Study On Preparation And Application Of Nanozymes Based On Iron And Manganese In Biochemical Analysis

Enzymes are catalytically active proteins and RNA produced by cells.However,the development and application of natural enzymes are severely restricted by the following shortcomings:(1)high cost of preparation and purification;(2)Poor stability;(3)sensitive to the reaction environment;(4)Difficult to recycle and reuse,etc.In recent years,with the continuous development of nanotechnology,Nanozymes,as artificial enzymes,have become a new research field.Nanozymes not only have physicochemical properties specific to nanomaterials,but also have catalytic properties similar to those of natural enzymes.In addition,nanozyme catalysis is not only of high stability,low cost,easy recovery and storage,but also can be controlled remotely by different external stimuli such as magnetic field,light,ultrasonic and heat.According to their catalytic activity,they are widely used in chemical analysis,environmental monitoring and other biomedical fields.Although it has been reported that a large number of nanomaterials can simulate a variety of natural enzyme activities,the catalytic activity of nanomaterials is still not comparable to that of natural enzymes.The reported nanozymes mainly focus on peroxidase,oxidase,catalase,superoxide dismutase,etc.Therefore,further development of nanozymes with higher activity and better selectivity and new enzyme-like activities has become the main direction of future research.In this paper,three types of nanozymes were prepared based on transition metal elements iron and manganese,including manganese-based nanozymes,iron single-atom nanozymes,biomimetic nanozymes,etc.,and their enzyme-like activities were studied.It was found that these nanozymes have oxidase-like,peroxidase-like and catalase-like activity.Next,a visual colorimetric sensor was established to detect the contents of H₂O₂,glutathione(GSH)and glucose.In addition,biomimetic nanozymes were also studied on tumor sonodynamic therapy,which expanded the application scope of nanozymes.Firstly,MnO₂/PS nanozyme was prepared by template method,and its structure was characterized by scanning electron microscopy(SEM),transmission electron microscopy(TEM)and X-ray photoelectron spectroscopy(XPS)and other techniques.Through the systematic study of enzyme-like activity,it was confirmed that the MnO₂/PS nanozyme has good OXD-like activity in a near-neutral environment at room temperature,and can directly oxidize 3,3',5,5'-tetramethylbenzidine(TMB)to blue ox TMB in the absence of H₂O₂.The kinetic studies showed that MnO₂/PS exhibited an excellent affinity for TMB with a K_m value of 0.068 m M.Radical trapping experiments showed that MnO₂/PS produced two kinds of reactive oxygen species(ROS)during the catalytic process,namely O₂^·-and ¹O₂.On account of the inhibiting effect of glutathione(GSH)to TMB oxidation,the blue ox TMB is reduced to colorless TMB.Based on this,a colorimetric sensor of MnO₂/PS was established for the first time for the detection of GSH.The experimental results showed that there was a strong linear relationship between the content of GSH and?A in the range 1-50?M,and the LOD was 0.08?M,indicating the method has a wide linear range and high sensitivity.In addition,interference experiments and stability experiments of coexisting components were also carried out,and the results confirmed that the method has good selectivity and stability.The method was used to determine the content of GSH in tablets and injections and then the results were statistically comparing with those of HPLC.These results were consistent,which confirmed that the MnO₂/PS colorimetric sensor designed in this paper can be successfully used for the detection of glutathione.Secondly,single-atom iron-modified carbon-based nanodiamonds(Fe-NDs)were prepared by a one-pot in situ reduction method.The structures of Fe-NDs were characterized by SEM,TEM,X-ray diffraction(XRD)and spherical aberration corrected transmission electron microscope(ACTEM),and the existence of single-atom Iron in Fe-NDs was proved.Through the systematic study of enzyme-like activity,it is confirmed that Fe-NDs have dual mimetic enzyme activities,namely peroxidase-like activity and oxidase-like activity.Compared with horseradish peroxidase(HRP),Fe-NDs are well tolerated in acidic and high