The Research On High Sensitivity And Rapid Detection Of Norfloxacin Based On Click Chemistry

The overuse of antibiotics in the world has caused a large amount of antibiotics to accumulate in the animal body and living environment,and long-term intake may cause serious harm to human health.In recent years,state’s supervision of food quality and safety has continued to deepen,and the standards for the limit of antibiotic residues in animal foods have become more stringent.However,there are still unscrupulous businesses that illegally use antibiotics in animal farming and try to evade supervision.Therefore,the development of highly sensitive and rapid detection methods for antibiotics in food is crucial for ensuring food safety.Traditional large-scale instrument detection methods are overly dependent on large-scale equipment and technicians,and it is difficult to meet the increasing detection demand.Immunological detection methods have the advantages of high sensitivity and specificity,and play an important role in the detection of antibiotics.However,as a hapten,the process of synthesizing a complete antigen from an antibiotic-coupled carrier protein is very complicated the stability of the product is very low.In view of the above deficiencies,this study took the fluoroquinolone antibiotic norfloxacin as a typical representative,and innovatively used the click chemistry synthesis method with high yield,high product stability and modular characteristics for the modification of hapten.The molecular weight and immunological activity were verified by fourier transform infrared spectroscopy,mass spectrometry and immunological methods,and two detection methods were established based on immunological analysis technology.The specific research contents are as follows:(1)Construction of norfloxacin modular detection system based on click chemistry.Norfloxacin-azide conjugate was synthesized by modifying the azide group at the carboxyl terminus of norfloxacin by the condensation reaction of amino and carboxyl groups.Norfloxacin-Biotin and norfloxacin modified primers were synthesized by coupling norfloxacin-azide conjugates with alkynyl-modified biotin or alkynyl-modified isothermal amplification primers using click chemistry(azido-alkynyl cycloaddition reaction).A highly sensitive immunological detection method for norfloxacin was developed by using nanomaterials to load biological enzymes and catalyzing substrates for signal amplification and a sensitive and rapid method for the detection of norfloxacin was developed by combining immunology with isothermal amplification techniques.(2)A high-sensitivity dual-signal detection technology for norfloxacin in milk based on click chemistry.In this chapter,a direct competitive immunoassay method was established to realize the chemiluminescence/fluorescence dual-signal detection of norfloxacin residues in milk.First,the bifunctional conjugate of norfloxacin-biotin was prepared by click chemistry.During the detection process,norfloxacin-biotin competes with free norfloxacin for the solid-phase antibody.And the norfloxacin-biotin immobilized on the antibody is coupled to the quantum dot microsphere(labeled with streptavidin-alkaline phosphatase)through the biotin-avidin system.Because of large specific surface area,the quantum dots were loaded with a large amount of alkaline phosphatase to amplify the signal,so as to achieve highly sensitive output of chemiluminescence and fluorescence dual signals.The detection limits of the two output signals(chemiluminescence and fluorescence)reached 3.40 pg/m L and 8.80 pg/m L,respectively,with a linear range of 0.013 ～ 12.48 ng/m L and 0.042 ～ 39.87 ng/m L.This method has good specificity and can distinguish different types of antibiotics.The spiked recovery test showed that the detection method could effectively detect norfloxacin in milk.At the same time,compared with other methods reported in the literature,this method has certain advantages of high sensitivity.(3)Research on the rapid detection technology of norfloxacin based on click chemistry and antibody-controlled isothermal amplification reaction.The innovation of this chapter lies in the combination of immunological technology and isothermal amplification technology to construct a novel norfloxacin biosensor.Norfloxacin was modified at a specific site at the 3’ end of the isothermal amplification primer by click chemistry reaction,and the end of the primer contained the specific recognition sequence of the nicking endonuclease Nt.Bsm AI.When the sample does not contain norfloxacin,the antibody specifically binds to the norfloxacin modified at the end of the primer.Due to its steric hindrance,it prevents the recognition and nicking of Nt.Bsm AI.The DNA chain cannot be nicked,and the DNA polymerizes enzyme Klenow Fragment cannot catalyze the synthesis of new DNA chains,thereby preventing the SDA reaction.On the contrary,the free norfloxacin in the sample competes with the norfloxacin modified at the end of the primer to bind with the antibody,resulting in the dissociation and release of the antibody from the primer.Therefore,Nt.Bsm AI smoothly binds to its specific recognition site and nicks,and the DNA polymerase Klenow Fragment with strand displacement activity catalyzes the synthesis of new DNA strands,thereby triggering the SDA cycle.The G-rich sequence generated by SDA reaction can be folded into a G-quadruplex,which binds to the dye thioflavin T,resulting in enhanced fluorescence signal.The constructed biosensor has a linear range of 0.1 ～ 500 ng/m L,and a detection limit of 0.04 ng/m L(S/N=3).Other types of commonly used antibiotics can be distinguished with good specificity.This method can effectively detect norfloxacin in food,environmental and biological samples.The detection system has high storage stability at 4 °C after freeze-drying,and can detect norfloxacin in only two hours,which has the potential for rapid on-site detection.Based on the modularity of click chemistry,norfloxacin was modified with azide groups,and biotin and DNA were modified with alkynyl groups,respectively and two detection systems for norfloxacin were established.All of them had high responsiveness to norfloxacin,realizing high sensitivity and rapid detection of norfloxacin,respectively.This study provides a new idea for the highly sensitive and modular detection of small molecules,and has certain application prospects.