Micro-or Nano-capillary Based Single-Cell Electrochemical-Mass Spectrometric Analysis

Cells are the basic units of structure and function of organisms.The study of cellular structures,functions and behaviors can help to explore physiological and biochemical phenomena of bodies,elucidate life activities of complex organisms and the nature of life.Moreover,it can also provide important information for diagnosis and treatment of diseases,such as cancers.Due to individual differences between cells,the signal from individual abnormal cells is obscured by the traditional analysis at cell population.Therefore,the analysis at single cell level has attracted much attention in recent years providing the information of special cells and their interactions in cell population.Accordingly,single cell analysis is of great importance for early diagnosis and prevention of cancer.Among various methods for single cell analysis,mass spectrometry and electrochemical analysis are two main label-free methods.Mass spectrometry has the advantages of high sensitivity,high selectivity,the ability to label-free detect multiple species simultaneously and the detection of unknown samples.Meanwhile,electrochemical analysis can provide high sensitivity,high temporal-spatial resolution and quantitative analysis.Micro-or nano-capillary that is pulled from borosilicate or quartz glass tubes have been applied in both fields of single cell mass spectrometry and single cell electrochemical analysis,and thus,become one of the most important tools in this analysis.In this dissertation,novel single cell electrochemical-mass spectrometric analysis based on micro-or nano-capillaries is developed for single-cell analysis.The main work is as following:1.Molecular Profiling of Single Axon and Dendrite in Living Neurons using Electrosyringe-assisted Electrospray Mass SpectrometryIn the second chapter,electrosyringe-assisted electrospray mass spectrometry was firstly established to achieve the intracellular sampling from one axon or dendrite in living neurons for mass spectrometric analysis.The key was to insert a?130 nm capillary tip into one axon or dendrite for the loading of the cytosol from these two compartments through electro-osmotic flow.The ionization efficiency from the nano-capillary was enhanced to guarantee the successful mass spectrometric analysis of multiple species from the axon and dendrite.Higher levels of pyroglutamic acid and glutamic acid were revealed in axon than those in the body and dendrite,which was in accordance with the accumulation of neurotransmitters in the axon for information delivery.The achievement of electrosyringe-assisted electrospray MS unveils the molecular distribution in the whole living neuron,which offers the feasibility for the deep investigation of molecular communication between the axon/dendrite and the body inside the neurons.2.Resistive Analysis of Hydrogen Peroxide in one Axon of single Neuron with Electrosyringe-assisted Nano-pipettesIn the third chapter,a resistive analysis based on acrylic acid polymerization in the nanocapillaries was established to measure hydrogen peroxide in one axon of single neuron.A nanocapillary(the orifice:?130 nm)containing acrylic acid,ferrous ions and phosphate buffer was inserted in the axon of one living neuron,and hydrogen peroxide was electrokinetically loaded into the pipette under the potential applied at the metal wire inside the capillary,to react with ferrous ions generating hydroxyl radicals.These radicals initialed the polymerization of acrylic acid for the elevation of capillary resistance,as reflected by the drop of the ion current.0.3?0.5 nA of current drop was observed supporting the successful analysis of intracellular hydrogen peroxide in the axon.Similar current drops were observed at the body and the axon after the physical loading of hydrogen peroxide or physiological stimulation by.phorbol ester(PMA),which suggested even distribution of hydrogen peroxide in the neuron.As compared with the classical amperometric analysis using nanoelectrodes,this new strategy avoided the complexity in the electrode fabrication and the measurement that should facilitate electrochemical analysis of intracellular molecules at subcellular level.3.Electrochemical-Mass Spectrometric Analysis of Oxidative Stress at Single Cells using microcapillaryIn the fourth chapter,an electrochemistry-mass spectrometry integrated micro-capillary was designed for the analysis of protein kinase C(PKC)activity and reactive oxygen species(ROS)during oxidative stress at single cells.The micro-capillary was platinized and insulated at the outer wall that could electrochemically measure hydrogen peroxide in individual HeLa cells after PMA stimulation.The hollow capillary could retain the solution with the peptide substrate that reacted with intracellular PKC generating the phosphorylated product.By nano-electrospray ionization from this capillary,the activity of PKC associated with this oxidative stress could be quantified.This novel probe enables the integration of electrochemical-mass spectrometric analysis at single cell level,which is expected to provide a deeper understanding of oxidative stress at single cells in the viewpoint of molecules.