Functionalization Of Peroxidase Nanoenzymes And Their Application To The Detection Of Antibiotics In Food

Nanozymes are a kind of nanomaterials with intrinsic biological enzymatic characteristics.Due to their great potential in solving the inherent defects of biological enzymes such as variability,high cost,difficulty in purification and difficulty in mass preparation,they have attracted great attention from researchers.Besides,because of the complexity of food substrate,the detection of harmful substances in food has always been a problem for researchers.It is still challenging to develop a simple,reliable and sensitive method for the analysis of harmful substances in food.Encouragingly,nanozymes have begun to offer opportunities to address some of the challenges of food safety.The construction of biosensors by nanozymes provides a cheap,convenient,efficient,rapid and sensitive detection method for food contamination detection.Compared with traditional methods,nano-enzyme biosensors have the advantages of high selectivity,high sensitivity and high specificity of target recognition,short detection time and good signal readout effect.This paper selected noble-metal nanoclusters and metal-organic frameworks(MOFs)to simulate the activity of peroxidases.In order to improve the catalytic activity and specificity of the nanozymes,noble metal nanoparticles and simulated antibodies were modified to their surfaces,and three quantitative analysis methods of antibiotics were developed.Finally,tetracycline(TCs)antibiotics,chloramphenicol(CAP)and metronidazole(MNZ)were detected in food matrix by these methods.The specific work is as follows:(1)Based on intrinsic peroxidase-like activity of gold nanoclusters(AuNCs),this work used TC-specific aptamers(Apt)to improve the catalytic activity of AuNCs toward the peroxidase substrate 3,3',5,5'-tetramethylbenzidine(TMB)oxidation by H2O2,and established a colorimetric sensing platform for TCs.The catalytic enhancement by Apt allows for sensitive colorimetric detection of TCs,and Apt as molecular recognition elements can specifically combine with TCs leading to high selectivity.This developed sensing platform can quantitatively detect TCs in the concentration range of 1 to 16 ?M with a limit of detection(LOD)as low as 46 nM.Interestingly,the naked-eye detection capability of this method is estimated to be 0.5?M.Finally,the detection of TCs in real samples like drugs and milk was validated.(2)In this study,based on the combination of AuPtNPs synergistic catalysis and Apt high specificity to enhance the enzyme activity and target recognition of MIL-88(Fe),a fluorescence detection method was developed for the simple,highly specific and sensitive detection of CAP.In this method,Fe(?)and terephthalic acid(PTA),which was a peroxidase substrate and an organic ligand were used to synthesize metal organic skeleton materials[MIL-88(Fe)],avoiding interference caused by the introduction of additional enzyme substrates.AuPtNPs was modified to the MOFs surface by in situ generation method,and formed a synergistic catalytic effect with MIL-88(Fe),which greatly promoted the peroxidase-like activity of the composite.The probe was used to detect CAP with a wide detection range,presenting a good linear range in the concentration range of 5-600 ?M,and LOD could be as low as 2.5 ?M.Finally,the effectiveness of the probe was verified by analyzing CAP in honey samples.(3)In this work,a complex[MIL-53(Fe)@MIP]with good peroxidase-like activity and specific target recognition function was synthesized by using molecular imprinting technology to coat polydopamine(PDA)to MIL-53(Fe)surface.Based on this material,a metronidazole(MNZ)fluorescence sensor was constructed.During the synthesis of MIL-53(Fe)@MIP,the substrate(PTA)has been constructed in the skeleton,so the probe does not require an additional enzyme substrate.This avoids the interference of substrate to the enzyme sensing system and improves the accuracy of the results.The data demonstrate that the sensor could quantitatively analyze MNZ with detection concentrations ranging from 1 to 200 ?M and LOD as low as 22.9 nM.The recoveries of the standard addition method were 93.2%-102%and the relative standard deviation(RSD)did not exceed 3%.This shows that the sensor could detect reliably MNZ in complex real samples.