Application Of Electrochemistry/mass Spectrometry In The Analysis Of Biomolecules

Cells are the basic building blocks of organisms that are quite different in the morphology and composition.Therefore,the detection of biomolecules in a single living cell is significant to understand life activities at the cell level,such as cell growth and differentiation,lesions,etc.All the information obtained will be useful for the early diagnosis and prevention of diseases.In the past fifty years,the technologies for single cell analysis,including fluorescence imaging,electrochemical analysis,mass spectrometry and microfluidic technology,have made great progress.In the first part of this thesis,the detection of phosphate ions in single cells was realized by the nanokit based electrochemical analysis that has the advantages of non-labeling,low detection limit and simplicity.Because the electrochemical analysis lacks the information about the molecular structures during the redox process,mass spectrometry that can directly determine the composition of the mixture is adopted into the electrochemical analysis.In the second part of this thesis,a combination of electrochemistry and mass spectrometry in a capillary is developed that can simultaneously obtain the redox information and structural information in an electrochemical reaction to realize the understanding of molecular mechanism.The third part of this thesis couples the nanokit and nano-electrospray to realize the enzymatic hydrolysis of cytochrome C in the micro-capillary and the following structure analysis using MS.This preliminary data will be useful for the future development of single cell proteomics using the nano-capillary.The first work in this thesis uses the nano-capillary with an opening of?130nm to hold the phosphate assay kit that could be loaded into an individual cell for the detection of intracellular phosphate ions with minor interruption of cellular activity.Phosphate assay kit includes the biologically specific maltose phosphorylase that converts maltose(in the presence of phosphate ions)into glucose-1-phosphate and glucose.Then,glucose oxidase included in the kit reacts with glucose to generate gluconolactone and hydrogen peroxide.A small region of Pt layer is exposed at the tip of capillary that behaves as the electrochemical surface for the detection of hydrogen peroxide.Accordingly,intracellular phosphate ions at single cells are successfully detected using this electrochemical approach.The possible interruption of other phosphates in the detection of phosphate ions is investigated,and the result exhibits a good selectivity for phosphate.Moreover,the contribution of intracellular glucose on the signal is excluded resulting in an accurate quantification of the phosphate level at single cells.To obtain the structural information during this electrochemical process,in-situ molecular characterization of an electrochemical reaction is achieved in the second work.The micro-capillary behaved as both of the electrochemical cell and electrospray ionization source so that the products in the reaction could be identified in mass spectroscopy.The key is to position both of the working and reference electrodes in the capillary,which are applied with a low and high voltage in serial to initial the electrochemical reaction and electrospray in two steps.Different products after the electrochemical oxidation of dopamine in aqueous solutions with different pHs,and the redox process of bis(diarylmethylene)type dyes are obtained,which provides the molecular information to elucidate the reaction mechanisms of these reactions.This successful realization of in-situ molecular characterization using this setup should facilitate the deep understanding of the electrochemical process that is significant in the field of electrochemical analysis.The third work in this thesis couples the nanokit and nano-electrospray to detect cytochrome C in a micro-capillary.Dithiothreitol(DTT),and iodoacetic acid required for protein enzymatic hydrolysis are pre-loaded into the micro-capillary following the loading of trypsin and cytochrome C.Cytochrome C exposes the disulfide bond through the reaction with urea,which is then oxidized by DTT and cleaved by Trypsin in the next step.After the enzymatic hydrolysis is completed,the composition of the enzymatic hydrolysate peptides is analyzed by ESI-MS.The successful realization of the detection of cytochrome C is expected to be applied for the intracellular protein analysis in the future.