temperature environments and exhibit excellent catalytic performance.Kinetic studies showed that Fe-NDs exhibited a good affinity for H₂O₂ compared with HRP(K_m=3.7 m M)with a K_m value of 0.87 m M.The experimental study of free radical trapping showed that Fe-NDs,as peroxidase-like enzymes,produced mainly·OH and a few O₂^·-in the catalytic process;as oxidase-like enzyme,produced mainly O₂^·-,¹O₂and·OH in the catalytic process.Based on this,a colorimetric sensor based on Fe-NDs was established for the detection of H₂O₂ and GSH with the dual-enzyme property.The experimental results showed that there was a strong linear relationship between the content of GSH and?A in the range 1-25?M,LOD was 0.3?M;the content of H₂O₂a n d?A i n t h e r a n g e 1-60?M,LOD was 0.072?M respectively.In addition,interference experiments and stability experiments of coexisting components were also carried out,and the results confirmed that the method has good selectivity and stability of the Fe-NDs.The content of GSH in glutathione tablets and injections determined by this method was also compared with that by HPLC.The two methods were basically consistent,which confirmed the high accuracy and sensitivity of this method.Fe-NDs nanozyme colorimetric sensor can be used for the detection of glutathione and H₂O₂.Then,three biomimetic nanozymes were successfully synthesized by self-assembly of amphiphile Fmoc-histidine and Hemin,namely His@Hemin,Fe-His@Hemin and Mn-His@Hemin respectively.Their structures were characterized by SEM,XPS,inductively coupled plasma-optical emission spectroscopy(ICP-OES).Through the systematic study of the enzyme-like activities,it was confirmed that the three nanozymes can mimic two kinds of enzyme-like activities,peroxidase-like and catalase-like respectively.The comparison of enzyme-like activities and kinetic studies show that Fe-His@Hemin has the highest catalytic activity and exhibits good catalytic activity in a neutral environment.Radical trapping experiments showed that Fe-His@Hemin generated three kinds of reactive oxygen species during the catalytic process,O₂^·-,¹O₂ and·OH respectively.Based on the synergy of the dual enzymes of Fe-His@Hemin,two colorimetric sensors were successfully constructed for the detection of H₂O₂ and the one-step detection of glucose respectively.The experimental results showed that there was a strong linear relationship between the content of H₂O₂ and?A in the range 0.25-120?M,LOD is0.16?M;the content of glucose and A in the range 0.5-400?M,LOD was 0.25?M respectively.In addition,interference and stability experiments showed that this method has excellent selectivity and good stability of Fe-His@Hemin.At the same time,the glucose sensor in this paper was applied to the determination of glucose in human serum with high accuracy and good reproducibility.Finally,based on the sonosensitive characteristics of hemoporphyrin and the high catalytic activity and excellent bionic structure of Fe-His@Hemin nanozyme,reactive oxygen species(Fe-His@Hemin+US)triggered by ultrasound was designed in this paper.The in vivo and in vitro sonodynamic tumor inhibition of Fe-His@Hemin nanase was investigated using 4T1 tumor cells.In vivo sonodynamic therapy experiment showed that the tumor inhibition rate was more than 90%(volume inhibition rate 92.4%and mass inhibition rate 94.8%)under the condition of external ultrasonic radiation.The results of photoacoustic imaging and ROS fluorescence staining in mice showed that Fe-His@Hemin nanozyme could rapidly produce high concentration of ROS in tumor tissue,effectively inducing tumor cell apoptosis.In addition,under the action of Fe-His@Hemin with CAT-like,O₂ in situ enhances the oxygen content in the tumor environment and improves the efficiency of Fe-His@Hemin nanozyme sonodynamic therapy.The results of blood biochemical test showed that Fe-His@Hemin nanozyme has good biosafety.This study provided a new idea for the application of nanozyme in sonodynamic therapy of tumors.In conclusion,MnO₂/PS,Fe-NDs and Fe-His@Hemin were used as colorimetric sensors for the detection of GSH,H₂O₂ and glucose,then the sonodynamic therapeutic effect of Fe-His@Hemin on tumor and its mechanism were investigated.These results of this study provided a new scientific basis for the application of nanozymes in the fields of chemical/biological sensing,biomedicine and nanomedicine.