| The official release of the Announcement No.194 of the Ministry of Agriculture and Rural Affairs on July 10,2019 marked the withdrawal of 12 growth-promoting feed additives such as chlortetracycline from the stage of history.Chlortetracycline(CTC)is a broad-spectrum tetracycline antibiotic with the advantages of wide antibacterial range,low price and significant effect.This becomes the main reason for the abuse of CTC in the breeding industry,leading to food safety issues such as excessive residues in livestock and poultry products.In today’s society,many testing methods for chlortetracycline residues in foods reveal many shortcomings such as reduced sensitivity,long operation time,many false positives,weak selectivity,and so on.Thus,developed a good sensitivity,good selectivity chlortetracycline rapid detection is important for controlling residue chlortetracycline food.Carbon quantum dots are a kind of new nanomaterials,which have the advantages of strong fluorescence,non-toxicity,green,and strong biocompatibility.The use of food safety testing is also increasing.In this study,hawthorn was used as a raw material to prepare nitrogen-doped carbon dots(N-CDs),and to understand their various properties through a series of characterization.Using the excellent fluorescence properties of N-CDs and immunoassay technology,two methods for rapid detection of CTC have been established.The main research contents and results are as follows:1.Preparation and characterization of N-CDs.A one-step hydrothermal method was used to prepare and characterize N-CDs with hawthorn as the carbon source and diethylenetriamine(DETA)as the nitrogen source.By optimizing the amounts of hawthorn and DETA,reaction time and temperature,the optimal preparation conditions were determined to be 0.25 g of hawthorn powder,300 μL of DETA,20 mL of ultrapure water and heating at 180℃ for 10 h.The N-CDs were characterized by transmission electron microscope,X-ray photoelectron spectroscopy and other equipment,and it was found that the N-CDs glowed blue under ultraviolet light,the best excitation and emission wavelengths were 370 nm and 447 nm,and the quantum yield was as high as 22.96%;it has good dispersibility,uniform size distribution and an average particle size of 3.17 nm.Approximately spherical;with good water solubility,the main components are C,N,O,and rich in oxygen and nitrogen functional groups.The best fluorescence performance is when BR buffer,pH 8 and diluted 100 times.2.A method for rapid detection of CTC was established based on the N-CDs fluorescence quenching technology.In this study,factors such as pH value,BR buffer dosage,N-CDs concentration,reaction temperature,and reaction time were optimized to explore the best detection conditions.The best detection conditions were 3 μg mL-1 N-CDs,1 mL pH 8 BR buffer and room temperature.The linear detection range of this method was between 0.4～20 μg mL-1,and the detection limit was 73 μg L-1.The detection recovery rate of CTC in pork samples was 93.62%～103.18%.This method was green,simple,fast,and highly sensitive,but it has poor specificity in tetracycline drugs.3.A method for rapid detection of CTC was established based on N-CDs fluorescent enzyme-linked immunosorbent technology.Based on the development of N-CDs fluorescence quenching technology in the early stage,this study combined with enzyme-linked immunoassay technology,by optimizing the concentration of the original coating and antibody dilution,blocking solution,diluent and other factors,the original concentration of the coating was determined to be 5 μg mL-1,the antibody dilution factor was 6000 times,the blocking solution was 0.5%skimmed milk,and the detection effect was best when the dilution solution is PBST.The detection range of this method was between 0.032～20 ng mL-1,the linear range was 0.6～2.8 ng mL-1,and the detection limit was 0.82 ng L-1.This method greatly improved the sensitivity and specificity,and was used for the rapid detection of CTC residues in pork.Conclusion:N-CDs prepared with hawthorn as raw material had many advantages such as green environmental protection,simple preparation,wide source,low cost,strong water solubility,good biocompatibility and good fluorescence performance,and had huge application potential;Based on the phenomenon that CTC can quench the fluorescence of N-CDs,the established method for detecting CTC with N-CDs had the advantages of environmental protection,simplicity,rapidness,high sensitivity and good selectivity;Based on the combination of enzyme-linked immunoassay technology and fluorescence quenching technology,the product of TMB chromogenic solution catalyzed by HRP can cause fluorescence quenching of N-CDs.The established fluorescence enzyme-linked immunoassay method for detecting CTC improved the performance of ELISA method.The detection signal was used for the detection of CTC residues in pork.It had the advantages of environmental protection,simplicity,rapidity,good sensitivity and high specificity.It proposed new ideas in the preparation and application of carbon quantum dots and the expansion of the field of food safety detection. |
