Preparation Of Potassium Ion-Selective Microelectrode Combined With Potentiometric Mode In Scanning Electrochemical Microscopy For In Situ Detection Of Extracellular K⁺ At Single Cell Surface

Potassium channels are the most widely distributed ion channels on the cell membrane,and they can regulate a variety of cellular biological processes by controlling the flow of potassium ions.In common tumor tissues,potassium channels are abnormally expressed and participate in tumor development,such as cell proliferation,invasion,migration,and differentiation.Hence,the detection of extracellular K+to reflect the expression of potassium channels is significant for the early diagnosis and prognosis of cancer.In recent years,fluorescence sensing,photoacoustic imaging and electrochemical methods have been developed for the determination of extracellular K+.These traditional methods are stable and reliable,however,it is difficult to detect extracellular concentrations at precise locations,so more accurate and specific information about the physiological process cannot be obtained.Scanning electrochemical microscopy can control the position of the tip by drawing the approach curves,and realize the in-situ measurement of the electrochemical signal in the micro-area.The potentiometric mode could provide a higher selectivity for ion detection,relying on the specific recognition of analytes at ion-selective membranes,and has been used for in situ detection of H+,Ca2+,and Mg2+.Therefore,scanning electrochemical microscopy provides a useful method for in-situ detection of K+at cell surface.In this paper,the K+at cell surface has been detected in situ based on the potentiometric mode in scanning electrochemical microscopy.For the first time,a dual-functional potassium ion-selective microelectrode tip was prepared by using multi-walled carbon nanotubes as a solid-state transition layer.The K+concentrations at the surface of three different breast cancer cell lines were then in situ detected.The K+outflow induced by electrical stimulation and pH changes of the culture environment was further explored.And the possible mechanism of K+outflow was preliminarily explored by using 4-aminopyridine(4-AP).The research content of this paper is divided into four chapters,and the specific contents are as follows:Chapter One:IntroductionThis chapter first introduces the research significance and analysis methods of potassium channels and K+at cell surface,as well as the effects of electrical stimulation and pH changes of the culture environment on potassium channels and K+concentration at cell surface.And it introduces the device and principle of scanning electrochemical microscopy,the tips and the application in biosensing based on the potentiometric mode in scanning electrochemical microscopy.Chapter Two:Preparation and characterization of all-solid-state potassium ionselective microelectrode tipIn this chapter,the dual-functional potassium ion-selective microelectrode tip(K+-ISE tip)was prepared.And its electrochemical performance and analytical performance were characterized and tested.The K+-ISE tips were prepared by drop-casting the ion selective membrane cocktail doped with multi-walled carbon nanotubes.Since the multi-walled carbon nanotubes acted as the transduction layer,the conductivity and stability of the K+-ISE were improved.Then the tip can record the potential signal and draw approach curves.Cyclic voltammetry,electrochemical impedance spectroscopy and drawing approach curves were used to evaluate the electrochemical characteristics of the tip.Its analytical performance was then investigated,showing that its linear range was 10 ?mol/L-0.10 mol/L,the detection limit was 7.9 ?mol/L,and the slope was is 54.0 ± 2.0 mV/decade,and it has good selectivity and stability.This study provides an effective way for the detection of potassium ions.Chapter Three:In situ detection of potassium ion concentration at cell surface based on the potentiometric mode in scanning electrochemical microscopyIn this chapter,the dual-functional K+-ISE was used as the scanning electrochemical microscopy tip for in situ detection of K+concentrations at the surface of different breast cancer cells(MCF-7,MDA-MB-231,and SK-BR-3).The distance between the tip and the cell was determined by drawing approach curve,and the tip was positioned at 15 ?m above the cell.Then,the cell was scanned by scanning electrochemical microscopy to determine the range of a single cell,and the tip was positioned above the single cell to in situ detect the K+concentrations at the surface([K+]o)of three breast cancer cell at 3 ?m above the cell.The results showed that[K+]o of MDA-MB-231 cells was higher than that of MCF-7,and[K+]o of SK-BR-3 cells was the smallest.This study provides a new method for in situ detection of K+concentrations at the cell surface.Chapter Four:In situ detection of potassium outflow induced by external stimulation based on the potentiometric mode in scanning electrochemical microscopyIn this chapter,the dual-functional K+-ISE tip was used to in situ detect K+outflow induced by electrical stimulation or pH changes of the culture environment(?[K+]o),and the possible mechanism of K+outflow was preliminarily investigated.It was found that with the increase of the stimulation potential,the K+outflow increased.MCF-7 cells present the largest value of?[K+]o,followed by MDA-MB-231 cells and SK-BR-3 cells at all the stimulation potentials.In cell culture environment with different pH values(8.00,7.40,6.50,6.00,5.50),pH 6.50 showed the greatest impact on K+outflow of the three cell lines.As the pH becomes lower,the K+outflow of the three cell lines was suppressed to varying degrees.By comparing the changes of K+outflow caused by external stimulation with and without 4-AP pretreatment,it could be concluded that voltage-gated potassium channels participate in the regulation of changes in K+outflow caused by external stimuli.This study is of great significance for exploring the mechanism of potassium channels involved in cellular regulation.