Design And Application Of Multi Functional Light Addressable Cell-based Biosensors

Living cells'responses to stimulation changes in several cellular parameters, such as cell metabolism, action potential and cell impedance changes. Cell-based biosensors (CBB) use living cells as sensing elements, and convert cellular response to electrical signals that can be detected by a secondary transducer. Light addressable cell-based biosensors (LACBB) can simplify the cell culture process by using a laser to address cells. The light addressable potentiometric sensor (LAPS) is the secondary transducer in LACBB.This thesis studied the multi-functional light addressable cell-based biosensors based on LAPS. A light addressable cell impedance sensor (LACIS) was proposed for cell adhesion based on LAPS and impedance techniques. Stem cells were used as a novel cell source for LAPS cell chip. Extracellular action potential and drug screening were studied using such stem cell chip. Simultaneous detection of extracellular potential and acidification were realized with a duel function LAPS. Extracellular microenvironment detection system was designed and fabricated as an instrument for microenvironment monitoring. The ideas and contributions of this thesis are given as the following aspects.1. A distinguished way of light addressable impedance detectionCell impedance is an important physiological parameter, which can show cell migration, proliferation, decay and other physiological processes. Conventional metal electrodes detect the average impedance of all cells upon the electrodes. A new method was proposed for light addressable cell impedance detection using an EIS structure device LACIS. This method is based on the light addressable principle of LAPS and electrochemical impedance detection. With a focused laser beam addressing the target cells, LACIS detects localized cell impedance at illuminated area instead of the average impedance of all cells on the sensor. This light addressable cell impedance detection can be an effective way to monitor the impedance of low density cells or even a single cell. LACIS can also be a potent candidate for scanning cell impedance image.2, Design of LAPS-based stem cell chip and applications in drug screening.Embryoid stem cells (ES cells) were used as a novel cell source. Stem cell chip based on optimized LAPS was designed for extracellular action potential detection and drug screening. Stable and reliable extracellular action potential signals have been acquired with cardiomyocytes differentiated from ES cells. Effects of four drugs were evaluated with the stem cell chip on cardiac effects. Stem cell chip can solve the problem in cell culture and positioning, which is usually encountered in traditional cell chips such as MEA and FET.3. Multi functional LAPS design and application in extracellular detection of potential signal and microenvironment.To precisely defining cellular status, it is necessary to evaluated different cell activities. Cell based biosensors, which can detect multi parameters of cellular response, showed great importance. A multi functional cell-based biosensor based on LAPS was designed for detection of both extracellular potential and acidification. Experiments on cardiomyocytes showed that cellular electric status and cell metabolic activities could be simultaneously evaluated.4. Design and manufacture of an extracellar microenvironment monitor.Traditional microphysiometer suffered from single testing parameter (only pH), expensive equipment and difficulty to achieve high-throughput detection. Combining sensitive membrane technology, sensor packaging technology, fluidic design, circuit design and prototype process technology, an extracellular microenvironment monitor was designed and processed for extracellular measurement of cell metabolites, such as H+, Ca2+ etc. This system can detect multi-parameters with eight-channels, which effectively expands the LAPS cell-based biosensors to detect multi cellular parameters.