Real Time And Continuous Electrochemical Sensing Of Three Kinds Of RONSS Small Molecules In Biological Liquid

Reactive oxygen species,reactive nitrogen species and reactive sulfur species（RONSS）in biomarkers participate in the cellular maintenance of redox equilibrium,which has important physiological significance.Understanding and clarifying the interaction between RONSS and its regulation mechanism on redox balance can avoid the oxidative damage of nucleic acid and protein caused by stress response,and can also be used for disease screening,auxiliary diagnosis and evaluation of the safety and effectiveness of new drugs and new therapies.These studies are inseparable from the accurate and rapid detection of RONSS,but the real-time and continuous detection of RONSS in biological liquid is very challenging due to its high reactivity,rapid diffusion and transient transformation characteristics.The biosensor based on electrochemical voltammetry has the advantages of high sensitivity and easy miniaturization,which makes it possible to measure the species of RONSS in biological liquid environment in real time and continuously.The application of composite nanomaterials and biological recognition elements in the construction of sensing interface is a common signal amplification strategy of sensors,and is the only way to improve the sensitivity and selectivity.Noble metal nanomaterials,carbon nanomaterials,transition metal and its oxide nanomaterials and conductive polymer materials are the most suitable modified electrode materials.The electrode surface modifier can be deposited in many different combinations,and the strict analysis of the characteristics of individual modification is very important for the wise selection of appropriate modification materials.The ideal sensor ultimately depends on the practical application.Sensitivity,selectivity,long-term stability and biocompatibility are equally important in qualitative and quantitative assurance.Specifically,the electrochemical sensing of three kinds of bioactive small molecules of hydrogen peroxide（H₂O₂）,hydrogen sulfide（H₂S）and nitric oxide（NO）is studied in five parts.（1）1-Aminopyrene was used as an intermediate to construct an electrochemical biosensor for monitoring H₂O₂.For it can covalently linked with HRP by amino group at the sensing interface,and the conjugated aryl group can be fixed with reduced graphene oxide（RGO）byπ-πconjugation on the other end.The covalent bonding of enzyme can prevent the leaching of enzyme during the use of electrode,whileπ-πconjugated bonding can accelerate the electron transfer,after then,efficient and directional immobilization of the enzyme can be realized.The electrochemical performance of HRP-AP/RGO sensing interface was determined by cyclic voltammetry（CV）,electrochemical impedance spectroscopy and constant potential amperometry（CPA）.Under the optimal conditions,the linear range of the sensor was 1.5μM to 28.5μM,and the limit of detection was 0.5μM.The sensor also had good stability and high selectivity,and was suitable for monitoring the concentration of H₂O₂ in biological liquid.（2）Manganese dioxide nanoflakes were synthesized by biomineralization method using bovine serum albumin as template.The composition and two-dimensional morphology of the materials were characterized by scanning electron microscopy（SEM）,transmission electron microscopy,energy dispersive X-ray spectroscopy and attenuated total reflectance method fourier transform infrared spectroscopy（ATR-FTIR）.Then it was hybridized with RGO with good conductivity for electrochemical sensing of H₂O₂ in biological environment.Experiments results show that the composite has good catalytic performance,high sensitivity and low detection limit.Sensor perform linear response to H₂O₂ concentration in the range of 20 n M to5μM and 5μM to 800μM,and the limit of detection was 14.92 n M（s/N=3）.The constructed sensor was stable,reproducible and selective,and could be used to monitor the content of H₂O₂in real biological environment.（3）Biomass derived carbon material（Bcn）was prepared from lotus leaf by high-temperature pyrolysis,and nano-Cu Fe₂O₄ was prepared by high-temperature solid-state reaction.The synthetic material was characterized by X-ray photoelectron spectroscopy,SEM and ATR-FTIR.A H₂S electrochemical sensor was prepared by combining Bcn with Cu Fe₂O₄on GCE surface.CV,DPV and CPA were used to investigate the sensitivity,selectivity and stability of the sensor.It was found that the composite can avoided the deposition of sulfur on the electrode surface and can refrain from the interference of electroactive substances in the biological environment.The Cu Fe₂O₄/Bcn sensor can sensitively and accurately respond to H₂S in the concentration range of 5 n M-10μM,and has high sensitivity retention rates to H₂S in simulated wound fluid.（4）Triple pulse amperometry（TPA）can provide discrete cleaning and measurement pulses,eliminating the passivation of electrode surface caused by sulfur deposition.The performance of the sensor can be stabilized and the selectivity can be improved by modifying noble metal materials or conductive polymer films on the electrode surface.Au nanoparticles,Pt nanoparticles and PEDOT modified electrodes were used to construct electrochemical sensors for the detection of H₂S.Based on the current response of CPA and TPA method to H₂S and four kinds of main interferences of AA,DA,UA and EP,the sensitivity and the limit of detection were calculated,and the selectivity coefficients were comprehensively compared to determine the optimal electrode modification scheme which is PEDOT/Au composite film modified electrode and TPA method.The sensor also had good repeatability and stability,and could be used in simulate real biological environment for the continuous electrochemical monitoring of H₂S.（5）The direct electrochemical oxidation of NO occurs at a high potential,so it is necessary to quantitatively investigate the selectivity of the sensor.CNT sensing interface（CNT/GCE）,Ti₃C₂ sensing interface（Ti₃C₂/GCE）,Au nanoparticles sensing interface（Au/GCE）and poly（1-amino-5-naphthol）sensing interface（P1A5N/GCE）were prepared by drop coating method,electrochemical deposition method and electrochemical polymerization method,respectively.The voltammetry responses of four sensing interface to NO and three interfering compounds of AA,CYS and NO₂^-were studied by DPV method.The corresponding sensitivity and selective interference coefficient were calculated.The optimal sensing interface was Ti₃C₂/CNT/GCE,and its good performance was evaluated in simulated real biological liquid.This dissertation contains 68 figures,9 tables and 315 references.