Transition Metal-based Biomimentic Enzymes Preparation Andapplications For Electrochemical Detection Of Biologically Active Small Molecules

Some bioactive small molecules such as glucose,dopamine?DA?,hydrogen peroxide?H₂O₂?,nitric oxide?NO?and uric acid?UA?play essential roles in a variety of important biological processes,the imbalance of which can cause many fatal diseases,including diabetes,gout,Alzheimer's disease and cancers and severely affect human health.Therefore,it is of importance to timely and accurately monitor the concentrations of these biomolecules in the human body for diagnostic analysis and fundamental medical researches.Electrochemical methods have been broadly applied in clinic diagnosis and biomedical researches.In particular,enzyme-based electrochemical sensors have already been commercialized.However,the enzyme activity is extremely susceptible to environments,tedious operation and high expense severely limiting the further development of enzymatic sensors.Enzyme-free electrochemical biomimetic sensors have attracted great interest in the past decades due to their excellent stability under harsh environment and relatively low expense.Nevertheless,the performance of the enzyme-free biomimetic electrochemical sensors is mainly dependent on the electrode materials with unique chemistry and physics.The studies on the biomimetic sensors was begun first focused on precious metal nanomaterials,but their high price and scarce reserves severely restrict their application.Transition metal-based nanomaterials hold great promises to be alternatives of the precious metals for the enzyme-free biomimetic electrochemical sensors because of their high catalytic activity,abundant reserves,low cost,as well as simple and controllable fabrication.Among them,nickel,cobalt,and manganese-based nanomaterials have been particularly fuelled up recently for high-performance biomimetic sensors.Functional design of nanomaterials is an effective way to improve catalytic performance.The introduction of heteroatoms can change the electronic structure of the materials,in particular the surface electronic structure,to improve the adsorption and conversion of biomolecules,achieving good catalytic performance.The construction of special interfaces?such as heterojunctions?can effectively reduce the charge transfer resistance and provide more sufficient reactive sites,thus improving the surface reaction kinetics.In addition,the composite of multi-components can achieve the enhancement of the catalytic performance by integrating the advantages of the each component.In this thesis,a series of transition metal?nickel,manganese,cobalt?-based nanomaterials were synthesized through functional design strategies.Moreover,enzyme-free biomimetic sensors constructed from these nanomaterials were successfully applied to electrochemically detect bioactive small molecules in biological samples.The main research contents and results are as follows:?1?The high concentration of blood glucose mainly leads to diabetes.Therefore,accurate and convenient blood glucose determination is of great significance for the diagnosis and treatment of diabetes.Up to now,NiO has been used in enzyme-free electrochemical detection toward glucose.However,there is still a great demand to improve and optimize the detection performance of plain NiO.Herein,we have successfully synthesized Mn-doped NiO nanomaterials with tremella-like structure through hydrothermal and calcination processes using doping and morphology control methods,along with the study of the glucose detection performance in NaOH solution.Results of scanning electron microscope?SEM?and transmission electron microscope?TEM?show that tremella morphology is assembled from abundant nanosheets.The results of energy dispersive X-ray spectroscopy?EDS?,X-ray diffraction?XRD?,and X-ray photoelectrons energy spectroscopy?XPS?and inductively coupled plasma mass spectrometry?ICP-MS?confirm the successful doping with Mn element.Further results show that the tremella structure provides a sufficient specific surface area,which is conducive to the rapid charge transfer,while Mn doping results in the alteration of the electronic structure,which increases the reactive sites and enhances the catalytic performance.Due to the synergistic effect between structure and doping,the Mn-NiO modified electrode exhibits faster current response?response time<5 s?,higher sensitivity(3212.52?A·mM^-1·cm^-2),good stability and reproducibility,along with well feasibility in blood serum analysis.?2?As a pivotal signal molecule in the body,H₂O₂,which performs the strong oxidizing property,can damage biomolecules such as DNA in the cell and cause disease.Therefore,accurate and real-time detection of H₂O₂ is essential.In this chapter,the MnOOH-MnO₂ heterogeneous structures with different morphologies were successfully synthesized by controlling the reaction time in the liquid phase,and further applied to electrochemically detect H₂O₂.SEM,TEM,XRD and XPS were used to characterize and study the formation mechanism of MnOOH-MnO₂ heterostructures.The systematic electrochemical test shows that compared with MnOOH and MnOOH/l-MnO₂?layered distribution structure?,MnOOH/i-MnO₂?island-shaped distribution structure?exhibits better catalytic performance,specifically manifested in higher response current,larger electrochemical active area?ECSA?,more efficient charge transfer and electrocatalytic reactions.In addition,the MnOOH/i-MnO₂ modified electrode is used for H₂O₂ detection,showing high sensitivity(265.40 and 97.90?A·mM^-1·cm^-2),wide linear range?10?M-19.44 mM?,excellent selectivity,reproducibility and stability,and successfully in situ detect the H₂O₂ released from human prostate cancer cell DU145.?3?Based on the above studies,in order to overcome the shortcomings of poor conductivity and high overpotential of the oxide,MnO@C nanowire composites were successfully prepared by in-situ polymerization coating and carbonization process by using the excellent adhesion of polydopamine,and the coating amount was further optimized.The electrochemical results show that when the mass ratio of dopamine to MnOOH is 3:4,the obtained MnO@C nanowire composite has the better catalytic performance for H₂O₂?higher response current,larger electrochemical specific surface area,faster charge transfer and catalytic reaction rate?.Moreover,the linear detection range of the composite modified electrode for H₂O₂ is 1?M–1.731 mM and 1.731–5.831 mM,the detection limit is 0.45?M,and the response time is less than 5 s.In addition,the electrode also exhibited excellent stability and anti-interference,and successfully achieved in situ detection of H₂O₂ released from human prostate cancer cell DU145.?4?Superoxide anion(O₂^·-)is another small molecule with strong oxidative properties in the cell,which is an important mediator molecule in the body.Abnormal levels of O₂^·-can cause oxidative stress in the body,which in turn can damage cells and cause lesions.Therefore,real-time quantitative detection of O₂^·-is extremely significant for the diagnosis and prevention of diseases.Herein,Co₃?PO₄?₂ nanomaterials with spherical structures were successfully prepared by a simple one-step hydrothermal process,whose structure and morphology were also characterized.It is found that urea is an essential condition for the synthesis of spherical Co₃?PO₄?₂ nanomaterials,but its amount has no significant effect on the morphology and size of the nanomaterials.Thanks to the fast interfacial electron transfer rate and large electrochemical surface area,the spherical Co₃?PO₄?₂ nanomaterial modified electrode has excellent performance in the enzyme-free detection of O₂^·-,such as a wide linear detection range?27 nM–6.132?M?,lower limit of detection?1 nM?,higher sensitivity(1280.14?A·?M^-1·cm^-2),and excellent stability and immunity.In addition,the nanomaterial-modified electrode successfully achieved real-time detection of O₂^·-in situ released from human prostate cancer cell DU145.In summary,the electrocatalytic performance of biomimetic enzymes was effectively improved through using functional design approaches such as doping,compositing,and interface regulation,which results in the smart use of the synergy of structure and chemistry.Meanwhile,the prepared enzyme-free electrochemical biomimetic sensors exhibit excellent detection performance.These approaches could provide an effective way for the subsequent design of materials for high-performance enzyme-free electrochemical sensors.