Fabrication And Application Of Hydrogen Peroxide Electrochemical Sensor Based On Platinum Nickel-doped Nitrogen Graphene Nanocomposites

Background and Objective:Cancer is a serious threat to human health and the frequently-occurring disease.With the growth of the population and the coming of the population ageing,the number of cancer patients in the world has a rising trend year by year.However,the incidence and mortality of cancer in China are above the average level,the source of which is lower rate of early detection and treatment.The cancer initial period may be no obvious disease symptoms,most patients with the disease are diagnosed in a late stage accompanying with specific clinical symptoms.Therefore,the early diagnosis of cancer has attracted great attention in the medical field.With the development of detection technology,the clinic diagnosis methods of cancer are primarily based general examinations,imaging tests and biopsy at present.General examinations include blood and urine test etc.It is usually not easy to find abnormalities from these clinical examination findings.The imaging tests including computerized tomography scan,magnetic resonance imaging,have some shortcomings,such as relying on large and expensive equipment,requiring professional operating technicians and high cost of patients’examination.In most cases,the results of imaging examination cannot be confirmed and they can only be used as an auxiliary examination.At present,pathological biopsy is the"gold standard"for clinical tumor diagnosis.But this method has some limits for complicated steps and long diagnosis time.Because the biopsy tissue needs to be fixed,embedded,sectioned,stained and observed under microscope.Further observation of the section is also based on the pathology knowledge and the actual experience of the pathologist to get the subjective diagnosis results.However,the development of pathologic section in our country’s basic hospitals is relatively backward.There are some disadvantages such as shortage of pathological talents,simple equipment and poor working environment,which lead to low diagnosis efficiency.Therefore,it is great significance to develop a simple and cheap tumor diagnosis method without the operation of professionals for the diagnosis of tumor diseases,particularly in basic hospitals.Hydrogen peroxide is one of the most important reactive oxygen species in organisms and by-product of a variety of oxidase-catalyzed reactions.H₂O₂ also plays an essential role in physiological and pathological regulation of organisms as a signaling molecule.Based on the advantages of relatively stable and easy to obtain,H₂O₂ can penetrate the cell membrane.Previous studies have shown that there are some differences in the content of H₂O₂ released by tumor cells and normal cells.Therefore,H₂O₂ can be used as a molecular marker for tumor detection.However,in practical application,the development of H₂O₂ detection technology is relatively slow.The classic methods mainly include fluorescence spectroscopy,chemiluminescence,and so on.They are always faced with the problems of complex detection steps,expensive equipment,low sensitivity and failure of continuous monitoring on site.Aiming at the existing problems in current methods,electrochemical sensor detection method has supplied a new selection for sensitive detection of H₂O₂ as a simple,rapid,sensitive and cheap technology.Therefore,we propose for the first time that the electrochemical sensor can be used to detect H₂O₂ in tumor biopsy tissue,which is a potential tumor diagnosis strategy.Based on the above problems,we have developed a highly sensitive hydrogen peroxide electrochemical sensor for tumor diagnosis.In the first chapter,this thesis develops a kind of nanocomposite material based on platinum nickel nanoparticles and doped nitrogen graphene,which is improving the electrocatalytic performance through synergism effect.At the same time,the synergetic mechanism of materials is discussed.In the second chapter,the hydrogen peroxide electrochemical sensor is constructed and possesses the characteristics of good selectivity,stability,high sensitivity,and fast analysis.Then,the sensing mechanism are discussed.In the third chapter,H₂O₂ as the tumor diagnostic marker,biosensor is used to detect H₂O₂ from some tumor cells,normal cells,tumor tissues,para-carcinoma tissues and normal tissues successfully.Further,the H₂O₂ content of lung tissues with tumor metastasis and