Preparation Of Scanning Electrochemical Microscopy Probes And Study On In Situ Monitoring Of Cell Membrane Permeability And Extracellular PH

Scanning electrochemical microscopy(SECM)has been applied to a variety of biological analysis problems since its invention.It has made considerable progress in the analysis of biological samples,including living cells and immobilized biological macromolecules,such as enzymes,antibodies and DNA fragments.Compared with other scanning probe techniques,SECM can be used to observe the morphology of biotargets,and more importantly,chemical information can be collected on biological surfaces with high spatial resolution.As an imaging technique,improving the resolution of SECM scanning imaging has always been one of the goals pursued by researchers.On the one hand,smaller and smaller micro-nano probes has been prepared.On the other hand,the factors that affect the imaging resolution of SECM are theoretically discussed to obtain the best conditions in the imaging.Therefore,using mathematical formulas to describe the SECM imaging resolution can clarify more accurately the relationship between the SECM imaging resolution and the experimental parameters,thus helping to guide the acquisition of the best imaging resolution in the experiment,and promote its further application in biological imaging.Meanwhile,SECM could achieve in situ cell monitoring,showing strong advantages and extensive application prospects in biological sample analysis.For cells,the membrane permeability and extracellular pH are closely related to the physiological activity,which can reflect the physiological state of cells.Therefore,it is necessary to prepare the probes with different functions and develop in situ SECM monitoring methods for the investigation of changes in cell membrane permeability and extracellular pH,which shows great significance in observing the physiological state of cells and monitoring the cytopathic process in time.In this thesis,SECM probes with different functions were prepared by etching glass encapsulation.A series of self-made gold disk microelectrode probes at different sizes were prepared to discuss and verify the scanning imaging resolution theory in the SECM feedback mode proposed by us.The effect of copper ions on cell membrane permeability was in situ monitored with a self-made copper-gold dual electrode probe.Using a self-made gold-polyaniline modified platinum dual electrode probe,the effect of electrical stimulation on extracellular pH of different cells was investigated using SECM potentiometric method.The main research work of this thesis is as follows:(1)Gold disk microelectrode probes at different sizes and dual-electrode probes with different functions were prepared by the etching glass encapsulation method.To explore the influence of probe size on SECM imaging resolution,a series of gold disk microelectrode probes at different sizes were prepared.To in situ monitor the effect of Cu²⁺on cell membrane permeability,a copper-gold dual-electrode probe was prepared.In this dual-electrode probe,the copper electrode was used to generate copper ions by electrolysis,and the gold electrode was used to measure the approach curve.To monitor the extracellular pH in situ,a gold-polyaniline modified platinum dual-electrode probe was prepared.In this probe,the gold electrode was used to measure the approach curve for determining the distance between the probe and the cell,and the polyaniline modified platinum electrode was used as a pH-selective electrode to monitor the extracellular pH using the potentiometric method.The prepared probes were characterized and their properties were tested.The effective radius of the gold disk microelectrode was calculated using the steady-state limiting current formula.The size of the dual-electrode probe was characterized by cyclic voltammetry and SEM.The open circuit potential method was used to test the pH response characteristics of the polyaniline modified platinum electrode in the gold-polyaniline modified platinum dual-electrode probe.The preparation of these probes laid the foundation for the subsequent research of SECM.(2)Based on the feedback current theory,the mathematical relationship formula between the scanning current of the SECM tip electrode and the imaging resolution was defined and derived.This formula described various factors that affect the SECM imaging resolution,such as the size of the tip electrode and the tip-substrate distance.It was verified by scanning gold spot arrays at a certain size with self-made tip electrodes at different sizes and different tip-substrate distances.The experimental results were in good agreement with the results obtained by the formula simulation.Moreover,this formula showed that when the tip-substrate distance was determined,it should choose the tip electrode at the size with the value equal to that of distance,not the smallest size,to get the best imaging resolution.The experimental results proved that the proposed imaging resolution formula can provide theoretical support and guidance for improving the imaging resolution in SECM feedback mode.(3)Based on the self-made SECM copper-gold dual-electrode probe,an in situ monitoring method was developed to investigate the influence of Cu²⁺on the membrane permeability of living cells.Cu²⁺is one of the essential trace elements in the organism,but excessive Cu²⁺may be harmful to human health.Herein,Cu²⁺was generated by electrolyzing the copper electrode in the dual-electrode probe above the cell to stimulate the cell in situ.Potassium ferricyanide was used as the redox mediator to measure the change of the probe approach curve(PAC)when the probe approached the cell,thus in situ monitoring the influence process of Cu²⁺on cell membrane permeability.By overlapping the experimentally obtained PAC with the theoretical simulation PAC,the membrane permeability coefficient(Pm)value of the cell membrane change process was obtained.This work provides a way for SECM to monitor the cytopathic process and the treatment process of drugs on cells.It is also of great significance in exploring the complex mechanisms of acute Cu²⁺poisoning and could provide some references for the determination of the optimal time for the treatment of acute Cu²⁺poisoning.(4)An in situ monitoring strategy based on the SECM potentiometric method was developed to study the influence of external stimuli on the extracellular pH(pHe)of different cells with the self-made gold-polyaniline modified platinum dual-electrode probe.The gold electrode in the dual-electrode probe was used to measure the approach curve,and the distance between the probe and the cell surface was calculated by negative feedback theory.The SECM shear force method was used to verify the reliability of the distance between the probe and the cell surface obtained by the approach curve.The polyaniline-modified platinum electrode in the dual-electrode probe was used as the pH-selective electrode,and the pHe changes of normal cells and cancer cells under electrical stimulation were measured in situ by the SECM potentiometric method.It was found that normal cells and cancer cells responded differently to environmental stimuli,showing significantly different pHe changes.Through the measurement of cell membrane permeability changes under the same conditions,the hypothesis that pHe changes were related to cell membrane permeability changes was verified,and this hypothesis was also confirmed by fluorescent staining of calcein-AM and propidium iodide.The results revealed that this method is very useful for monitoring the stimulation response of cells and exploring the physiological activity state of cells.