Researches On The Cell-Based Biosensor And The Application For Analysis Of Drugs

With the development of Biomedicine Engineering, the research on biomedical sensor technology has reach on the cellular level and molecular level. Cells provide and express an array of naturally evolved receptor, ion-channels, and enzymes that may be the targets of biological or biologically active analytes. When stimulated, the living cell response 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. Such biosensors have numerous applications including pharmaceutical screening, cellular physiological analysis, toxin detecting, peripheral nerve regeneration and environment monitoring.Recently, there are many types of secondary biosensors that couple with living cells to construst the cell-based biosensors. Among these secondary biosensors, the light addressable potentiometric sensor (LAPS) has many characteristics, such as high sensitivity, fast response, easy fabrication techniques, easy to monitor the metabolic action of a population cells and the electronic action of a single cell, which attract the biomedical engineers and researchers. Thus, this thesis focuses on the theoretical design, fabrication technology and the design of detection system of cell-based biosensor following the principles of LAPS. In addition, we take the experiments for drugs detection and analysis using such biosensors.The major contents and contributions of this thesis are given in the following aspects:1. The model of the cell-silicon interface and the detection model of 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 interface on the cell-silicon interface model. Then, the neuron-FET model established by Fromherz group in German has been deduced into the cell-silicon device model in general, and the latter is simplified into a point contact model. Afterwards, the detection theory and model of LAPS has been discussed in detail, which are the theoretical foundations of cell-based biosensor design and provide the premise to explain the experiment results.2. We design a novel microphysiometer based on MLAPS (multi-light addressable potentiometric sensor) and a set of automatic fluid injection system, which can controlthe process of experiment by software, avoiding the jamming, detect and analyze the ionic concentration changes of the extracellular microenvironmental K~+, Ca`(2+),H~+ ions under some drugs. Many ionic sensitive membranes are deposited on the surface of LAPS, and then the different sensitive membranes are illuminated in parallel with multi-light sources at different frequencies (3KHz, 3.5KHz and 4KHz). The response amplitude of each frequency component can be measured on-line by parallel processing algorithm. Thus the changes rate of ionic concentration can be calculated.3. The design and the method of fabrication of cell-based biosensors following the principle of LAPS are provided. The surface of the LAPS is soaked in the phosphate-buffered saline with 100μg/ml poly-L-ornithine and 8μg/ml laminin to enhance the attachment of cell on LAPS. When the cultured cells are stimulated and generate action potential, the peripheral circuit is able to detect the response of LAPS and the extralcellular action potentiometric can be extracted. We use this device to monitor the action potential of rat cortical neurons and design the primary experiment of olfactory chip and taste chip.4. According to the data obtained from cell-based biosensors following the principle of LAPS, we provide a method to analyze and evaluate the drugs, and investigate the method to acquire and process the experimental data. Through the time-domain analysis and frequency-domain analysis, we have calculated the metabolic products and the extracellular action potential of living cells, and studie...