High Performance Cell-based Biosensors And Their Applications In Pharmaceutical Cardiac Safety And Marine Biotoxin Analysis

Cell-based biosensor is a novel biosensor that living cells as the primary sensitive elements and physical or chemical sensors as the secondary transducers. Cell-based biosensor employs living cells to sense the stimuli, which can induce the change of cellular physiological parameters, such as cellular metabolism, cellular impedance, and cellular action potential. These response changes can be converted into the electric signals by physical or chemical sensors, and electric signals can be detected by sensor circuit system. Cell-based biosensor is sensitive, real-time, dynamic, long-term, non-invasive, and label-free, which is widely applied in the cell physiological research and other relevant fields.In this dissertation, based on the sensor fabrication, cell culture, and signal detection technologies, several types of cell-based biosensor systems are established for cellular physiological detection and drug analysis. The high-sensitivity light-addressable potentiometric sensor (LAPS) and sensitive cellular metabolism sensor system are designed and fabricated to detect the extracellular acidification rate. The high-throughput electric cell-substrate impedance sensor (ECIS) and multifunctional impedance sensor system are designed and fabricated to detect the cell impedance and cardiomyocyte beating. Integrated LAPS and ECIS sensor and integrated sensor system are designed and fabricated to simultaneously detect these two physiological parameters. In the study, cardiomyocyte-based biosensors are used to verify the drug function and cardiac safety assessment. Futhermore, the signal beating profile function of cardiomyocyte-based biosensor is developed to specifically recognize the hERG channel inhibitors. Besides, cell-based biosensors are used to perform marine diarrhetic shellfish toxin analysis. With the development of cell-based biosensor technology, cell-based biosensor and detection system can provide a utility and rapid platform for the toxin detection and drug safety analysis.The main content and innovations of this dissertation are given as the following aspects.1. A high-sensitivity cellular metabolism sensor and its application in extracellular acidification rate detection. Cell metabolism plays a important role to maintain the activities of living cells. In order to maintain homeostasis of the intercellular environment, cells release the acidic metabolic products, causing acidification of the extracellular microenvironment. In this dissertation, a high-sensitivity LAPS are designed and fabricated by optimization of the substrate material selection and the sensor structure. And a high-sensitivity cellular metabolism sensor system is established, combining LAPS with cell culture microchamber. The cellular metabolism sensor system is tested by the sensor unit performance experiments and extracellular acidification experiments.2. A multifunctional cellular impedance sensor for cellular growth and cardiomyocyte beating simultaneous detection.Cellular impedance can reflect the cellular adhesion, proliferation, apoptosis, and other processes of cell cultured on the ECIS. Traditional ECIS is commonly used to detect cellular growth. In this dissertation, a high-throughput ECIS and its high speed impedance sensor system is used to establish a multifunctional ECIS system, which can not only detect the cellular growth, but also detect the cardiomyocyte beating. The multifunctional ECIS system is tested by the mouse neuroblastoma growth experiments and primary neonatal rat cardiomyocytes beating experiments.3. A integrated cellular metabolism and impedance sensor for cellular growth and extracellular acidification rate simultaneous detection.In order to reflect the physiological parameters of cells in response to external stimuli, more physiological parameters need to be detected,so it is important to develop a multiparameter integrated cell-based biosensor system. In this dissertation, LAPS and ECIS are integrated to fabricate the integrated cellular metabolism and impedance sensor and detection system. And the integrated cell-based biosensor system is tested by basic sensor performance experiments, cellular growth and metabolism experiments under drug stimulation.4. Further study of cardiomyocyte-based biosensors and their applications for drug cardiac safety assessment.Traditional drug cardiac safety analysis method has a tradeoff of throughput and clinical relevance. For example, the ex vivo or in vivo animal experiments usually have high clinical relevance, low throughput, high cost. While the hERG protein inhibitor binding experiments have high throughput and low clinical relevance. In this dissertation, primary neonatal rat cardiomyocytes and human induced pluripotent stem cell derived cardiomyocytes are used to fabricate the cardiomyocyte-based biosensor combining with ECIS. The cardiomyocyte-based biosensor is applied for the function analysis of ion channel drug. It is first to develop the profile recognition function of human iPSC-CMs-based biosensor. By analysis the hERG positive inhibitor and withdrawal hERG inhibitor drugs from market, the beating signal profiles of this drugs is similar, which indicates that iPSC-CMs-based biosensor can specifically recognize the hERG inhibitors. Therefore, iPSC-CMs-based biosensor can provide a high-throughput platform for rapid detection of drug cardiac safety analysis.5. Further study of electric cell-substrate impedance sensors and their applications in marine biotoxins high-sensitivity and rapid detectionTraditional detection methods of marine biotoxins have a variety of disadvantages, which are difficult to overcome. For example, large individual differences exist in the mouse bioassay which induces inaccurate results. And LC-MS/MS detection instrument is expensive and its application cost is high. Traditional cell-based biosensor is commonly used in cellular physiology and drug study. In this dissertation, cell lines, primary cardiomyocyte and cell impedance sensors are used to establish high-performance cell-based biosensors. And cell-based biosensors are applied in marine biotoxins detection. Cell-based biosensor function is verified by detection of diarrhetic and paralytic shellfish toxin. Cell-based biosensor is a effective complement and development for the existing marine shellfish toxin detection methods.