Study On Fluorescence Sensing Based On Luminescent Metal-organic Frameworks For Detection Of Antibiotics TC/SMZ/AMP In Pork

Antibiotics are widely used as feed additives and drugs for the treatment of bacterial infections in the pig breeding process owing to their low cost,easy absorption and board-spectrum bactericidal activity.However,the residual antibiotics in pork have seriously affected meat quality and consumer health,which have become a prominent issue of concern.Traditional methods such as high-performance liquid chromatography（HPLC）and liquid chromatography-mass spectrometry（LC-MS）have good accuracy and high reliability,but there are shortcomings such as cumbersome pretreatment procedures,high analytical cost,and time-consuming.Therefore,there is an urgent need to develop simple and efficient methods for the detection of antibiotic residues in pork.In recent years,fluorescence analysis has received substantial attention due to its high sensitivity,easy operation and good reproducibility.In particular,the ratiometric dual-channel fluorescence analysis method overcomes the interference of single-channel-based model susceptible to environmental and instrumental factors to significantly improve the reliability and accuracy of the analysis of the target to be measured,however,traditional fluorescence materials have limitations in luminescent form,structure,and performance,which are not conducive to the adsorption,enrichment,and trace detection of target molecules.Luminescent metal-organic frameworks（LMOFs）have gradually shown great potential for food analysis due to their large specific surface area,high porosity,diverse structures and excellent luminescence properties.Therefore,in this study,three different fluorescent sensing systems were constructed using LMOFs as the main body and aptamers as biometric elements to achieve sensitive detection of three typical antibiotics（tetracycline,sulfamethazine and ampicillin）in pork samples,as follows:（1）Construction of a fluorescence sensing system based on intrinsic dual-emitting lanthanide-MOFs and its detection of tetracycline.To address the issue of susceptibility to interference in single-signal systems and complicated construction of ratiometric systems,a dual-emitting design principle for integrated organic ligands and lanthanide ions was proposed using post-functionalization modification treatment.In this chapter,Ag⁺/Eu³⁺@Ui O-66（COOH）₂（AEUC）was synthesized using Zr⁴⁺and Eu³⁺as metal souces,1,2,4,5-benzenetetracarboxylic acid（H₄btec）as organic ligands,and Ag⁺as sensitizer.The energy transfer from organic ligand and Ag⁺to Eu³⁺,that is the"antenna effect",plays a crucial role in the luminescence of this material.Gaussian simulations showed that the lowest unoccupied molecular orbital（LOMO）of H₄btec ligand is higher than the lowest unoccupied molecular orbital energy of tetracycline,and thus the transfer of electrons from the ligand to the tetracycline can be achieved,suppressing the"antenna effect".In addition,the excitation spectrum of H₄btec has a large overlap with the UV-vis absorption spectrum of tetracycline antibiotics.Therefore,the internal filtration effect（IFE）and photoelectron induction effect（PET）jointly lead to the fluorescence quenching of the emission peak of AEUC at 614 nm,while the intensity of the emission peak at 421 nm remains constant during this process,serving as a reference signal.Based on the above principles,the ratio of dual-signal fluorescence(I₆₁₄/I₄₂₁)of AEUC was linearly correlated with the tetracycline（TC）concentration in the range of 0.1-200μM under the optimal detection conditions,acquiring a detection limit of 12.8 n M.The method was used for the detection of TC in pork samples,with recoveries ranging from 96.01 to 107.73%,and the detection results were consistent with those of high-performance liquid chromatography（HPLC）.The experimental results showed that AEUC possess advantages of rapidity and accuracy for the determination of TC residues in pork,providing a novel idea for the detection of TC residues in pork.（2）Construction of a fluorescent sensing system for bimetallic LMOFs based on specific recognition strategy and its detection of sulfamethazine.To achieve specific recognition of antibiotic molecules,a bimetallic LMOFs aptamer sensing system was designed by utilizing the high specific recognition and high affinity binding properties of nucleic acid aptamers to sulfamethazine molecules.In this chapter,Zn/Fe-MOF@PDANSs was synthesized by surfactant-assistant epitaxial growth method using Zn²⁺and Fe³⁺as metal sources and 5,10,15,20-tetrakis（4-carboxyphenyl）porphyrins as organic ligands.The dye-modified aptamer（FAM-ss DNA）can be adsorbed on the surface of Zn/Fe-MOF@PDANSs throughπ-πstacking,hydrogen bonding and coordination and the fluorescence quenching of FAM-ss DNA can be realized by synergistic effect of fluorescence resonance energy transfer（FRET）and photoelectron-induced transfer（PET）.Owing to the conformational change of the aptamer to form a G-quadruplex structure caused by specific binding of FAM-ss DNA and sulfamethazine,FAM-ss DNA detached from the surface of Zn/Fe-MOF@PDANSs and thus the fluorescence of FAM-ss DNA restored.Meanwhile,the fluorescence intensity of Zn/Fe-MOF@PDANSs remains unchanged,serving as reference signal.Based on above principle,the specific detection of sulfamethazine（SMZ）in the range of 0.5-100 ng·m L^-1 was achieved with a detection limit of 0.025 ng·m L^-1.In addition,the introduction of the aptamer significantly enhanced the specificity of the sensing system,which can effectively overcome the influence of other co-existing antibiotics with strong anti-interference ability.Using the spiked recovery experiments,the method was applied for the detection of SMZ in pork samples,with recoveries of 92.69～108.48%,and the relative standard deviations（RSD）less than 5%,which were consistent with the results obtained by HPLC method,demonstrating the advantages of high accuracy,specificity and sensitivity of this sensing system for the analysis of SMZ and providing a technical reference for the detection of SMZ in pork.（3）Construction of multi-layer heterogeneous LMOFs fluorescence sensing system based on sensitization strategy and its detection of ampicillin.To meet the demand of trace detection,the pre-concentration effect of heterogeneous LMOFs on ampicillin molecules was exploited to enhance the enrichment efficiency and detection sensitivity by increasing the interaction sites.In this chapter,3D hierarchical NH₂-MIL-53（Al）@Mo/Zn-MOF was synthesized by internal diffusion growth method to modulate the structure,and the aptamer was coupled with NH₂-MIL-53（Al）@Mo/Zn-MOF to obtain LMOFs-ap complex as reference signal while Cd Te QDs@Si O₂ as response signal by EDC/sulfo-NHS method.Due to the wide overlapping area between the UV-vis absorption spectrum of mitoxantrone（MIT）and the emission spectrum of Cd Te QDs@Si O₂ and the electron transfer process between them,MIT can be used as a quenching agent for LMOFs-ap-Cd Te QDs@Si O₂ fluorescent probe under the synergistic effects of IFE and PET.Meanwhile,the fluorescence intensity of LMOFs-ap keep constant.The pre-concentration effect and analytical performance of the material were investigated through batch experiments and fluorescence analysis experiments,and the results showed that I₆₀₇/I₄₃₇ was linearly correlated with AMP concentration in the range of 5～1000 n M with recoveries of 94.25～107.76%.By virtue of the preconcentration effect of the synthesized probe on AMP,the constructed sensing system possess the advantage of low detection limit（0.69 n M）and strong anti-interference ability,providing a reliable method for sensitive and accurate detection of AMP in pork.In summary,the fluorescent sensing system based on LMOFs constructed in this paper has exhibited advantages of high sensitivity,strong anti-interference ability and high accuracy,which successfully realized the rapid detection of antibiotics and provided a new solution and research idea for the detection of typical antibiotic residues in pork.