In Situ Detection Of Bionic Enzymes And Reactive Oxygen Molecules Released By Cells In Carbon-based Nanocomposite

As the most abundant element in the earth,the research of nanomaterials composed of carbon are still rising.At present,carbon nanomaterials are used in various commercial products,ranging from rechargeable batteries,auto parts and daily necessities to military equipment.Researchs progress of material synthesis,purification,and modification have enabled carbon materials to be integrated into miniature electronic devices and biosensors.Although new materials have been explored to provide compelling electrical conductivity and flexibility for many biosensing applications,carbon materials still occupy an important position in biosensing.The ubiquity and importance of reactive oxygen species（ROS）in cells in living organisms has aroused great interest in their detection and quantification.ROS have a role in regulating the tumor environment,which affecting various stromal cells that provide metabolic support,blood supply,and immune responses to tumors.Although ROS play an important role in tumorigenesis,it is difficult to reliably predict the effect of ROS regulation.Among these common analytical techniques,electrochemical sensors are stand out in detection of ROS due to their high sensitivity and miniaturization.More importantly,the ROS released from cells are extremely low and their half-life is short,in situ and real-time detection of ROS is urgently needed.Biomimetic enzyme-based electrochemical sensors constructed by carbon nanomaterials have shown great advantages in terms of biocompatibility,selectivity,stability,and unique catalytic activity,which can be used to rapidly detect ROS in different cell populations.In this dissertation,two novel biomimetic enzyme sensing materials with specific physicochemical properties were designed and constructed based on carbon nanofibers（CNF）combined with surface interface regulation and functional modification.Firstly,functional group-modified carbon nanofibers（FCNF）were prepared by acidizing CNF,and then a biomimetic enzyme of FCNF co-modified with Pt and CuO was designed and constructed by doping metal platinum（Pt）and modifying copper oxide（CuO）.Pt-CuO@FCNF),the biomimetic enzyme can specifically catalyze hydrogen peroxide（H₂O₂）molecules and exhibit excellent charge transfer ability,finally realizing the rapid and highly sensitive detection of H₂O₂molecules released by living cells.Furthermore,imidazole-induced manganese oxide crystals（Mn Ox）were formed in situ on the surface of hydroxylated CNFs（ACNFs）by utilizing conjugated five-membered cyclic imidazole molecules to form coordination bonds with carbon and their dual functions as nitrogen-chelating ligands.Then,gold nanoparticles（AuNPs）were loaded on it to construct Au-Mn Ox/Im@ACNF bifunctional biomimetic enzyme,and the bifunctional biomimetic enzyme-based flexible sensing electrode was constructed by screen printing technology,and then realized the release of cells.Real-time monitoring of two molecules,H₂O₂and superoxide anion(O₂^·-).1.The surface of FCNF was regulated by metal Pt doping and CuO modification,and a biomimetic enzyme（Pt-CuO@FCNF）functionalized with Pt and CuO was prepared in batches by one-step high-temperature firing method.Characterization results such as transmission electron microscopy,Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy proved the successful preparation of the Pt-CuO@FCNF biomimetic enzyme.The sensing platform constructed with this biomimetic enzyme exhibits excellent performance for H₂O₂detection,including a wide linear detection range of 0.4μM-11 m M and a detection limit as low as 17 n M.sensing mechanism.The electrochemical sensor chip was fabricated by screen printing technology,which finally realized the rapid and highly sensitive detection of H₂O₂molecules released by living cells.2.Taking advantage of the dual functions of conjugated five-membered ring imidazole molecules,which can form coordination with carbon and function as nitrogen chelating ligands,imidazole-induced Mn Ox was formed in situ on the surface of ACNF,and then AuNPs were modified in situ to construct a Au-Mn Ox/Im@ACNF bifunctional biomimetic enzyme.The sensing platform based on the bifunctional biomimetic enzyme exhibits excellent sensing performance for both H₂O₂and O₂^·-,and the linear detection ranges for H₂O₂and O₂^·-are 0.8μM-300 m M,respectively.and 6 n M-13μM,with detection limits as low as 70 n M and 0.3 n M,respectively.Furthermore,a printing paste was prepared based on the bifunctional biomimetic enzyme,and a flexible electrochemical sensor chip was constructed by a screen printing process,thereby realizing in situ real-time monitoring of two molecules of H₂O₂and O₂^·-released by cells.