Construction Of Optical/Electrical Micro-nano Probe And In Situ Detection Of Single Cell Bioactive Molecules

As the basic unit of organism morphological structure and life function,cell can generate two types of important bioactive molecules,including reactive oxygen species?ROS?and reactive nitrogen species?RNS?.They produced and consumed constantly in the cell,while controlling the differentiation and proliferation of a cell,mediating intracellular signal transduction and determining the survival and apoptosis of the cell,thus play a key influence on the pathological and physiological processes of organisms.On the one hand,they are used as signal and functional molecules to maintain the conventional functions and help the cell to self-repair.On the other hand,they are unfriendly to induce the cell death in abnormal procedure and lead to lesion of organism.However,the interests mainly depend on the sites,types and concentration of ROS and RNS generated in the cell.In addition,the nature characteristics of short half-life,low production,fast diffusion rate and high reactivity about ROS and RNS make the capture and detection full of challenges.What's more,comparing to the general investigation of population cells,only analyzing the individual cell one by one can get the information about the difference and particularity of generating locations,operating sites and content in single cell,which are covered up by the average resulting from traditional methods,thus we can get a new understanding of the mechanism during the physiological and pathological processes,providing a deeper insight to some particular functions and pathogenic factors.What's more,single cell analysis can identify and interrogate the subcellular structure at nanoscale,mapping the spatial distribution about production and release of ROS and RNS at the subcellular levels,investigating the expression and distribution of associated protein molecules,exploring the nanodomains for biochemical reactions and revealing the precise spatial architectures of organelles,which are not only render an important references for the development of cell biology and pathology,but also play a guiding significance for the prevention,diagnosis and treatment of related diseases caused by ROS and RNS,especially the design of drug delivery routes and the determination of effect sites to targeted drugs.Thus requires us to construct novel sensoring probes to real time detection of ROS and RNS generated and released from single cell with high temporal resolution,excellent sensitivity and selectivity.This PhD research project focuses on the fabrication of different types of sensing probes to detect the generation and/or release of ROS and RNS in single cell and even subcellular level through the optical and/or electrochemical modes with the ability of high spatial resolution,sensitivity and anti-interference,while resolving the dynamic change process to obtain temporal and spatial heterogeneity during the generation and/or release process,as well as speculating the spatial distribution and working process about the related enzymes,proteins and signal transduction,further explaining the biological mechanism associated with the physiological processes in single cell and providing scientific opinions for the corresponding pathological processes.The contents and results of the thesis are summarized as follows:?1?In this study,an optical nanofiber probe was constructed through the conventional method of physical vapor deposition with high optical resolution and applied to real time monitor the changes of ROS in a single HepG2 cell under the stimulation of different concentrations of graphene oxide?GO?,which is a signal molecule of oxidative stress.At the same time,reduced thiol level,a biomarker of intracellular antioxidant level was also detected to confirm the existence and change of oxidative stress.In addition,the physiological morphology of cells exposed to GO for a long time have changed significantly.All the results showed that GO can induce the oxidative stress of cell and indirectly proved the nanotoxicity GO to organism,thus further explaining the mechanism of toxicity on cell growth and providing parameters for the safety application of GO in the biological field.The study offer a reliable and rapid assessment mean for the preparation of nanomaterials with high safety and their further application in the biological fields.?2?Based on the independent optical detection for single cell,we fabricated an optical-electro bifunctional nanoprobe through direct plasma enhanced chemical vapor deposition?PECVD?growth of graphene nanowalls?GNWs?on the tip of nanoprobe with uniformly distributed and continuous free-standing graphene nanosheets,which can collect optical and electrochemical signals simultaneously.At the same time,this preparation method has good reproducibility and stability.The functional GNWs electrode with integrated nanoring,processing excellent structural and electrocatalytic properties,applied to detect NO with low detection limit,fast response time,high sensitivity and excellent selectivity.Under the induction of ultraviolet B?UVB?,the core of the nanoprobe will transmit the excitation light to a specific cellular domains and stimulate the NO-specific fluorescent molecular probe to obtain NO-induced intracellular oxidation state,while the modified GNWs nanoring electrode can real-time record the electrochemical signals and quantify the released NO molecules from single cell.The bifunctional nanoprobe can not only monitor the generated and released NO molecules from normal skin and cancer cells,but also acquire the localized detection on the cell membrane.The results showed that the cancer cell compared to normal cell and the nucleus domain contrasted to the peripheral domain display higher oxidative stress,reflecting the expression of nitric oxide synthase?NOS?in different cells and the heterogeneity of spatial distribution about NOS in the same cells.The designed nanoprobe on the one hand make sure the optical detection confined in evanescent field,on the other hand achieve the electrochemical detection with high signal to noise ratio and non-interference from UVB light,thus further applied to describe the mechanisms of UVB-induced oxidative damage.?3?Directly grown vertically-oriented graphene?VG?onto nanoprobe through PECVD,also calling GNWs,was adopted as not only an electrode substrate but also an efficient drug carrier,thus can deliver the drug to localized subcellular sites and monitor the dynamic changes of biomarker molecules to evaluate the subcellular site of action.We have loaded quercetin onto the VG covered nanoprobe successfully by?-?stacking and hydrophobic interaction and further investigated the amount of loading and release.The drug loaded nanoprobe was applied to deliver quercetin to three selected subcellular locations:nucleus,nucleus edge and cytoplasm,and simultaneously monitor the real-time dynamic changes of ROS production and Ca²⁺concentration under the existing of oxidative stress.The results show that the VG modified nanoprobe can payload the quercetin efficiently and demonstrates the cytosol is the best site of drug action to counteract the oxidative stress,which can be explained by the mechanisms of drug action properly.The constructed method provides a new platform for subcellular cellular drug loaded and delivered,and display a powerful insight for the design of targeted drug delivery routes in clinical research.?4?Based on the previously constructed sensoring probes that can only be used for detection of single components,we prepare a ring-plate electrochemical microsensor for real-time simultaneous detection of two kinds of bioactive small molecules generated from a single cell.The disk-shaped electrode fabricated through carbon nanotube?CNT?fiber with large surface area was electrodeposited with the nanocomposites of CTAB-nafion to detect NO molecule,whereas the ring portion consisted of gold film sputtered by magnetron sputtering was applied as a H₂O₂ electrode and further modified with Pt nanoparticles to improve the sensitivity,selectivity and response time.The prepared dual-electrode sensor has high temporal resolution and independent detection capability without chemical crosstalk effect between each other.Then,it was used to detect the real-time dynamic changes of H₂O₂ and NO produced by single cell under the stimulation of ochratoxin A?OTA?,and the amount of release,the time beginning release and the time reaching peak current were analyzed and calculated to reveal the relationship between two species during the produced progress.This work could be extended and applied to other dual analytes detection in many basic research areas with excellent performance.The sensing probes prepared in my PhD project research,on the one hand,is used to detect the bioactive molecules under particular induction conditions with high temporal resolution and sensitivity.On the other hand,it is mainly used to elucidate the generated mechanisms of these molecules,exploring the diseases factors,prevention,diagnosis and drug treatment for these molecules induced diseases,providing scientific insight and guidance for biology and pathology?...