Single Cell Electrochemical Analysis Of Cell Membrane Cholesterol And Intracellular Phosphate

Cells are the basic structural units of living organisms.Due to certain differences between single cells,achieving the analysis at single cells becomes a huge challenge.Currently,single cell analysis can be achieved mainly by fluorescence imaging,electrochemistry,mass spectrometry,etc.Since the electrochemistry could offer high sensitivity and spatial-temporal resolution,it is popular for single cell analysis.This thesis is developing novel micro/nanoelectrodes to detect some molecules that are hardly electrochemically detected at single cells.The first work of the thesis is to develop cholesterol oxidase/Triton X-100 parked microelectrodes for the detection of plasma membrane cholesterol in single living cells.The classic electrochemical analysis of plasma membrane cholesterol at single cells utilizes cholesterol oxidase modified microelectrode that oxidizes local cholesterol efflux from plasma membrane to generate hydrogen peroxide for the electrochemical quantification.In this letter,a mixture of cholesterol oxidase and Triton X-100 was filled in the micro-capillary that could park at Pt layer coated tip due to slow hydrodynamic flow.During the contact of the tip with cellular membrane,Triton X-100 at the tip permeabilized the contacted membrane to release cholesterol for the reaction with cholesterol oxidase.As compared with the linkage of cholesterol oxidase at the electrode surface,the oxidase parked in aqueous solution at the tip had a higher turn-over rate resulting in larger electrochemical signal for single cell analysis.More charge collected at acyl-coA:cholesterol acyltransferase(ACAT)inhibited cells supported that this novel detection strategy could monitor the flunctation of membrane cholesterol at single cells.The successful detection of plasma membrane cholesterol at single cells using oxidase parked microelectrode will provide a special strategy for the fabrication of biosensor that permits the integration of more molecules without function groups at the electrode to measure active and inactive molecules in plasma membrane.Moreover,the larger electrochemical signals collected could further increase the spatial resolution for single cell electrochemical analysis.The second work of this thesis is to apply the nanoelectrode with a diameter of?100 nm for the detection of phosphate in single cells.After a certain electrochemical voltage is applied inside the capillary,the solution containing maltose,maltose phosphorylase and glucose oxidase is electrochemically egressed at the tip of capillary.Once exposed to phosphate,it could react with maltose producing glucose under the catalysis of maltose phosphorylase,and then generate hydrogen peroxide through the catalysis of glucose oxidase.Eventually,hdrogen peroxide is detected by the nanoelectrode for the quantitation of phosphate.To validate this assay,we firstly tested the reaction system using fluorescence test and nano-ESI detection.All the experimental results verify the feasibility of the reaction system.The following electrochemical experiments using nanoelectrodes showed more electrochemical signals with the increased concentration of phosphate.Furthermore,the electrochemical signal generated from intracellular phosphate reaction with the enzymes at the tip of nanoelectrodes was also detected.After eliminating the background electrochemical signal from the non-specific adsorption and intracellular glucose,the intracellular phosphate content can be estimated.The perfusion of the enzyme solution at the nanoelectrode can effectively ensure the activity of the enzyme.Moreover,the nanoscale tips reduce the damage to the cell and ensure the activity of the cell in the detection process.This work could provide a new,simple,and universal technology to monitor intracellular biological molecules.