Regulation Of Enzymatic Activity Of Fe₃O₄-based Nanozyme And Its Assay Application Study

Nanozymes are a class of nanomaterials with enzyme-like activity,which have the advantages of simple preparation process,low cost,easy preservation and good stability compared with natural enzymes.Since iron tetroxide nanoparticles（Fe₃O₄ NPs）with paramagnetic characteristics were first discovered to have peroxidase-like activity in2007,they have been a hot topic and focus in the field of nanozyme research.Despite the great progress in the fields of analytical detection,tumor therapy,antibacterial and bioimaging,the field of Fe₃O₄-based nanozyme research is still constrained by its own low enzymatic catalytic activity and low affinity for substrates at this stage.Therefore,how to prepare Fe₃O₄ nanozymes with high substrate affinity and excellent catalytic activity is an important challenge for them to be able to be more widely used.Nanozymes enzymatic activity modulation technique is generally proven as a solution.In this paper,Fe₃O₄-based nanozymes are used as a template to modulate Fe₃O₄-based enzyme-like catalytic activity by surface modification,doping and complexation techniques.The main contents of this paper are as follows:Firstly,hemein chloride（Hemin）and multi-walled carbon nanotubes（MWCNTs）were assembled with Fe₃O₄ NPs by one-step hydrothermal method to finally obtain ternary composite Fe₃O₄@MWCNTs/Hemin nanozymes.A systematic study of enzyme-like activity experiments confirmed that Fe₃O₄@MWCNTs/Hemin nanomaterials have good peroxidase-like activity.The results of steady-state kinetic experiments revealed that Fe₃O₄@MWCNTs/Hemin had a good affinity for TMB with a K_m value of 0.13mmol/L compared to other Fe₃O₄-based nanomaterials.The catalytic mechanism of Fe₃O₄@MWCNTs/Hemin was subsequently confirmed to follow a peroxidase-like catalytic mechanism（i.e.,·OH）by probing the generation of reactive oxygen species（ROS）in the system.The modified nanozyme has increased affinity for H₂O₂ and can recognize the trace amount of H₂O₂ produced by oxidation of xanthine by xanthine oxidase.A Fe₃O₄@MWCNTs/Hemin-mediated enzyme cascade sensor was developed for the detection of xanthine and xanthine oxidase activity based on the property that nanozyme can oxidize 3,3’,5,5’-tetramethylbenzidine（TMB）in the presence of H₂O₂ to produce the blue product ox TMB,and simultaneously a smartphone-based visual colorimetric sensor was established.The results showed that the concentrations of xanthine and xanthine oxidase showed good linearity with the absorbance of the system,and the limits of detection（LOD）were 0.25μmol/L and 0.216 m U/m L,respectively.The sensor was used for the determination of xanthine and xanthine oxidase in serum samples,and the experimental results showed that the sensor has good stability and reproducibility.Secondly,in order to further improve the catalytic activity of Fe₃O₄ nanozymes,modification on the surface of Fe₃O₄ nanozymes was attempted to introduce oxygen vacancies.The Mo-doped Fe₃O₄-based nanozymes was prepared by co-precipitation method,and after optimizing the Mo to Fe ratio,P was loaded on the Fe₃O₄/Mo surface by co-heat method,and finally the Fe₃O₄/Mo/P nanozymes was obtained.The presence of oxygen vacancies in the Fe₃O₄/Mo/P was confirmed by characterization.It is found through steady-state kinetics analysis that the Fe₃O₄/Mo/P under NIR light excitation has a faster reaction rate compared to Fe₃O₄/Mo and Fe₃O₄/Mo/P under normal conditions.The increased enzymatic activity of Fe₃O₄/Mo/P under NIR light was demonstrated by using a radical trapping agent,which was mainly due to the increased production of·OH and increased electron transfer efficiency during the catalytic process.Interestingly,glyphosate can inhibit the catalytic activity of Fe₃O₄/Mo/P enzymes,and in this way,a colorimetric assay platform for glyphosate based on the activity of Fe₃O₄/Mo/P-like enzymes was established,and the concentration of glyphosate was linearly and positively correlated with the absorbance value of ox TMB at 654 nm with an LOD of 0.039 mg/L in the concentration range of 0.124-12.4 mg/L.Interference experiments demonstrated the excellent selectivity of the colorimetric detection platform,which was also applied to the detection of glyphosate in different types of coffee with high accuracy and good reproducibility.Finally,in order to enhance the substrate affinity,carbon dots and polymer modifications were attempted in the synthesis of Fe₃O₄ NPs,using carbon dots with enzyme-like activity and abundant functional groups on polymers.Fe₃O₄/Cu-CDs were obtained by controlled synthesis of Fe₃O₄ NPs by hydrothermal method using Cu-CDs,and the surface of Fe₃O₄/Cu-CDs was modified by wrapping with poly epinephrine（PEP）,and finally Fe₃O₄/Cu-CDs@PEP with core-shell structure was obtained.The peroxidase-like activity of Fe₃O₄/Cu-CDs@PEP was confirmed by enzyme-like activity experiments,and it was also found that the catalytic activity of the nanozyme was further improved under ultrasonic conditions.After steady-state kinetic analysis,it was found that Fe₃O₄/Cu-CDs surface-modified by aggregated epinephrine had good affinity for TMB and H₂O₂ with K_m values of 0.21 mmol/L and 0.28 mmol/L,respectively,compared with Fe₃O₄/Cu-CDs@PEP that were not surface-modified.The increase in the catalytic activity of the enzyme stems mainly from the increase of the main free radicals（·OH and singlet oxygen（¹O₂））in its catalytic process.Fe₃O₄/Cu-CDs@PEP can oxidize dopamine to the corresponding catechol derivative o-quinone intermediate in the presence of H₂O₂,and 1,3-naphthalenediphenol reacts with o-quinone as an indicator to produce intense fluorescence.Through this phenomenon,a fluorescence sensing platform based on Fe₃O₄/Cu-CDs@PEP was established for dopamine detection.It was also applied to the dopamine assay of human serum,and the results were accurate and reproducible,and the fluorescence sensing platform has high specificity and good resistance to interference.In summary,in this paper,three nanozymes,Fe₃O₄@MWCNTs/Hemin,Fe₃O₄/Mo/P,and Fe₃O₄/Cu-CDs@PEP,were applied to detect xanthine,xanthine oxidase,glyphosate,and dopamine,respectively,and the effects of different enzyme activity modulation on the enzyme-like activities of the three nanozymes were investigated.The results of this paper provide an experimental and theoretical basis for the construction of Fe₃O₄-based nanozymes with high catalytic activity and high substrate affinity,and provide some new scientific basis and ideas for expanding the preparation process of Fe₃O₄-based nanozymes and their applications in the fields of analytical assays and biosensing.