC novel single-atom nanozyme.Fe³⁺and Ce³⁺were introduced into ZIF-8 by chemical doping method,and a novel FeCe/NC single-atom nanozyme with high activity was prepared by adjusting the calcination temperature.Then,FeCe/NC was tested and characterized by various methods.Then,the enzyme activity type and catalytic mechanism to which FeCe/NC belonged were studied.The study found that FeCe/NC belongs to the class of oxidases,and the mechanism of catalyzing 3,3’,5,5’-tetramethylbenzidine（TMB）is to directly oxidize O₂ in the air into·OH,O₂^·-and ¹O₂.Then,comparing the enzyme reaction kinetics of FeCe/NC and Fe/NC,it was confirmed that the newly prepared FeCe/NC single-atom nanozyme had higher catalytic activity.In addition,FeCe/NC was found to have an inhibitory effect on sulfhydryl groups,and the preliminary construction of a colorimetric biosensor realized the sensitive detection of cysteine（Cys）.The linear response range of Cys was 5.0-50.0μM,and the R² was 0.993.（3）A fluorescent colorimetric dual-signal biosensor based on UCNPs and FeCe/NC was used to detect the content of antioxidant tannic acid in feed.The detection mechanism is that FeCe/NC has oxidase-like properties,can catalyze O₂ to generate·OH,O₂^·-and ¹O₂,and oxidize colorless TMB to blue ox TMB,and has an absorption peak at 652 nm.At the same time,the generated ox TMB will have an inner filter effect with UCNPs,which will reduce the fluorescence of UCNPs.When tannic acid exists,it can scavenge free radicals and inhibit the generation of blue ox TMB,resulting in a decrease in the absorbance at 652 nm and a recovery of the fluorescence of UCNPs.The reduction of absorbance and the recovery of upconversion fluorescence have a linear relationship with the concentration of tannic acid,and the fluorescence colorimetric dual-signal biosensor constructed on this basis can realize the sensitive detection of tannic acid.Under the optimal conditions,the linear response range of tannic acid was 1.0-6.0μM,the R² of the colorimetric method was 0.995,and the detection limit was 0.27μM;the R² of the fluorescence method was 0.993,and the detection limit was0.19μM.The sensor has good selectivity to tannic acid,and was successfully used to detect tannic acid in real sample feed with high recovery rate.