Tailoring Biomimetic Enzymes In Nanoscales To Catalyze Electrochemical Oxidation Behaviors Of Superoxide Anion

The superoxide anion(O₂^·-)plays essential roles in various biological processes.Fast and sensitive detection of O₂^·-offers a promising strategy for the rapid related disease detection,but faces great challenges due to the low concentration,high activity and short lifetime of O₂^·-released from living cells.Electrochemical methods demonstrate numerious prominent advantages,including fast response,simplicity,relatively low cost,and in real time detection of O₂^·-released from living cells.Conventional electrochemical O₂^·-sensors mainly rely on specific enzymes modified onto the electrode surfaces,but they often suffer from poor reproducibility,short shelf time and high expense,and require well-trained personnel to operate.Synthetic biomimetic enzyme sensors could overcome the shortcomings of the enzyme-based ones,and thus are in high demand to replace the latter.This thesis introduces four biomimetic enzymes of high catalytic activity toward O₂^·-aiming at solving the problem of electrochemistry sensors on lack of effective enzyme substitute.Through tailoring the biomimetic enzymes in nanoscales to catalyze electrochemical oxidation behaviors of O₂^·-,the sensors based on them exhibit high performance in monitoring O₂^·-released from living cells.The main contents are as follows:1.Cobalt phosphate?Co₃?PO₄?₂?and iodine-doped reduced graphene oxide?I-rGO?materials were synthesized by chemical precipitation and one-step hydrothermal reaction to construct Co₃?PO₄?₂/I-rGO nanocomposites.Compared to reduced graphene oxide?rGO?,the I-rGO can bind with Co₃?PO₄?₂ closer.This may be due to the fact that I in the I-rGO plays an important role in promoting the binding.The sensor based on Co₃?PO₄?₂/I-rGO biomimetic enzyme achieves low detection limit(1.67 nmol·L^-1),short response time?2.99s?,high sensitivity(177.14?A·(?mol·L^-1·cm²)^-1),a wide linear range(1.67 nmol·L^-1-2.195×10³ nmol·L^-1)and high selectivity,and was futher used to monitor O₂^·-released from prostate cancer cells?DU145?under drug stimulation.2.Cobalt?Co?nanoparticles were deposited on nitrogen-sulfur-doped graphene?NSG?as an efficient biomimetic enzyme for O₂^·-detection.The Co atom was identified as the active site of the biomimetic enzyme.The Co-NSG biomimetic enzyme sensor exhibits excellent performance towards O₂^·-detection,reaching good anti-jamming ability,low detection limit(1.67 nmol·L^-1),short response time?1.35 s?,high sensititvty(628.86?A·(?mol·L^-1·cm²)^-1),wide linear range(1.67 nmol·L^-1-575 nmol·L^-1)and long-term stability,and was futher used to monitor O₂^·-released from DU145 cells under drug stimulation.3.Porous electrodes possess a large surface area and are often used to greatly improve the sensor sensitivity.However,how to tailor the pore structure,especially the pore size distribution to further improve the sensitivity and selectivity of a biomimetic enzyme sensor,has not been investigated yet.Herein,the pore structure of carbon nanofibers?CNFs?was tailored by pyrolysis to obtain an optimal mesopore structure for strong adsorption of DNA,followed by guiding the growth of manganic phosphate?Mn₃?PO₄?₂?as a biomimetic enzyme toward highly sensitive detection of O₂^·-.The Mn₃?PO₄?₂-DNA/CNF sensor achieves low detection limit(1.67 nmol·L^-1),short response time?3.17 s?,excellent sensitivity(249.29?A·(?mol·L^-1·cm²)^-1),wide linear range(1.67 nmol·L^-1-747.5 nmol·L^-1)while possessing good selectivity.The enhancement mechanism is also investigated,indicating that the mesopore ratio of CNFs plays an essential role in the high sensitivity and selectivity due to their strong adsorption of DNA for guiding the growth of a large amount of uniform sensing components,Mn₃?PO₄?₂,toward high sensitivity and selectivity.This discovery shedding a light on the enhancement mechanism of the pore structure,especially the pore size distribution of a porous electrode for high performance sensing processes.The Mn₃?PO₄?₂-DNA/CNF sensor was further used to monitor O₂^·-released from human keratinocyte cells and human malignant melanoma cells under drug stimulation,showing high sensitivity to quantitative detection of O₂^·-.4.Phytic acid?PA?is an organic compound with high density of negatively charged phosphate groups and broad source,allowing forms stable complexes with positively charged manganese ions.Herein,a Mn-PA biomimetic enzyme with uniformity and good coverage was obtained on MWCNTs by self-assembly,and the sensor based on Mn-PA-MWCNTs achieved excellent selectivity,low detection limit(1.25 nmol·L^-1),short response time?2.15 s?,wide linear range(1.25 nmol·L^-1-175 nmol·L^-1)and the highest sensitivity(757.16?A·(?mol·L^-1·cm²)^-1)so far.Moreover,explanation was proposed for clarifying the behaviors observed.In brief,the biomimetic enzymes based electrochemical sensors can offer great sensitivity and high selectivity for O₂^·-detection,which mainly due to the unique functions of the unique functional groups for the high selectivity and the large surface area of the nanomaterials to attached for the high sensitivity.Thus demonstrating universal schemes to design better biomimetic enzyme biosensors for O₂^·-released from living cells detection and for applications in biological research and diagnosis.