normal lung tissues is further detected.Finally,the evaluation criterion of tumor diagnosis is preliminarily deduced.MethodsFirstly,graphene oxide（GO）was synthesized by improved Hummers method.The morphology,microstructure and components of as-prepared graphene oxide were characterized by means of SEM,EDS,XRD and XPS.Secondly,N-doped reduced graphene supported Pt Ni nanoparticles（PtNi-N-rGO）were in-situ synthesized by a simple one-step method,with Pt（acac）₂ and Ni（acac）₂ as precursors,and DMF as solvent and reducing agent.Morphology,components and electrochemical response of PtNi-N-rGO were characterized by TEM,EDS and electrochemical methods.The effects of the temperature,reaction time and ratio of precursors were studied determining the optimum process conditions.The morphology,crystal structure and element composition of Pt Ni nanoparticles were observed by TEM,HRTEM,SEAD and EDS-mapping.Qualitative analysis of nanocomposites was carried out by FT-IR and UV for subscribing to reduction and doping of graphene oxide.Finally,the elemental composition and chemical state of the nanocomposites were observed by XPS.The mechanism of the synergistic effect of nanocomposites was discussed.Thirdly,the hydrogen peroxide electrochemical sensor was constructed.PtNi-N-rGO nanocomposites were modified on glassy carbon electrode.The electrochemical performances of the PtNi-N-rGO with different nanocomposite concentrations were investigated through Cyclic Voltammetry.The effect of different loading amounts of PtNi-N-rGO on the performance of sensor was studied.To find the optimized potential for the electrochemical detection of H₂O₂,different potentials were measured by amperometric i-t curve.Fourthly,the performances of the PtNi-N-rGO electrochemical sensor were characterized by electrochemical methods.The conductivity of PtNi-N-rGO sensors was investigated by electrochemical impedance spectroscopy（EIS）.The electrocatalytic activity of the PtNi-N-rGO toward H₂O₂ was investigated via Cyclic Voltammetry.The sensitivity and adsorption of the electrochemical sensor to H₂O₂were investigated by changing the concentrations of H₂O₂ and scan rates of CV.Amperometric response and anti-interference ability of the electrochemical sensor were measured by amperometric i-t curve.The reproducibility and stability of the electrochemical sensor were also investigated.Furthermore,dynamic process was also explored by chronoamperometry.Finally,the catalytic mechanism of the sensor was discussed.Fifthly,the feasibility of hydrogen peroxide electrochemical sensor was validated.Firstly,Calcein-AM and propidium iodide（PI）co-staining were used to assess cell viability before and after sensor detection.Based on normal cells and a variety of tumor cells used as models,real time monitoring of H₂O₂ released by cells were disposed by amperometric i-t curve.At last,we established 4T1 lung metastasis model,LLC lung cancer model and HCT116 colorectal cancer model.We detected the content of H₂O₂ in tumor tissues,para-carcinoma tissues,normal tissues and metastasis lung tissue,analyzed the detection effect,and initially deduced the evaluation criteria of tumor diagnosis.ResultsIn this article,we constructed a novel hydrogen peroxide electrochemical sensor.The optimization and characterization of nanocomposites,the performance optimization and characterization of the sensor,and the unique application of the sensor were studied,all of results were as follows:First chapter was preparation and characterization of PtNi-N-rGO nanocomposites.We had successfully prepared a uniform and rich-wrinkle morphology of graphene oxide（GO）by improved Hummers method,which had loose and disordered lamellar structure.PtNi-N-rGO was in-situ synthesized successfully by a simple one-step method.It was confirmed that Pt Ni nanoparticles were uniformly distributed on doped N-reduced graphene oxide under the optimal reaction condition.The size of Pt Ni nanoparticles was 5.0±1.0 nm and it was proved that the morphology of Pt Ni nanoparticles features the exposure of（111）and（200）facets.N element had been doped into grapheme lattice successfully.In this composite system,layer structure of graphene provided the backbone for nanocomposites.The local electronic structure of graphene had been changed by graphite nitrogen structure,which improved the electron transport capacity and adsorption of H₂O₂.Pt Ni nanoparticles could be effectively loaded on the skeleton structure of N-r GO,which had