Glass Conical Nanopore For Detection Of Hydroxyl Radicals In Single Cell

As is known to all,hydroxyl radical(·OH)was the best offensive reactive oxygen species(ROS),is thought to induce neurodegenerative diseases,such as alzheimer's disease(AD)plays an important role.It is reported that A?amyloid protein fragments,especially A?_1-42,can be induced by mitochondrial oxidatie stress(MOS)toxicity on neurons.However,due to the extremely short life expectancy(in vivo?10^-9 s)and very short diffusion distance(?10^-9 m)of·OH,it is difficult for·OH produced by mitochondria to diffuse outside the cell.Therefore,in situ detection of mitochondrial oxidative stress product·OH induced by A?_1-42in a single cell is of great significance for monitoring intracellular activities,and will help reveal the role of·OH in the pathogenesis of AD and search for specific drugs for it.In this paper,a biosensor is constructed based on the principle of ion rectification of glass tapered nanopores to detect the concentration of·OH in single cells in situ.The first work is to design a sensor based on the influence of wetting degree on ion rectification.By self-assembling a hydrophobic molecular layer on the inner wall of the nanopore,the interaction between·OH and the molecular layer was used to change the wettability degree of the inner wall of the nanopore,and the change of ion rectifying was analyzed to determine the concentration of·OH in the unknown sample.The second task is to design a sensor based on the influence of surface charge on ion rectification.By self-assembling a charged molecular layer on the inner wall of the nanopore,the surface charge of the inner wall of the nanopore was changed by the interaction between·OH and the molecular layer.Analyze the change of ion rectification to determine the concentration of·OH in the unknown sample.Specifically,this paper is divided into the following three parts:Charpter 1:OverviewThis chapter first summarizes the existing analysis methods of·OH,including electrochemical methods and other methods,and compares the advantages of these methods.Then it mainly explains the principle of ion rectification of nanopores and chemical and biological sensing based on ion rectification.In addition,the electrochemical analysis of reactive oxygen species in single cells is discussed from the perspective of nanoelectrodes and nanoporous electrodes.Finally,clarify the main meaning and content of this thesis.Charpter 2:In situ detection of hydroxyl radicals in single cells by functionalized nanopores with adjustable wettability based on 1-hexanethiol modificationIn this work,we first uniformly plate a layer of gold film on the inner wall of the glass tapered nanopore,and then use the strong interaction of the Au-S bond to modify the 1-hexanethiol(HAT)to construct an adjustable inner surface wettability The nanopore sensor is used to detect the content of·OH near mitochondria in situ with high selectivity and sensitivity.Since·OH can degrade HAT at an ultra-fast reaction rate(rate constant:?10¹⁰ M^-1S^-1),the inner wall of the nanopore changes from hydrophobic to hydrophilic,leading to changes in the ion rectification signal,thereby realizing·OH Highly selective detection,the lowest detection limit can be as low as 1 n M.In addition,with the help of a micromanipulator and a confocal microscope,the nanopore sensor can easily penetrate the cell wall and be positioned near the subcellular unit.Based on the above advantages,we inserted the nanopore sensor near the mitochondria of mouse monocyte macrophages,and detected the·OH produced by the mitochondrial oxidative stress stimulated by A?_1-42.Charpter 3:In Situ Detection of Hydroxyl Radicals in Single Cells Based on Protoporphyrin Modified Functionalized Nanopore with Adjustable Surface ChargeIn this work,we first used the strong interaction of the Au-S bond to modify the inner wall of the gold-plated nanopore with cysteamine,then through the amide reaction,the protoporphyrin was connected,and finally the protoporphyrin was coordinated and charged.The metal zinc ions,construct a nanoporous sensor with adjustable surface charge,which is used to detect·OH near mitochondria in situ with high selectivity and green biomimetic ground.Based on·OH,the carbon chain of cysteamine can be quickly cleaved,causing the charged self-assembled molecular layer to leave the inner wall of the nanopore,resulting in a change in the ion rectification signal,thereby realizing the highly selective detection of·OH.The electrode was finally used to detect LPS-induced·OH bursts in single mouse macrophages.