The Research Of Automated Multifunctional Cellular Physiological Analyzer With Integrated Chip

Cell-based Biosensors (CBB) exploit the naturally evolved sensitivity of cells in order to detect a broad range of biochemical agents as a cellular response. This cellular response is detected by a secondary transducer and converted into an electronic form suitable for analysis. Depending on the measured physiological parameters, commonly used secondary transducers are mainly Micro-electrodes Array (MEA), Light Addressable Potentiometric Sensor (LAPS), and Electric Cell-substrate Impedance Sensor (ECIS). During cellular physiology experiments, single parameter monitoring is often only from one aspect reflects the physiological activities of cells. And the complexity of test environment (various noise sources, long-term testing of the corrosion medium on the electrodes, etc.) may interfere with experimental results. So revealing the mechanism of cellular physiological activities needs the comprehensive analysis of a variety of related physiological parameters.An automated multifunctional cellular physiological analyzer based on integrated chip, was developed using electronic technology and biomedical technology. This instrument achieved the simultaneous detection of three kinds of physiological parameters, such as extracellular ion concentrations (micro-environment detection), extracellular action potentials, and electrophysiological impedance. The instrument has several advantages such as shorter analysis time, low sample consumption and high efficiency. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. At the same time, the fully automated analysis was achieved by the flow analysis system. The instrument can be widely used in cell physiology analysis, drug screening and environmental monitoring and other fields. In addition, the instrument enables real-time quantitative detection of the conduction of single cells and cell networks, and the cellular physiological state after the effects of drugs. Based on the unique design of chip-instrument interface and circuit board, the instrument is applicable to a variety of cells and different designs of multifunctional integrated chip. The major contents and innovations of this thesis are given as the following aspects.1.Numerical design and simulation of multifunctional integrated chipA multifunctional integrated chip was designed, which integrates three kinds of sensors as MEA, LAPS, and ECIS. The chip can simultaneously detect the extracellular action potentials, the state of cell growth and adhesion and extracellular micro-environment.Theoretical analysis of multifunctional integrated chip was used to understand the basic theory of cell-based biosensor detecting physiological parameters. We analyzed how cells receipt the stimulation signals as primary transducers. Here, we focused on the basis of cellular functions as they relate to cellular metabolism, electrical activity in cells, cellular membrane impedance characteristics, and cell adhesion and motion. The model of cells coupled to the secondary transducers was established by analyzing cellular responses to the outside stimulation. Based on these results, the model of cell-based biosensor was established.We analyzed the different functional units of the integrated chip, and established the electrical model of MEA sensor unit, ECIS sensor unit and LAPS sensor unit. Numerical model and simulation help us design of multifunctional integrated chip and cellular physiological analyzer.2.Design and processing of automated multifunctional cellular physiological analyzerThe corresponding detection circuit of MEA, LAPS, ECIS sensing unit on the integrated chip, was designed. Temperature, humidity and CO2 concentration in the measuring cavity can be automatically adjusted during the measuring process, in order to meet environmental requirements in cell growth. Flow control circuit controls on-off and speed of valve, injection pump and output pump. Thus automatic analysis is achieved, by auto sampling the culture medium and drugs as well as washing the waste liquid.The central control circuit controls the process of physical signal acquisition, preliminary analysis of the signal data, and transfer to computer by USB interface. The designed software on PC enables data collection, storage, and post-analysis. Integrated chip cultured with cells was coupled to the instrument by a special interface. At last, a prototype of multifunctional cellular physiological analyzer was completed, which can achieve the needs of cellular multi-parameter analysis.3. Detection and analysis of cellular physiological parameters for drugsEvaluation of the instrument, mainly include calibration of sensing unit detection circuit calibration, as well as the testing of environment and drug control system. The results indicate that the instrument can be used for analysis of cell physiological parameters.Based on multifunctional integrated chip, several experiments were conducted to detect cellular parameters for typical experimental test. LAPS sensor unit on the integrated chip was used to detect acidification rate changes of the kidney cells under the action of 5-fluorouracil. MEA sensor unit on the integrated chip was used to detect extracellular action potential changes of SD rat myocardial cells under the action of TTX. ECIS sensor unit on the integrated chip was used to detect cell extension, growth, proliferation, and death process. At last, the instrument is used to detect the extracellular action potential, cell metabolism, and cell impedance simultaneously. It is used to study the acute toxicity effect of adriamycin acting on the HL-1 cardiac cell line on the electrophysiological point. The results indicated that the multifunctional cellular physiological analyzer can be widely used in future based on the capability of simultaneous detection of three kinds of parameters.