high conductivity and catalytic activity.The synergistic effect of nanocomposites presented high specific surface area,fast electron transfer rate and excellent catalytic performance.In the second chapter,hydrogen peroxide electrochemical sensor based on PtNi-N-rGO nanocomposites was constructed.Concentration and volume of PtNi-N-rGO were 3.0 mg/ml and 4.0μl in the optimal condition respectively.The optimized potential was-0.6V by current-time curves.The good conductivity of PtNi-N-rGO made it competitive catalyst in electrochemical sensor fabrication by EIS.It was found that the PtNi-N-rGO sensor displayed excellent catalytic activity,rapid response for H₂O₂ and high sensitivity.The detection limit of this method was 0.0028μM（S/N=3）and the sensitivity was 207.1μA·μM^-1·cm^-2.The relevant linear ranges for the electrochemical detection of H₂O₂ was 0.00001～8.632 m M.Further the sensor had good anti-interference ability,reproducibility and stability.At the same time,the results indicated that the sensor to H₂O₂had good response characteristics,and the reduction process of H₂O₂ on the electrode surface was the diffusion-controlled.The favorable catalytic performance of sensors could be characterized by the kinetic parameters such as reaction rate constant(8.5×10⁵cm³·mol^-1·s^-1)and diffusion coefficient(3.6×10^-5cm²·s^-1).The sensing mechanism of sensor was introduced basing on effectively chemical adsorption.First the H₂O₂ dissociated and adsorbed on Pt Ni sites via non electrochemically,the adsorbed OH moiety on Pt Ni surface was not stable during-0.6 V potential scan,the regeneration of fresh Pt Ni surface sites was created rapidly in an electrochemical reduction H₂O₂ period,and subsequently the fresh Pt Ni sites became accessible to dissociate other H₂O₂ molecules.Due to the good conductivity of doped nitrogen graphene skeleton and Pt Ni nanoparticles,the synergistic effect of nanocomposites was improved the electron transfer rate of the whole catalytic process,which shortened the response time,improved the sensitivity and selectivity.The third chapter mainly mentioned applicable research of PtNi-N-rGO sensors.Based on various cancer cells and tissues as a model,PtNi-N-rGO sensors could further achieve the real-time detection of traced H₂O₂ released.The results showed that there were striking differences between normal cells and tumor cells,para-carcinoma tissues,normal tissues and tumor tissues.Additionally,there existed a significant difference between normal lung tissue and metastasis lung tissue.The evaluation criteria for rapid diagnosis of tissues cancerous by sensors was summarized.It was preliminarily determined that H₂O₂ could be used as a molecular marker for early diagnosis of tumor.It achieved effective signal quickly and took only 0.5 hours for measurement of one sample.The application experiment had achieved the purpose of rapid and efficient monitoring.The detection environment of the sensor had little effect on the cell survival rate through the characterization.Electrochemical sensors provided a suitable microenvironment for cells,which indicated that cells could maintain cellular activity under hypoxic conditions for a short time.PtNi-N-rGO sensors could achieve good selectivity,stable,high sensitivity and fast real-time response for the H₂O₂ molecules from tumor cells,which would provide a safe,fast,simple and cheap assistant diagnosis method for clinical biopsy diagnosis.This sensor might have potential application value in early diagnosis,physiological and pathological analysis of tumor.ConclusionIn this study,PtNi-N-rGO nanocomposites were prepared by a simple one-step method,and hydrogen peroxide electrochemical sensor based on PtNi-N-rGO was constructed.The sensor had excellent catalytic activity and rapid response for H₂O₂,with high sensitivity,selectivity,good anti-interference and stability.Finally,H₂O₂ was used as tumor molecular marker and sensor was applied in the detection H₂O₂ of tumor cells and tissues.It had realized the application in the diagnosis and detection of tumor biopsy tissue.In conclusion,this paper preliminarily proved that H₂O₂ can be used as a molecular marker to realize the application of tumor diagnosis through sensors.This study provides a new strategy for tumor diagnosis and as a safe,fast,simple and cheap assistant diagnosis method for clinical biopsy diagnosis,which has good applicational prospect in the diagnosis of tumor diseases in basic hospitals.