Preparation Of Sheet-like Peroxidase Mimetic Enzymes And Their Application In Colorimetric Sensing

Nanozymes are nanomaterials that exhibited high enzymatic activities.Nanozymes are considered a better alternative to natural enzymes due to their high catalytic activity,simplicity of preparation,low cost,and good stability.Colorimetric sensing using nanozymes with peroxidase-like activity has become widely adopted in recent years.In this paper,several kinds of sheet-like peroxidase mimetic enzymes were synthesized,and the kinetic model,catalytic mechanism and the factors affecting enzyme activity of their catalytic oxidation of 3,3’,5,5’-tetramethylbenzidine（TMB）by hydrogen peroxide（H₂O₂）were examined.New colorimetric methods for the determination of uric acid（UA）,organophosphorus pesticides（OPs）,biothiols and glucose based on sheet-like peroxidase mimetic enzymes were established.（1）MoS₂ nanosheets with peroxidase-like activity were prepared with sodium molybdate dihydrate（Na₂Mo O₄·2H₂O）and L-cysteine by one-step hydrothermal method.The obtained MoS₂ nanosheets could catalyze the H₂O₂-mediated oxidation of TMB to produce a corresponding blue oxidized product（ox TMB）.Therefore,a simple method for detecting H₂O₂ has been developed.Increasing H₂O₂ concentration led to an increase in ox TMB absorbance under p H 3.7,temperature 35°C,MoS₂ 1.0μg·m L-¹ and incubation time 40 min were maintained.In view of the fact that H₂O₂ played a crucial role in the oxidation of UA catalyzed by uric acid oxidase,a method based on MoS₂nanosheets-catalyzed and H₂O₂-mediated TMB oxidation was developed for the indirect detection of UA.The increase of ox TMB absorbance enabled the sensitive determination of UA in the linear range of 2.5～40μmol·L^-1 and 40～100μmol·L^-1,respectively,and the detection limit（LOD）was 0.093μmol·L^-1（3σ/k）.A recovery rate of 92.6%～102.0%demonstrated the possibility of using the method to detect UA in urine and serum samples.（2）MoS₂ nanosheets（MoS₂ NSs）which can catalyze TMB oxidation by H₂O₂ for a color change,were prepared by a one-step hydrothermal method using Na₂Mo O₄·2H₂O and thiourea.Acetylcholine（ACh）is hydrolyzed to choline by acetylcholinesterase（ACh E）,and choline is oxidase by choline oxidase（CHO）to produce H₂O₂,which is oxidizing TMB under the catalysis of MoS₂ NSs.However,the addition of OPs inhibited ACh E activity,which decreased the generation of H₂O₂ and the oxidation of TMB.OPs can be quantitatively detected by measuring the decreased value of ox TMB absorbance after adding OPs.Under optimal experimental conditions,the good linear ranges of OPs detection range from 5 to 300 ng·m L^-1,and the LOD of OPs is as low as0.68 ng·m L^-1.The established method has been applied to the analysis of fruit and vegetable samples,and the detection results are consistent with that of gas chromatography（GC）.In addition,it has great potential for immediate on-site analysis of pesticide residues and biosecurity research in the agricultural field by collecting and analyzing visual signals of OPs through a smartphone.（3）Au@MoS₂ nanocomposites with high peroxidase-like activity were prepared by reducing HAuCl₄ with sodium citrate and used for the colorimetric determination of biothiols.The synergistic effect between Au NPs and MoS₂ NSs promoted the color conversion of TMB from colorless to blue ox TMB in the presence of H₂O₂.As a result of the addition of biothiols homocysteine（Hcy）,glutathione（GSH）,and cysteine（Cys）,the solution’s blue color faded and its absorbance decreased.At 652 nm,the decrease in absorbance was linear with d Hcy,GSH,and Cys concentrations in the range of 1～100μmol·L^-1,and the LOD is 0.93,0.71,and 0.73μmol·L^-1,respectively.In addition,the combination of the proposed method and smartphone provides a simple and convenient strategy for the on-site detection of biothiols.In the practical assay of Hcy levels in serums,the results matched the clinical values.（4）WO₃ nanosheets（WO₃ NSs）were prepared with sodium tungstate dihydrate（Na₂WO₄·2H₂O）and nitric acid at room temperature.The obtained WO₃ NSs had peroxidase-like activity and catalyzed TMB to blue oxidation product（ox TMB）in the presence of H₂O₂.Based on this,a convenient and sensitive colorimetric method for the determination of H₂O₂ and glucose was established.The linear ranges of H₂O₂ and glucose detection were from 1～200μmol·L^-1 and from 1～100μmol·L^-1,respectively.And the LOD of H₂O₂ and glucose were 0.79 and 0.96μmol·L^-1,respectively.Using this highly efficient method,glucose in human urine has been successfully determined,and the results are consistent with those of the clinical method.