The Detection And Removal Of Reactive Oxygen Based On Nanoenzymes

Reactive oxygen species(ROS)is a kind of metabolite involved in electron transport,which participates in various physiological and pathological processes.Under physiological conditions,antioxidant defense mechanisms(including enzymatic antioxidants and non-enzymatic molecules)can maintain the ROS content in cells at normal levels.The enzymatic antioxidants mainly include:peroxidase(POD),superoxide dismutase(SOD),catalase(CAT),glutathione peroxidase(GPx)and so on.Non-enzymatic molecules mainly include small molecules such as reduced glutathione.At medium and low levels,ROS has beneficial effects,participating in many physiological processes,such as defensing against pathogenic microorganisms,mitotic response and redox regulation.But,the ROS concentration imbalance can lead to oxidative stress,which may cause diseases such as rheumatoid arthritis,cardiovascular disease and cancer.Hence,detection and regulating the intracellular ROS concentration plays crucial role in the treatment of these diseases.Although we can produce natural enzyme to realize the diagnosis and treatment of this kind of disease,there are some shortcomings of natural enzyme,such as sensitive to environmental conditions,complex production process and complicated purification process.Therefore,it is necessary to develop enzymatic materials with simple preparation method and not harsh to the environment.Nanoenzymes are a kind of simulated enzymes with unique properties of nanomaterials and catalytic properties of natural enzyme.Nanoenzymes are defined to meet two conditions.First,they belong to nanomaterials.Second,they have enzymatic properties and can simulate the enzymatic properties in redox reactions.Compared with the single enzymatic properties of natural enzymes,nanoenzymes can have multi-enzyme active sites.What's more,nanoenzymes are low production cost,acid and alkali resistance,high temperature resistance and easy to achieve mass production.At present,nanoenzymes have been widely used in material science,environmental protection,biomedical applications and so on,which connects nanotechnology with biology and environment,and provides a variety of functional platforms.However,nanoenzymes can not accurately mimic the catalytic active center structure of natural enzymes,and usually exhibit lower enzyme-like activity than natural enzymes.Therefore,to realize nanoenzymes playing better role in these fields,it is important to improve the catalytic performance and develop multi-enzymatic activities of nanoenzymes.Based on the above research background,we prepared two kinds of nanoenzymes,the specific research contents are as follows:(1)Hemin and phytic acid functionalized porous conductive polymer hydrogels for electrochemical detection of H₂O₂released from living cells.We used PA to induce polyaniline(PANI)gelation to form PA-PANI conductive hydrogels polymer.Subsequently,to obtain Hemin/PA-PANI,PA-PANI was co-incubated with Hemin.PA-PANI provided stable scaffold for Hemin,which maintained or even improved Hemin catalytic activity,because it can prevent Hemin aggregation.To realize real-time monitoring of H₂O₂released from living cells,Hemin/PA-PANI was modified onto glassy carbon electrode(GCE)to obtain Hemin/PA-PANI/GCE.Then,we further prepared Hela/Hemin/PA-PANI/GCE by dropping Hela cell suspension onto the Hemin/PA-PANI/GCE.Finally,the Hela/Hemin/PA-PANI/GCE was used as the working electrode and ascorbic acid(AA)was added to stimulate cell releasing H₂O₂,which realized electrochemical real-time detection of H₂O₂releasing from living cells.This provided ideas and methods for the detection and diagnosis of molecules released by living cells.(2)ROS removal of graphitic carbons doped with Mn?Fe nitridesIn this work,we fabricated Mn Fe PBA-700-HCl nanoenzymes using K₃Fe(CN)₆as atemplate by coprecipitation doping manganese ions,700?high-temperature pyrolysis and HCl etching.By XRD and XPS characterization,the nanomaterial was determined as graphite-like carbon nanoparticles doped with Mn?Fe nitride.The UV absorption spectroscopy and dissolved oxygen test in water showed that the nanomaterial had many enzyme-like activities,including POD,CAT,SOD,GPx and·OH removal ability.Moreover,compared with the single metal catalytic active sites of Mn PBA-700-HCl and Fe PBA-700-HCl,the Mn Fe PBA-HCl enzyme catalytic ability was stronger.The MTT experiments showed that the nanoenzyme had good biocompatibility.We used the enzyme to reduce oxidative stress damage to Raw-264.7 cells induced by H₂O₂.Finally,fluorescence imaging further proved the nanoenzyme can remove intracellular ROS.