Application Of Nanozyme Based Colorimetric Sensors For Detection Of Disease Markers

Objective:As people become increasingly concerned about their health,the early prevention and diagnosis of disease is receiving more and more attention.Abnormal expression of disease markers in body fluids（urine,serum,tissue extracts,etc.）,as"molecular markers"in specific physiological and pathological states,is often associated with a variety of human diseases.The development of a simple,rapid and accurate method for the detection of disease markers is therefore of great importance in the prevention,diagnosis and treatment of disease.In contrast to current complex and expensive clinical assays,colorimetric assays are of great interest because of their simplicity,selectivity,sensitivity,real-time monitoring and fast response time.With the rapid development of nanotechnology,nanomaterials that mimic enzymatic activity（nanozymes）have taken on a new life in disease diagnosis and treatment due to their high catalytic properties,stability,low cost and ease of recovery.Based on this,this study will combine the advantages of colorimetric methods and nanozymes to construct a colorimetric sensor based on nanozymes for the detection of disease-related markers.Methods:A variety of structurally stable nanomaterials were prepared by a one-step hydrothermal method,and 3,3’,5,5’-tetramethylbenzidine（TMB）was used as a chromogenic substrate to investigate the simulated enzymatic activity of the synthesized nanomaterials;Characterization of the synthesized nanomaterials in terms of morphology,crystallinity and elemental valence using scanning electron microscopy（SEM）,transmission electron microscopy（TEM）,X-ray diffractometry（XRD）and X-ray photoelectron spectroscopy（XPS）;By detecting the free radicals or electron transfer generated during the reaction,it is possible to determine whether the catalytic mechanism is a Fenton mechanism or an electron transfer mechanism,or other new mechanisms to be discovered;The inhibition of TMB catalytic oxidation by reducing small molecules is used to achieve their colorimetric detection.Result:In this thesis,two nanozyme-based colorimetric sensors were constructed to enable the detection of disease relevant small molecules such as H₂O₂ as well as glutathione（GSH）.The main findings are divided into two parts as follows:（1）In-situ Synthesis of Co-doped Mo S₂ Nanosheet for Enhanced Mimicking Peroxidase ActivityIn this study,a facile in-situ hydrothermal method for the preparation of various ultrathin transition metals（Fe,Cu,Co,Mn,and Ni）doped Mo S₂ nanosheets has been reported.Through the density functional theory（DFT）and steady-state kinetic analysis,the Co-doped Mo S₂ nanosheets exhibited the highest peroxidase-like catalytical activity among them.Furthermore,a typical colorimetric assay for H₂O₂ was presented based on the catalytic oxidation of colorless TMB to a blue product（ox TMB）by Co-Mo S₂.The proposed colorimetric method showed excellent tolerance under extreme conditions and a broad linear range from 0.0005～25 mmol L^-1 for H₂O₂ determination.Concerning the practical application,in situ detection of H₂O₂ generated from Si Ha cells was also fulfilled,fully confirming the great practicability of the proposed method in biosensing fields.（2）Selective detection of glutathione by flower-like Ni V₂O₆ with only peroxidase-like activity at neutral p HIn this study,flower-like Ni V₂O₆was synthesized and their enzyme-mimicking activity was investigated.Through the regulation of p H,Ni V₂O₆ nanozyme showed only peroxidase-like activity but not oxidase-like activity under neutral conditions,which could catalyze the oxidation of colorless TMB into its blue product in the presence of H₂O₂.Furthermore,based on the competitive effect of GSH on the catalytic activity of nanozymes,a semi-quantitative/quantitative colorimetric assay was established for GSH detection by using peroxidase-like Ni V₂O₆.The assay exhibited a good linear relationship in GSH concentration ranging from 3～100μmol L^-1,with a detection limit of 0.89μmol L^-1.Moreover,in the presence of formaldehyde as masking agent,this method showed satisfactory specificity for GSH under the interference of a variety of interfering substances and even biothiols.Concerning the practical application,the system was applied to monitor GSH level in fetal bovine serum,human serum and Si Ha cells.Satisfyingly,the obtained results were consistent well with those of Ultra performance liquid chromatography（UPLC）and assay kit,indicating the constructed assay has great potential in clinical application.Conclusion:It was shown that both nanomaterials synthesized by a one-step hydrothermal method exhibited excellent peroxidase-like activity and were able to catalyze the oxidative color change of TMB in the presence of H₂O₂.Based on the important role of H₂O₂ in the colorimetric system and the reducing effect of GSH on the colorimetric system,the colorimetric detection from standards to actual samples was achieved and showed the advantages of simple operation,good selectivity,high sensitivity,real-time monitoring and fast response time.This novel colorimetric sensing strategy using the peroxidase-like activity of nanozymes will lead to promising applications in the field of biomedical sensing and biomedicine.