Scanning Electrochemical Microscopy Of Single Live Human Bladder Cancer Cells

It is difficult to evaluate heterogeneity of cells using conventional biological analysis, people have to rely on single-cell analysis. With such demand as the driving force, people has developed various single cell analytical methods, such as, capillary electrophoresis, microfluidics, fluorescent microscopy, confocal microscopy, scanning probe microscopy etc. As one of the scanning probe microscopy, scanning electrochemical microscopy (SECM) has its unique strength in studying topography in micro-scale and interface processes. The core component of scanning electrochemical microscopy is an ultra-microelectrode. It possesses extraordinary spatiotemporal resolution and sensitivity thus it can image micro-scaled topography and detect trace electrochemically active species in the system. In SECM, the scanning tip does not need to contact the substrate, therefore it is a suited tool to scan over soft substrate for a long time, and hence it has been developed into single cell analysis area. Existing research results demonstrate that SECM exhibits superior performance in studying cellular topography, redox activity, active sites on cell membranes and transmembrane transportation processes. In this thesis, we apply SECM to analysis of single cancer cells; utilize the reactive oxygen speices (ROS) that release by the cell itself as the redox active mediator, invesgate the topography, temporal development of ROS, membrane permeability, and interaction with anti-cancer agents of human bladder cancer cells (T24). Main research contains following parts:1. Imaged T24 cells with extracellular ROS as the mediator using SECM. The current above the cell is higher than the background current due to ROS generation. The SECM images reflect the spatial distribution of extracellular ROS around a T24 cell. Formal potentials to reduce main ROS under physiological conditions were determined using ultra-microelectrode.2. SECM imaging of T24 cells in real time with ROS as the indicator. The regulation of temporal development of extracellular ROS of T24 cells was revealed, and the concentration of extracellular ROS was determined by SECM depth scan technique. We found that extracellular ROS of T24 developes periodically and we classified that one ROS releasing cycle consists of one active stage (releasing ROS) and one resting stage (not releasing ROS). Such information cannot be accessed with other single-cell analysis method. SECM can provide new insight into the physiology of cancer cells.3. Time-lapse SECM imaging cisplatin effect on the ROS releasing cycle of T24 cells, and quantification of ROS concentrations in apoptosis using SECM depth scan. It was observed that cisplatin can significantly shorten the ROS releasing cycle of T24 cells and in the early stage of apoptosis the concentration of extracellular ROS was elevated. It proved that SECM can effectively study the intereaction between anti-cancer drugs and cancer cells. SECM will become a promising method of drug screening with further development.4. The resting stage of ROS releasing activity of T24 cells can last for up to 5 h. In the resting stage, dissolved oxygen in the medium solution was used as the indicator in time-lapse SECM imaging and captured the dynamic morphological change of T24 cells. The gradual evolution process and details of cell membrane and volume can be vividly observed. Although such method is confined with the status of ROS releasing activity thus cannot be widely used, the observation obtained in this research is unaccessible with other methods. Moreover, since no additional redox active mediator was involved in this research, it minimized the interference of exotic chemicals to the system.5. Quantitatively determined the cisplatin effect on the membrane permeability of T24 cells using ferrocene methanol as the redox mediator. Instead of conventional SECM probe approach curve measurement, we introduced time-lapse SECM depth scan method, and mornitored the cellular topography and membrane permeability in real time. Beside the permeabilization as a result, we also realized observing the procedure of such permeabilization. Our results demonstrated that the administration of cisplatin can effectively permeabilize T24 cells in 5 min. Membrane permeability is very sensitive to anti-cancer drugs, therefore it is promising to use such method in drug-screen based on further development.6. SECM operation modes were applied to investigating the transient response of T24 cells to biological active stimulant from multiple aspects:cellular morphology, extracellular ROS profile and membrane permeability. And a series of transient response of T24 cells was concluded upon the addition of 1-phorbol-12-myristate-13-acetate. This research proved that SECM is a facile tool to analyze the interation between cancer cells and additional chemicals.