Development And Application Of Chloramphenicol Immunoassay Based On Fe-metal Organic Framework

Chloramphenicol(CAP),a broad-spectrum antibacterial antibiotic,has been widely used in animal husbandry breeding and food animal disease treatment.Due to its serious side effects for leading leukemia and the development of bacterial resistance,China,European Union,United States,and many other countries have banned the use of CAP as an antibiotic veterinary drug for food animals,and made it a mandatory detection item in the international trade of agricultural and sideline products.Using functional nanomaterials to construct biosensors have the advantages of good operability,high specificity,low cost,wide applicability,etc.And then it has been widely used in research fields including food safety,analytical chemistry and biomedicine.Metal organic Frameworks(MOFs)are a new type of functional nanomaterials with periodic network structure formed by the self-assembly of metal ions(metal clusters)and organic ligands,they possess the features of diverse chemical compositions,large specific surface areas,adjustable active sites,and easy modification of metal nodes/organic ligands,which are one of preferred choice for construction of biosensors.In this paper,based on two synthetic Fe-MOFs with good biocompatibility,colorimetry and fluorescence immunoassays were constructed for the detection of chloramphenicol residues in milk,respectively.The main contents are as follows:1.Construction of MOFs NH₂-MIL-101(Fe)based chloramphenicol colorimetric immunoassay and analysis of its signal amplification mechanismIn this chapter,we used ferric chloride as the metal ion source and diaminoterephthalic acid(NH₂-BDC)as organic ligand to synthesize MOFs NH₂-MIL-101(Fe)with peroxidase activity,then using it as the fixed carrier and loading horseradish peroxidase-labeled goat anti-rabbit antibody(HRP-IgG)-gold nanoparticles(AuNPs)composite structure,further successfully prepared immunoprobes with enhanced catalytic activity(MOFs-AuNPs@HRP-IgG).The Michaelis-Menten catalytic kinetic of the immunoprobes showed that the Michaelis constant of MOFs was 1/24 of HRP,and the maximum reaction rate of HRP was 3.6 times that of MOFs,revealing the mechanism of immunoprobes'catalytic signal amplification.We used CAP as the detection target,and the prepared immunoprobes were used as the enzyme-labeled secondary antibody to established the signal enhance indirect competition colorimetric immunoassay.The results showed that synthesized immunoprobes have good catalytic ability,and the LOD was 0.006?g·L^-1,which was decreased by almost 5 times compared with the ELISA method using the same antibody,the linear range was 0.008?0.108?g·L^-1,and the detection recovery rate of milk samples(0.006?0.300?g·L^-1)was 76.0?106.0%(3?),indicating that the method is suitable for ultra-sensitive detection of CAP.2.Construction of a chloramphenicol fluorescence immunoassay based on the pyrophosphate competitive hydrolysis of MOFs NH₂-MIL-88B(Fe)In this chapter,the high crystallinity and good dispersion MOFs NH₂-MIL-88B(Fe)were prepared through solvothermal synthesis method by introducing the non-ionic triblock copolymer Pluronic(F127)and acetic acid as stabilizer and proton-removing agent,respectively.We found and verified that the pyrophosphate(PPi)can form a competitive coordination effect with the carboxyl group of the organic ligand(NH₂-BDC)in MOFs,which can lead to the collapse of the MOFs structure and releases NH₂-BDC and Fe³⁺in 0.2 m M,then weaken the ligand-metal charge transfer(LMCT)of the MOFs network structure,thereby restoring the original fluorescence of NH₂-BDC.Based on this principle,we use synthetic high-stability MOFs as fluorescent signal sources to trace target recognition and prepare MOFs-labeled immunoprobes by chemically coupling CAP monoclonal antibodies through the active ester method(EDC/Sulfo-NHS).Then the direct competitive fluorescence immunoassay method was established on the microplate for the detection of CAP residues in milk samples.The LOD was 0.028?g·L^-1,the linear range was 0.05?0.75?g·L^-1,and the recovery rate was 91.8?112.3%(3?).This work uses a one-step method to release fluorescent signals,which is simple and time-saving,and the established analytical method also has practical value for the rapid and effective detection of CAP residues in food samples.In all,this researches have developed a new method for CAP detection,explored a new mechanism for MOFs to enhance sensing performance,expanded the application of MOFs in the field of food safety detection,and provided the valuable reference for the research and application of MOFs in related fields.