Microelectrodes For Analysis Of Cytosol In Single Cells And Flip-Flop Of Cholesterol In A Phospholipid Membrane

Cells are basic units of the life. Usually cells are analyzed as a group, it is hard to get the information of a single cell. However, it is known that there are individual differences between cells, so ensemble analysis restricted a lot of biological research. While the single cell research can provide the information of individual differences, which is very important. There is biological significance on the study of hydrogen peroxide and cholesterol movement in the cell membrane. Therefore this thesis is based on the microelectrode method, which is used to study the hydrogen peroxide and flip-flop of cholesterol in the plasma membrane of single cells. The main work is as following:(1) A microelectrode array has been applied for single cell analysis with relatively high throughput; however, the cells were typically cultured on the microelectrodes under cell-size microwell traps leading to the difficulty in the functionalization of an electrode surface for higher detection sensitivity. Here, nanoring electrodes embedded under the microwell traps were fabricated to achieve the isolation of the electrode surface and the cell support, and thus, the electrode surface can be modified to obtain enhanced electrochemical sensitivity for single cell analysis. Moreover, the nanometer-sized electrode permitted a faster diffusion of analyte to the surface for additional improvement in the sensitivity, which was evidenced by the electrochemical characterization and the simulation. To demonstrate the concept of the functionalized nanoring electrode for single cell analysis, the electrode surface was deposited with prussian blue to detect intracellular hydrogen peroxide at a single cell. Hundreds of picoamperes were observed on our functionalized nanoring electrode exhibiting the enhanced electrochemical sensitivity. The success in the achievement of a functionalized nanoring electrode will benefit the development of high throughput single cell electrochemical analysis. Microelectrodes are powerful tools for single cell analysis.(2) Cholesterol flip-flop in phospholipid bilayer of cell membrane have been reported, but most of the traditional research uses fluorescence analogue method, and it is not the direct detection of membrane cholesterol. So, We applied the electrochemical method to directly detect membrane cholesterol. DHPC was chosen to form a bilayer across a 50μm orifice in a lipid bilayer chamber filled with buffer solution. In this method, the microelectrode surface was modified with cholesterol oxidase to perform membrane cholesterol analysis. In order to keep the bilayer stable during the electrochemical detection, and meanwhile guarantee the detection efficiency, we investigated the minimum voltage for the detection of cholesterol and maximum voltage for the stability of the bilayer. The appropriate voltage value of 0.3 V was determined for cholesterol electrochemical detection in our system. The experimental results confirmed that the flip-flop rate of cholesterol across the lipid bilayer is 275 s. This is the first experimental data on the flip-flop rate of cholesterol using unlabeled method, which is significant for the study of cholesterol flip-flop across the cellular membrane.