Study Of New Characterizations Of Single Cell Electrochemiluminescence Imaging

Single-cell analysis is one of cutting-edge technologies in the field of life analysis,which provides the possibility to fundamentally understand the cellular structure and biological mechanisms of basic life processes.Therefore,it is important to develop analytical methods for viewing single cells at high resolution in order to fully understand cellular life processes,such as apoptosis and carcinogenesis,to address fundamental biological problems,and ultimately to provide unique insights into the early diagnosis and treatment of disease.At present,various methodologies are mainly focused on single cell imaging,such as fluorescence microscopy imaging,Raman spectroscopy imaging,scanning electrochemical imaging,etc.Among them,the electrochemiluminescence(ECL)microscopy as a new imaging technology has been used in the research of nanoparticles,cell membrane proteins,electrochemical processes and other fields.In recent years,ECL imaging has been gradually applied to single cell imaging,showing a very high signal-to-noise ratio and good time and space controllability.The discovery of new characterizations of single cell electrochemical luminescence imaging is very important for the further development of this technology in the study of single cell imaging.In view of this challenge,this thesis further developed the application of ECL technology in single cell imaging,achieved high spatial resolution,high stability of single cell ECL imaging,and found the single cell ECL photobleaching phenomenon for the first time.The specific research work is as follows:(1)High Depth-of-Field Resolution ECL Imaging Based on Chemical Lens:ECL imaging technology is characterized by high spatial resolution and high signal-to-noise ratio,but the problem of effectively adjusting the depth of field(i.e.the height of the ECL luminous layer)during imaging to form high depth-of-field resolution ECL imaging remains to be solved.In fact,the luminescent region of ECL has limited its further development in the field of cell microscopic imaging and biological determination.In the first chapter,we propose an original strategy based on chemical lensing effect to adjust the luminescent layer height of a classic ECL reaction system.In the ECL reaction model system,the microbeads modified by tri-ruthenium-pyridine(Ru(bpy)₃²⁺)and the co-reactant tripropylamine(TPr A)were composed.By adjusting the buffer capacity of the solution and changing the reaction rate of ECL,it is proved that this strategy can achieve the precise control of the ECL reaction layer.The ECL image reflects the optical characteristics of the concentration of the electrogenic TPr A free radical in the reaction process.The discovery of this novel characterization provides a new idea for studying the mechanism of ECL reaction,and opens up a new way for microscopic imaging and biological determination of ECL.More importantly,this chemical-lens-based approach can control the spatial expansion of the ECL reactive layer,which is conceptually similar to total internal reflection fluorescence microscopy(TIRFM),and will facilitate high-depth-of-field studies and imaging of objects or cells at different heights.(2)High Stability Single Cell ECL Imaging:ECL microscopic imaging has been successfully used to image electrochemical processes on cells,micron or nanometer scale objects and electrode surfaces.The classic ECL reaction system consists of luminophore Ru(bpy)₃²⁺and efficient co-reactant TPA.When recording ECL images continuously,the fast attenuation of ECL intensity has become the key to limit the development of this technology.In the second chapter of this thesis,we studied the changes of light intensity during ECL imaging of chinese hamster ovary cells(CHO-K1).Ru(bpy)₃²⁺derivatives were used to label the plasma membrane of cell,and images of single cells were recorded in the presence of TPA,a coreactant.The results show that the loss of ECL intensity is related to the decrease of TPA oxidation current.Then,the oxidation intensity of TPA was restored to an approximate initial state by cathodic regeneration treatment on the electrode surface,and a series of ECL images with stable light intensity were obtained.Therefore,a highly stable single-cell ECL imaging analytic method is established,which will lay a foundation for further advance of single-cell ECL imaging.(3)Single cell ECL bleaching imaging:In the third chapter of the thesis,we first investigated the effects of photobleaching on ECL.After the plasma membrane of CHO-K1 cells was labeled with Ru(bpy)₃²⁺derivatives,the regions of interest of the fixed cells were photobleached by using a confocal mode of continuous progressive illumination,then the photoluminescence(PL)and ECL images were recorded.The results showed that the photobleached regions of the membrane in PL mode also showed lower light emission level during ECL imaging.There was a linear correlation between the decrease of ECL intensity and the loss of PL intensity,which suggesting that the decrease was caused by photobleaching of ECL label on the plasma membrane of CHO-K1 cells.The discovery of this new characterization provides valuable information for further understanding of the fundamental principles of ECL and provides new opportunities for combining single-cell ECL microscopic imaging with photobleaching techniques.