Study On Colorimetric Sensing Detection Of Tetracycline In Animal-Derived Foods By Magnetic Biomimetic Antibody Nanozyme

Tetracycline（TC）is a broad-spectrum antibiotic,which has important application value in livestock and aquaculture,etc.If used for a long time or irregularly,it can lead to excessive TC residues in animal-derived foods.Such,it will pose a threat to human health.As a rapid detection technology,colorimetric sensing has made some achievements in the rapid detection of TC,but it usually relies on natural enzyme and the detection cost is relatively high.The discovery of nanozymes has successfully solved the above problems.However,nanozymes often suffer from poor substrate specificity.In this paper,magnetic biomimetic antibody nanozymes that can specifically recognize TC were prepared by using carboxylated Fe₃O₄（Fe₃O₄@COOH）nanozymes as magnetic carriers and surface molecular imprinting technique.A simple,rapid and sensitive chemical colorimetric sensing method was established for detecting TC residues in animal-derived foods based on the specific recognition,peroxidase-like catalytic activity,and superparamagnetic properties of the magnetic biomimetic antibody nanozymes.The research content and results are as follows:（1）Preparation and verification of catalytic activity of magnetic biomimetic antibody nanozymesThe Fe₃O₄@COOH nanozymes were synthesized by the chemical co-precipitation method.Then magnetic biomimetic antibody nanozymes with good specific recognition performance,peroxidase-like catalytic activity,and superparamagnetic were successfully prepared based on the Fe₃O₄@COOH nanozymes as the magnetic carrier and active center by surface molecular imprinting technique.The optimal conditions were obtained for the rapid adsorption of TC using magnetic biomimetic antibody nanozymes through static and dynamic adsorption experiments.The adsorption efficiency could reach 100%when magnetic biomimetic antibody nanozymes suspension（180μL,6mg/m L）and TC（1m L,3μg/m L）were mixed and shaken for 4 minutes at room temperature.The control experiments of the different colorimetric sensing systems showed that the magnetic biomimetic antibody nanozyme could catalyze the oxidation of 3,3,5,5-tetramethylbenzidine（TMB）in the presence of H₂O₂ to produce the blue oxidation product（ox TMB）,indicating that the nanozymes has peroxidase-like catalytic activity.Moreover,the catalytic activity of the magnetic biomimetic antibody nanozyme was higher than that of the magnetic non-biomimetic antibody nanozymes prepared without the addition of TC（template molecules）.The catalytic activity of the magnetic biomimetic antibody nanozyme was inhibited after the magnetic biomimetic antibody nanozymes adsorbed TC,resulting in a decrease in absorbance value.This result verified the feasibility of TC detection by colorimetric sensing based on the magnetic biomimetic antibody nanozymes.Additionally,the magnetic biomimetic antibody nanozymes could also catalyzed the oxidation of O-Phenylenediamine（OPD）,diammonium 2,2’-azino-bis（3-ethylbenzothiazoline-6-sulfonate）（ABTS）in the participation of H₂O₂.Under the same conditions,the order of rendering effects is TMB>OPD>ABTS.So,TMB with high safety was selected as the colorimetric substrate in this work.（2）Study of the peroxidase-like catalytic activity of magnetic biomimetic antibody nanozymesBy analyzing the catalytic activity of Fe₃O₄@COOH nanozymes and magnetic biomimetic antibody nanozymes,it was found that the presence of molecularly imprinted layers resulted in the decrease of Fe₃O₄@COOH nanozymes activity.However,it could provide specific recognition sites for absorption of TC.The optimal colorimetric detection conditions for the nanozymes were obtained.That is,700μL HAc-Na Ac buffer at p H=2,200μL of 0.06 mol/L H₂O₂,100μL of 0.02 mol/L TMB concentration,and 2 min of reaction at room temperature to achieve the rapid detection of TC.The Michaelis-Menten constants（Km）of magnetic biomimetic antibody nanozymes for H₂O₂ and TMB were respectively 3.66 mmol/L and 1.18 mmol/L by enzyme kinetics analysis showed that which were lower than that of HRP,indicating that the nanozymes had high affinity for H₂O₂ and TMB.However,its reaction velocity is lower than that of HRP.The stability experiments showed that the magnetic biomimetic antibody nanozymes exhibited good catalytic activity and adsorption effect even after 180 days of dry storage at 4°C.After five cycles of adsorption-colorimetry-desorption,the adsorption efficiency of the nanozymes lost 14%,and the absorbance value decreased by about 0.15,indicating that the nanozymes have good reusability.（3）Magnetic biomimetic antibody nanozyme coupled with colorimetric sensing technology for the detection of TC in actual samplesThe detection process of the constructed chemical colorimetric sensing method is divided into two steps:first,the TC in solution is adsorbed using magnetic biomimetic antibody nanozyme under optimal adsorption conditions.Then the TC-magnetic biomimetic antibody nanozymes complex is separated by a magnet.Second,700μL HAc-Na Ac buffer（0.1 mol/L,p H=2）,200μL H₂O₂（0.06 mol/L）,and 100μL TMB（0.02 mol/L）were added for the color development reaction.The linear concentration range of the method was 1-200 ng/m L,the linear regression equation was y=0.10827+0.00142x（R²=0.9971）,and the limit of detection（LOD）was 0.41 ng/m L.The specificity experiments showed that the difference in absorbance of the reaction system caused by the magnetic biomimetic antibody nanozyme after adsorption of TC(ΔA_652nm:blank absorbance value minus sample absorbance value)was the largest,which is 1.15 times,1.32 times,1.78 times,and 1.15 times for Oxytetracycline（OTC）,Chlortetracycline（CTC）,Demeclocycline（DMCT）and 6-Benzylaminopurine（6-BA）,respectively.Three different brands of pure milk,beef,chicken,pork,and pig liver samples were examined by this method,and no TC was detected.The results were consistent with the national standard method（GB/T 21317-2007）and the ELISA kit.Compared with the results of the spiked recovery test of ELISA,the recoveries of the established colorimetric sensing method for the above samples ranged from 74.66%to97.27%,and the relative standard deviation（RSD）was between 1.65%and 8.5%;the recoveries of the ELISA kit ranged from 81.08%to 99.08%,and the RSD between 1.18%and 8.2%.The above results indicate that the colorimetric sensing detection method constructed in this study has some potential for practical application.