Study On Novel Green Extraction Method And Rapid Detection Technology For Fluoroquinolones In Freshwater Fish

Fluoroquinolones are broad-spectrum bactericidal antibiotics that are widely used in aquaculture because of their strong antibacterial activity.However,if it is used unreasonably and not completely metabolized,it will enter the human body through the environment and food,posing a serious threat to human health.Therefore,it is necessary to develop efficient,green,sensitive and rapid detection methods for fluoroquinolone antibiotics.This project is based on the simultaneous determination of several fluoroquinolone antibiotics in freshwater fish using a novel low eutectic solvent（DES）combined with high-performance liquid chromatography and aptamer combined with gold nanoparticle colorimetric method for the rapid detection of ofloxacin.（1）A method based on hydrophilic DES combined with ultrasound-assisted extraction for the simultaneous determination of seven fluoroquinolone antibiotics in freshwater fish was successfully established.The method uses DES as the extractant and utilizes ultrasonic-assisted extraction.After high-speed centrifugation to separate the solids from the liquid,the supernatant is measured directly by high-performance liquid chromatography with a photodiode array detector（HPLC-PDA）at 280 nm.The optimum experimental conditions were established by optimizing the type of DES,the molar ratio of DES synthesis,the volume of DES,the ultrasonic power and the ultrasonic time.The experimental results showed that a fixed molar ratio of 1:2,with choline chloride as the hydrogen bond acceptor and acetic acid as the hydrogen bond donor,resulted in a higher extraction rate of DES for fluoroquinolone antibiotics than other solvents.Meanwhile the volume of DES 4 m L,the sonication power 240 W and the sonication time 20 min.Under optimal extraction conditions,the linear range of the method was 0.05～2.00μg/g（r²=0.9973～0.9997）.The limits of detection were0.011～0.015μg/g,and the recoveries were in the range of 75.4%～86.5%.The intra-day and inter-day relative standard deviations were less than 4%.The results show that the use of ultrasound-assisted DES combined with HPLC-PDA extraction for detection is a simple,efficient,economical and green method with high potential for routine detection of fluoroquinolone antibiotics in freshwater fish.（2）A colorimetric method was developed for the detection of ofloxacin using the specific recognition function of ofloxacin by aptamers and the aggregation function of gold nanoparticles（Au NPs）by the cationic surfactant hexadecyltrimethylammonium bromide（CTAB）.Au NPs were synthesized by sodium citrate reduction.The assay conditions were optimized by optimizing CTAB concentration and aptamer concentration.The optimum conditions were:CTAB concentration 0.95μmol/L and aptamer concentration 1.5 nmol/L.Under the optimal conditions,the absorbance ratio of the systemΔA₆₅₀/A₅₂₀showed a good linear relationship with the concentration of ofloxacin.The method was capable of detecting ofloxacin in the concentrations range of 5 ng/m L to 75 ng/m L with a limit of detection was 0.295 ng/m L.The method is simple,rapid and sensitive and is expected to be applied in practical testing.