Single Cell Electrochemiluminescence Analysis

Previously, our group has developed a strategy based on the luminol electrochemiluminescence to analyze the active membrane cholesterol in single cells. A cell-sized pinhole was placed below the electrode cultured with the cells so that only one cell was exposed to the detector (PMT). Cholesterol oxidase present in the solution reacted with the active membrane cholesterol in the cells to generate hydrogen peroxide, which initialized the luminol electroluminescence with a voltage. The luminescence recorded on PMT was only attributed to the target cell above the pinhole to achieve single cell analysis. A large deviation of active membrane cholesterol in single cells was observed, which exhibited the significance of single cell analysis. The "pinhole" setup was low-cost and the measurement was simple, however, it was impossible to analyze multiply single cells on one electrode that limited the analysis throughput.To overcome this drawback, we combine the microfabrication technology, and developed a multiply microelectrode array embedded with the cell-sized microwell traps was developed to retrain single cells in the microwells. Each cell was in register with one microelectrode. Cholesterol oxidase reacted with the active membrane cholesterol in single cells to generate hydrogen peroxide in the microwells simultaneously. The structure of the microwells slowed down the diffusion of hydrogen peroxide into the bulk solution. The equilibrium between the generation and the diffusion of hydrogen peroxide created a semi-steady state distribution of hydrogen peroxide on the microelectrodes in a time length, in which the serial analysis was achieved by applying voltage on the microelectrodes sequentially generating electrochemiluminescence. In this setup, the single cell analyzed was achieved by sequentially applying a voltage, and thus, no pinhole was needed. The absence of pinhole facilitated the measurement automation and increased the analysis throughput. The accuracy and throughput of serial analysis were discussed. Using this platform, a relative large number of single cells were analyzed for the further investigation of cellular heterogeneity on active membrane cholesterol.The second project is to develop the assay for lipid molecules at cell membrane. We activated cell membrane phospholipids by immersing the cells into low ionic strength buffer solution. PLD reacted with lipid molecules at cell membrane to generate high concentrations of hydrogen peroxide on the microelectrodes, which generates measurable electrochemiluminescence. The rapidly sequence analysis of membrane lipid molecules in single cells can be achieved by a multiply microelectrode array embedded with the cell-sized microwells.The third project is the development of carbon can point chemiluminescence. Carbon dots (CDs) whose chemical luminescence properties are still in the early stages of the study are low-toxic fluorescent nanomaterials. Currently, in order to achieve its chemiluminescence, strong oxidants or strong bases need to be added to the system, limiting the application of the material in biological detection. We developed chemiluminescence new Carbon dots (CDs) by using the good catalysis of Co3O4 nanomaterials to hydrogen peroxide for the analysis of intracellular hydrogen peroxide. The principle of the detection of hydrogen peroxide with new materials is that:hydrogen peroxide diffused into the complex and reacted with Co3O4 nanoparticle in the core to generate oxygen radicals, which reacted with the surrounding CDs to emit the luminescence.The result supported that the addition of hydrogen peroxide induced a rapid increase in the luminescence intensity with pH of 7.4. The generation of luminescence indicated that the complex generated the luminescence with hydrogen peroxide in the absence of any oxidative or alkane. For the analysis of intracellular hydrogen peroxide, Co3O4@CDs complex was loaded into cells solution, and the material can enter the interior of the cell. After PMA stimulates cells to produce hydrogen peroxide, chemiluminescence can be detected using PMT. Compared with the fluorescence assay of intracellular hydrogen peroxide, this chemiluminescence assay does not need the excitation light source leading to a better compatibility on the in vivo study.