Development And Application Of Portable Quantitative Detection System For Sulfonamides And Tetracyclines In Honey

Since the discovery and separation of the first antibiotic,penicillin,in 1945,antibiotics have broadly been used for medicine,animal husbandry,aquaculture and food processing industries over the past few decades.The extensive use of antibiotics in the field of animal breeding is a major contributing factor to drug residues in animal-derived foods.For a long time,honey is popular with people for its edible and medicinal values.For the high quality and high yield of honey,it is necessary to guarantee the health of honeybees.Therefore,beekeepers use antibiotics to prevent bees from diseases,which has been the main reason for antibiotic residues in honey.In addition,the discharge of untreated or partially treated medical and industrial wastewater contains a large number of antibiotics.These antibiotics as well as pesticides used in agriculture have caused ground and vegetation pollution,especially polluting the flowering plants,also resulting in the contamination of antibiotics in honey.Sulfonamides(SAs)and tetracyclines(TCs)are main antibiotics used in bee breeding.Long-term consumption of honey containing these antibiotics can cause damage to human health,thus many countries set the maximum residue limits(MRLs)for antibiotics in honey.For example,the MRLs of SAs and TCs in honey in China are both 50 μg/kg.In order to ensure the food safety of honey,many detection methods are used to monitor the antibiotic residues in honey,such as high performance liquid chromatography,capillary zone electrophoresis,microbiological assay,enzyme-linked immunosorbent assay and colloidal gold immunochromato-graphy assay.However,these methods either relies on large-scale analytical instruments and professionals,which cannot meet the requirements of on-site rapid detection,or lacks high sensitivity and capability of quantitative detection.In this study,we design a honey antibiotic detection system(HADS),which includes lateral-flow immunochromatographic assay(LFIA)-based strips,quantum dot microsphere(QDM)labeled antibody probes and a handheld sensor reader combined smartphone.The LFIA strip with one sample pad but two channels is designed supporting dual antibiotic simultaneous detections on SAs and TCs.It can specifically detect the targets since SAs and TCs in samples compete with SA-BSA antigen and TC-BSA antigen on the test lines for QDM probes conjugated with the SA monoclonal antibody(m Ab)and TC m Ab.Quantum dots(QDs)have the advantages of adjustable emission wavelength,wide excitation spectrum and narrow emission spectrum,high fluorescence intensity and strong anti-photobleaching ability.We adopted quantum dot microspheres(QDMs)as markers,which solved the problems of biological toxicity and poor water dispersion of QDs,and significantly amplified the fluorescence signal.The handheld sensor reader is designed for automatic image recording,fluorescence signal extraction and antibiotic quantification,thus ensuring the detecting accuracy.HADs can simply and rapidly detect antibiotic residues in honey at room temperature by only requiring honey dilution,QDM-honey mixture,sample addition,room temperature incubation and quantitative detection,without any large and expensive equipment.A single sample testing can be finished within 20 min,and only costs 75 cents(～5 RMB).According to the results,HADS can specifically detect TCs and SAs in honey.For sulfadiazine and sulfamethazine,the detection limits were both 0.4 μg/kg,and the linear detection ranges were 0.4 ～ 400 μg/kg and 0.4 ～ 100 μg/kg;for tetracycline,chlortetracycline and oxytetracycline,the detection limits were 0.4 μg/kg,0.8 μg/kg and 4 μg/kg,with the linear ranges of 0.4 ～ 200 μg/kg,0.8 ～ 400 μg/kg and 4 ～ 800 μg/kg,respectively,thus satisfying the honey SA and TC residue detecting requirements established by China and European Union.In addition,both the QDM probes and the LFIA strips can be well stored in vacuumsealed bags at 4 °C for at least 6 months.Considering the low detecting limit and the wide quantitative detecting range as well as the advantages as simple operation,fast speed,low cost,high sensitivity and accuracy,the proposed HADS can be potentially used in many rapid on-site testing occasions such as import and export screening,food safety inspections and even individual consumer detections.