Research On Novel Cell Based Biosensor Of MEMS And Its Application In Cellular Electrophysiology

With the development of Biomedical Engineering and micro electronic mechanical system(MEMS), the research on micromation of cell-based biosensor (CBB) has reached on the cellular and molecular level. Cells provide and express a series of elements such as naturally evolved receptor, ion-channels, and enzymes that can be the targets of biological active analytes. When stimulated, the living cell responds and take actions: induce electronic activity, excrete something or absorb something. Cell-based biosensors that treat cells as biological sensing elements have the capacity to respond to analytes in a physiologically relevant manner.CBB with MEMS technique, which include microelectrode array(MEA), field effect transistor array(FET), and the light-addressable potentiometric sensor(LAPS), can be applied as the secondary biosensor that coupled with living cells to realize the recording of extracellular electrophysiological signals. As a novel cell-based biochip, the principle of which is to culture varieties of cells on certain type of sensor array. When the cells are stimulated and electronically activated, the signals are transferred to the effective area of sensors, such as the metal electrodes of MEA and the gate of FET, and the two-way interface of cell-sensor can be constructed to make it feasible of transferring cellular signals to the processing amplifying units. Because of the advantage of extracellular CBB, e. g. long-term recording in non-invasive way, fast response, and easy fabrication, such biosensors have numerous applications including pharmaceutical screening, cellular physiological analysis, toxin detecting, peripheral nerve regeneration and environment monitoring, as well as in-vivo recordings, thus they are also promising in fields of neuronal prostheses and the reconstruction of damaged sense organs.Nowadays, a diversity of CBBs are already commercially available, however, the main problems lie in the singleness of parameters, the instability of guide line and the critical environmental requirements. Thus this thesis first introduced MEA designed by ourselves for extracellular action potential monitoring, mainly focusing on the theories of cell-microelectrode interfacial model, the design and fabricating process, and the extracellular signal transferring process with different cell types, etc., cardiac myocytes and olfactory bulb neurons. Based on this, an integrated chip for detection of cell physiology was designed to realize the parallel monitoring of different parameters such as the metabolite(according to the H⁺,K⁺,Ca²⁺ level), the action potentials and the impedance change due to cell-IDA electrode attachment. The integrated chip deepens and widens the new fields of applications in CBBs.The major contents and contributions of this thesis are given in the following aspects:1. The model of the cell-silicon, cell-metal electrode interface and the detection model of MEA, IDA and LAPS have been demonstrated deeply. Firstly, the characteristic equations of transmembrane ionic current are given based on the conductance and permeability of cellular membrane. Secondly, we analyze the influence of Si/SiO₂ and Au interface on the cell-silicon interface model. Then, the cell-electrode model established by Kovacs group and the neuron-FET model established by Fromherz group in German have been deduced into the cell-silicon or cell-metal device model and simplified into the design of our experiment. Afterwards, the detection theory and model of certain sensor type has been discussed in detail, which is the theoretical foundations of cell-based biosensor design and provide the premise to explain the experiment results.2. We designed and brought forward a novel MEA based on MEMS technique, including the theories, the design and fabricating process, and the system (hardware and software) implementation. Varieties of cell types, e.g. cardiac myocytes and olfactory bulb neurons, were cultured on the surface of MEA to validate the 8 parallel channels of CBBs. It offers the advantages of long-term recording in non-invasive way and easy fabrication, which facilitates the latter integration process, and can be applied in physiological analysis, peripheral nerve regeneration and environment monitoring.3. The design and fabricating process of integrated cellular chip including MEA/IDA/LAPS unites are provided. Our efforts are directed to the parallel development, fabrication and integration of different sensors into miniaturized biochips for a multiparametric cellular monitoring with the multi parametric chip. Parallel and on-line acquisition of data related to different cellular targets will be required for advanced stages of drug screening, and the chip includes 3 main units: The IDA with the impedance measurement of cells for attachment evaluation is firstly detected for cellular impedance detection; The MEA with the voltage measurement of cells for extracellular action potential detection is secondly tested; The LAPS for the sensitive ions, e.g. H⁺, K⁺, Ca²⁺, from cellular metabolites in micro environment is thirdly measured. A set of automatic fluid flowing system is also introduced to control the process of inlet and outlet by software by changing the direction, the drugs and the velocity. Thus the continuous monitoring of cells from normal shapes to changes of cellular attachment, action potentials, metabolite according to stimulation is available. The primary cellular physiological experiments are done for further developments in cellular and molecular sensors.