Microelectrodes Electrochemiluminescence For Single-cell Analysis

Cells are the basic units of organisms. Due to high cellular heterogeneity, the study at single-cell level will provide the information to understand physiological process at different cellular states. Hydrogen peroxide is a main component of intracellular reactive oxygen species (ROS). When the body is in the inflammatory or antimicrobial response, a large quantity of hydrogen peroxide is produced in some cells such as phagocytic cells. Therefore, the analysis of intracellular hydrogen peroxide at single-cells is significant to study physiological and pathological processes. At present, microelectrode electrochemistry is the main method to detect intracellular hydrogen peroxide directly, in which micrometer or sub-micrometer microelectrodes penetrate the cell and collect its electrochemical signals. However, specific care is needed to maintain small current noise so that the current response at the picoamp level from electrochemical detection is collected. Also, the bright-field imaging of the electrode in the cell might not provide clear observation of the microelectrode tip in some cell types. To solve the above problems, we developed luminol integrated microelectrode for electrochemiluminescence (ECL) analysis of single cells. ECL combines the advantages of high selectivity from microelectrodes and the visibility of optical observation. Also, no background optical signal is needed so that high sensitivity of ECL is guaranteed. Two parts were included in my thesis.In the first work, we designed a luminol-Au microelectrode ECL system, and achieved electrochemical visualization of hydrogen peroxide inside the cells. The innovation of this work was the delicate integration of luminescence reagent at microelectrodes. The capillary with the tip opening of 1-2 μm was filled with the mixture of chitosan and luminol, which was coated with the thin layers of polyvinyl chloride / nitrophenyloctyl ether (PVC/NPOE) and gold as the microelectrode. Upon the contact with the aqueous hydrogen peroxide, hydrogen peroxide and luminol contacted with gold layer were oxidized under the positive potential resulting in the luminescence for the imaging. Due to the small diameter of the electrodes, the microelectrode tip was inserted into one cell and the bright luminescence observed at the tip confirmed the visualization of hydrogen peroxide inside one cell. The further coupling of oxidase on the electrode surface could open the field in the electrochemical imaging of intracellular biomolecules at single cells, which benefited the single cell electrochemical detection.In the second work, we have studied luminol ECL at metal disc microelectrodes. Three microelectrodes (18 μm Pt electrodes,50 μm Au electrodes and 50 μm Au electrode with thin glass cover) were fabricated, and the electrochemical properties, mainly ECL, of these microelectrodes were investigated. Furthermore, glucose oxidase was modified at the electrodes so that the ECL response of the electrode to glucose was detected. Although the ECL stability of metal microelectrodes was a little worse than indium tin oxide electrodes (ITO), its easy-to-operate feature in single-cell analysis made it a significant work to develop stable and sensible ECL microsensors.