Integrated Microfluidic Device For Cell Culture And Real-Time Electrochemical Detection

Cell is the basic structural and functional unit of all living organisms, and most vital activities were accomplished through intercellular communication. Signal transduction by various secretions from cells is a common intercellular communication manner in multicellular organism. Therefore, monitoring an infinitely minute number of released molecules from single cells on accurate, real-time and dynamic level is of great importance to understand the mechanism of cell communication.Up to now, various techniques have been applied in real-time monitoring cells-released chemical signal molecules and gained significant progresses. Ultramicroelectrodes (UMEs), with high sensitivity, selectivity, temporal-spatial resolution and quick response ability, play an important role in real-time dynamic monitoring chemical secretion from cells. Currently, carbon fiber microelectrode and modified microelectrode have played an irreplaceable role in obtaining quantitative and kinetic information of cell secretion with high temporal-spatial resolution. However, this kind of electrodes must be precisely controlled by micromanipulators, which is difficult to realize high throughout and automatic detection. Recently, plane microelectrode array (MEA) with controllable size, shape, distance and material can trap cells onto its surface automatically. It has been widely applied in real-time, dynamic electrochemical detection and drug screening, as its distinctive superiorities of easy integration and manipulation for high throughout assay. Moreover, developing a novel MEA possessing high sensitivity, refreshing ability and recyclability is very important for long-term detection of cells.Cells in vivo are in a complicated microenvironment. Precisely monitoring cells in vitro depends on the successful construction of cellular physiological microenvironment. Microfluidic chip can simulate cell microenvironment in vivo by well-designed microstructures, biofunctionalized surface and accurately-controlled extracellular matrix on a chip. Coupling MEA with microfluidics chip would provide the capability to probe cells during their cellular physiological process (e.g., cell growth, development, and cell signal transduction) in real time. However, it is still challenging to develop a3D-integrated chip with good biocompatibility, cost-effective, and easy-to-produce for cellular microenvironment rebuilding and real-time detection. In light of the superiorities and challenges of MEA, some innovative works were carried out in this thesis, including fabrication of high sensitive microelectrode, developing good biocompatibility microfluidic chip, establishing perfusion model for cell culture and construction of microfluidic chip integrated with MEA. The main works and conclusions are summarized as follows:1. A high aspect ratio multilayer poly(dimethylsiloxane)(PDMS) microdevice was developed for long-term automated perfusion culture of cells without shear stress and an independently addressable microelectrodes array (IAMEA) was used for electrochemical monitoring of as-cultured cells in real time. Novel design utilizing high aspect ratio between circular "moat" and ring-shaped micropillar array surrounding cell culture chamber combined with automated "bottom-up" perfusion model successfully provide continuous fresh medium for cells. Automated cultivation of human umbilical endothelial cell line (ECV304) and neuronal differentiation of rat pheochromocytoma (PC12) cells have been realized using this device. Furthermore, the dopamine release from individual PC12cells during their culture or propagation process was amperometrically monitored in real time.2. A Ti layer-based novel refreshable carbon-doped titanium dioxide nanotube microeletrode array (C-doped TiO2NTA MEA) was developed by rapid annealing of anodized nanotubes. Self-refreshment of the MEA sensor was studied. Electrochemical activity of fouling electrodes could be recovered eighty percent compared with fresh electrode after ultraviolet exposure. Moreover, exocytosis from PC12cells were amperometrically real-time detected on the C-doped TiO2NTA MEA.3. A vascular-like structure based on a well three-dimensional (3D) gelatin chip with good compatibility, cost-effective, easy fabrication was fabricated. The controllable lumen diameter and wall thickness enable close mimicking of blood vessels (artery, vein, even capillary) in vitro. The cultured ECV304cells proliferate on the gelatin lumen linings to form a vascular lumen. The hemodynamic behavior including adhesion, alignment of endothelial cells (ECs) under shear stress and pulsatile stretch were studied.4. A microelectrode composed of core-shell TiC/C nanowire array (TiC/C NWA) based on Ti6A14V wire with high sensitivity to nitric oxide (NO)was fabricated. The TiC/C NWA microelectrode has a NO detection limit of0.6nmol/L. Moreover, highly sensitive and real-time monitoring of NO generation and NO level changes from the vascular-like gelatin lumen was demonstrated using the TiC/C nanowire array.