Real-time Determination Of Extracellular PH By Potentiometric Method Based On Scanning Electrochemical Microscope

The regulating pH is critical to maintain a healthy balance in the biological environment.In biological systems,variations in pH may be the effect of disease and dysfunction in biological systems.Hence,the detection of pH is a hot topic in biomedical research.At the cellular level,The presence of pH deviations can indicate abnormal reactions and disease.For example,extracellular pH(pHe)of cancer cell is higher acidity comparing to normal cell.It is due to the metabolic pathway of cancer cell,which can accelerate to the production of lactic acid,result in the increase of local H+ activity.Hence,it is important to monitor pHe.In scanning electrochemical microscopy(SECM),the tip of ultramicroelectrode can be precisely positioned to the surface of the interested sample,and the local electrochemical activity of the sample surface can be observed.SECM has been used in biological systems,such as,living cells and immobilized biomolecules(antibodies,DNA fragments).Soild-state ion selective electrode has been widely paid much attention owning to the advantages of simple operation and won't change the composition of solution.The solid-state ion selective electrode can be used as the probe of scanning electrochemical microscope to detect H+,alkali metal or alkali earth metal ions.Therefore,scanning electrochemical microscopy provides a powerful tool for detecting pHe.In this paper,by preparing the scanning electrochemical microscope probe and using the scanning electrochemical microscope potentiometric method,the extracellular pH can be monitored.The research content of this paper is divided into four chapters,the specific contents are as follows:Chapter One: IntroductionIn first chapter,the basic principle,working mode and application of scanning electrochemical microscope were introduced.Then the method of detecting pHe and the effect of electrical stimulation on cells were introduced.At the end,the research significance and purpose of this paper were put forward.Chapter Two: Preparation of polyaniline solid-state pH selective microelectrode probe in scanning electrochemical microscope potentiometric methodIn this chapter,Solid-state pH selective microelectrode of polyaniline based on platinum successfully prepared.The pH selective microelectrode was used as a scanning electrochemical microscope probe to monitor the pH of solution.The pH selective microelectrode was obtained by depositing a polyaniline film in a hydrochloric acid solution containing aniline at a potential cycle of-0.1 ~ 0.9V.The preparation,stability and reversibility of polyaniline films were characterized and optimized.The electromotive force signal between the pH selective microelectrode and the Ag/Ag Cl reference electrode showed a linear response in the pH range of4.00~8.00,with a slope of 53.0± 0.4mv /pH and a good reversibility.The response time was 10 s,and the potential drift was 1.13% for the determination of 3 h,which can be used to detect the pH of the solution.Chapter Three: Preparation of dual-electrode in scanning electrochemical microscope and application to the real-time determination of extracellular pHIn this chapter,we investigate a method for preparing dual-electrode as scanning electrochemical microscope probe.The dual-electrode was fabrication by sealed gold/platinum wire with encapsulation method in a borosilicate glass tube,and the successful preparation of the dual-electrode is verified by cyclic voltammetry and scanning electron microscopy.The polyaniline film was modified on the platinummicroelectrode in the dual-electrode to prepare pH selective microelectrode and to monitor the pHe of human foreskin fibroblast HFF and breast cancer cell MCF-7.The distance between the dual-electrode and the cell was obtained by the approximation curve with the gold microelectrode and the pHe was measured by the potentiometric method with pH selective microelectrode.As a result,pHe of human preputial fibroblasts was higher than that of breast cancer cells,probably because of the different metabolic patterns of the normal cells and the cancer cells.This method provides a new idea for the study of pHe.Chapter Four: Using SECM potentiometric method to monitor extracellular pH from electrically stimulated cellsIn this chapter,the dual-electrode was used as a scanning electrochemical microscope probe and the change of extracellular pH(pHe)after electrical stimulation was monitored by the scanning electrochemical microscope potentiometric method.As the distance between the electrodes and the cells decreased,the pHe was decreased.With the increase of stimulation potential and stimulation time,pHe decreased gradually.The fluorescent staining results of calcein-AM /PI(propidium iodide)were used to verify that this phenomenon may be due to the increased permeability of cell membrane caused by electrical stimulation.The pHe of three different cells after electrical stimulation were compared,and the pHe comparison order was MCF-7<HFF<Hela.It provides a powerful tool for monitoring pHe at different distances under electrically stimulated cell.