Cnstruction Of Cellular Reactive Oxygen Species Monitoring And Regulatoion Platform And Its Performance Research

Reactive oxygen species(ROS)is a kind of substance produced by molecular oxygen in the process of redox in living organisms.It has the characteristics of short life,low concentration and high reactivity.Its dynamic changes can not only provide abundant physiological and pathological information,but also become a breakthrough in the diagnosis and treatment of diseases.However,the existing cell ROS monitoring platform has the problems of complicated preparation,low efficiency,and poor biocompatibility.In order to solve the problems in the above ROS monitoring and regulation,this paper used the functionalized short peptides and silica nanoparticles with excellent biocompatibility as the carrier,the enzyme and hemin as catalytic molecules to construct the electrochemical sensing interface and the enzyme-active nanoparticles.We realized the monitoring and regulation of cellular ROS.The specific research contents are as follows:1.Fabrication of self?assembled peptide hydrogel-based electrochemical biosensor for in suit detection of released H2O2.The self-assembled peptide nanofibrous hydrogel composed of N-fluorenylmethoxycarbonyl-diphenylalanine(Fmoc-FF)was used to construct a smart biointerface for enzyme-based electrochemical biosensing and cell monitoring.The Fmoc-FF hydrogel had two functions.One was as a matrix to embed an enzyme model,horseradish peroxidase(HRP),during the self-assembly of Fmoc-FF peptides.The other was use as a robust substrate for cell adhesion.Experimental data demonstrated that HRP was immobilized in a stable manner within the peptide hydrogel,and that HRP retained its inherent bioactivity toward H2O2.The HRP also can realize direct electron transfer in the Fmoc-FF hydrogel.The resulting third-generation electrochemical H2O2 biosensor exhibited good analytical performance,including a low limit of detection of 18 nM,satisfactory reproducibility,and high stability and selectivity.HeLa cells were then adhered to the HRP/Fmoc-FF hydrogel-modified electrode.The sensitive in situ monitoring of H2O2 released from HeLa cells was realized.2.Design and fabrication of three-dimensional(3D)cell cultured-based electrochemical biosensor for in suit detection of released O2·-.Distinctly different from the previously reported layering or separating fabrication of cell culture and sensing devices,herein living cells and enzymes as sensing elements are immobilized into a dipeptide-derived hydrogel matrix through simple one-pot self-assembly.The cells are then 3D cultured in the functional hydrogel,and the releasing superoxide anion(O2·-)is detected in situ by a cascade superoxide dismutase and horseradish peroxidase-based electrochemical biosensor.This novel design provides considerable advantages,including the possibility of capturing molecular signals immediately after they are secreted jfrom living cells,due to the close proximity of the enzymes and the 02·--producing cells.Furthermore,incorporating all components in a 3D matrix provides a confinement environment,that can lead to a concentrating effect of analysts.These properties allow the sensing device to achieve ultrahigh sensitivity and a precise response to a very low number of O2·-molecules.3.Preparation of self-assembling peptide artificial and its application as a detection probe for cancer cells and ROS scavenger.The hybrid nanoparticle(NP;fluorenylmethoxycarbonyl-arginine-glycine-aspartate and hemin,Fmoc-RGD/hemin NP)was developed for the simultaneous in vitro detection and inhibition of breast cancer cells.Hemin can regulate the levels of ROS while Fmoc-RGD acts as a scaffold for hemin nanocrystallization.Fmoc groups interact with the porphyrin groups of hemin through hydrophobic and ?-? interactions to form a hydrophobic core of NPs.The RGD groups of Fmoc-RGD also have a high affinity for binding integrin-rich tumor cells.Based on enhanced peroxidase activity,Fmoc-RGD/hemin NPs were developed as signal transducers in a facile and fast point-of-care cancer diagnosis platform.This platform is sensitive to breast cancer cells and hydrogen peroxide(H2O2),a biomarker for breast cancer.In addition,these Fmoc-RGD/hemin NPs can be used as nano-scavengers for ROS and for regulating the redox status of cancer cells.They also exhibit a targeted inhibitory effect on the epithelial-mesenchymal transition(EMT).4.Fabrication of a hollow nanomotor and explored its application as a ROS scavenger.Using hemin-coated mesoporous silica nanoparticles(MSNs)as a model,a large amount of ROS generated by oxidative stress in cells was used as fuel,the nanoparticles are self-powered by harnessing the chemical free energy of catalytic reactions to achieve autonomous intracellular walking.These hollow nanoparticles have exhibited average speeds up to 3.5-fold higher than those of solid nanoparticles.The ability of these movable hollow nanoparticles to scavenge ROS was verified both in vitro and in vivo using mice models.In this dissertation,the concentration of ROS in cells was monitored by a electrochemical platform based on the self-assembled peptides,providing a monitoring platform with high sensitivity,excellent biocompatibility and simple preparation.And through the assembly of organic molecules and the loading of inorganic nanoparticles,a highly efficient ROS scavenger was synthesized to achieve the regulation of ROS in cells.The ROS monitoring platform and ROS scavenger proposed in this paper can provide new ideas for the diagnosis and treatment of diseases.