| With the development of modern industry and agriculture, marine toxins which are mainly produced by microalgae, pose a serious threat to human health and environmental safety around the world, especially the ion channel marine toxins. They are the most serious and widely distributed toxins which account for more than 80% of the total toxins, and are likely to be involved in some ion channel diseases. Although mouse-bioassay (MBA) is the standard method, it is difficult to meet the need for routine monitoring of marine toxins due to some limitations (e.g., low sensitivity and poor repeatability). The structure-based method high performance liquid chromatography (HPLC), is difficult to completely replace MBA due to its expensive equipment and complex operation technology. Recently, some function-based assays have emerged, but the traditional cell-based assays are time-consuming and lose much dynamic and real-time information in toxin action.Cell-based biosensor, as a new functional cell-based assay, have attracted more and more reseachers’attention due to its advantages of non-invasiveness, high sensitivity and real-time monitoring. Currently, cell-based biosensor which possesses advantages of cell-based assay and biosensor, is rarely used in marine toxin research,, and it is a promising technology for detection of marine toxins. This paper focuses on development of high-stable and high-consistent cardiomyocytes-based biosensors for detection of the ion channel marine toxins.In this paper, the main contents and innovation work are as follows:1. High-throughput and multi-channel cell-based potential biosensors for detection of extracellular field potential (EFP).Two cell potential sensor chips are designed:single-well 32-channel microelectrode array (MEA) chip and 16-well and 64-channel MEA chip. Based the two MEA chips, three cell-based potential biosensor systems are developed: single-well 32-channel potential biosensor, single-well 8-channel Wi-Fi potential biosensor angd high-throughput potential biosensor. Then the three potential biosensors are tested by cardiomyocyte EFP experiments, the results demonstrate that they possess a good function for EFP detection. The two single-well biosensors realize multi-site EFP detection of cell network. Compared with single-well 32-channel potential biosensor, the 8-channel wireless potential biosensor is more suited for field detection of toxins due to its small and portable instrument. High-throughput potential biosensor realizes simultaneous EFP detection of multi-sample, greatly improving the detection efficiency, and high-throughput toxin screening.2. Study on the methods for enhancing the coupling of cardiomyovytes and sensor chips and successful development of high-stable and high-consistent cardiomyocyte-based impedance and potential biosensors.Protein modification and cell density optimization is studied for enhancing the coupling of cardiomyovytes and sensor chips. The results show that, for cell impedance sensor (ECIS) chip, cardiomyocytes seeded on the 96-well ECIS chip with gelatin modification at the proper cell density of 8.5×104 cells/cm2, prompting high-closely coupling between cardiomyocytes and ECIS. Under the optimal conditions, the 96-well cardiomyocyte-based impedance biosensor presents high-stable and high-consistent beating signals (amplitude of 0.045～0.050 and beating rate of 90～105 beats/min), which continues for 32 h (Best toxin monitoring period). For MEA chip, cardiomyocytes seeded on the single-well MEA chip with gelatin modification at the proper cell density of 1.2×105 cells/cm2, prompting high-closely coupling between cardiomyocytes and MEA. Under the optimal conditions, the single-well 8 channel wireless cardiomyocyte-based potential biosensor presents high-stable and high-consistent extracellular field potentials (EFP) signals (amplitude of 1.6～1.8 mv, firing rate of 150～180 beats/min), which continues for 60 h. In this work, two typical tool drugs isoprotenol and lidocaine are applied to test the analysis performance of the two biosensors. The results show that both of two biosensors perform correct and sensitive response to the two drugs, indicatig that cardiomyocyte-based biosensors posesses good detection function.3. A novel and high-throughput assay for detection of saxitoxin (STX) and tetrodotoxin (TTX) by cardiomyocytes-based impedance biosensor.High-performance cardiomyocyte-based impedance biosensor is used to study the toxicity of ion channel marine toxins, and a novel and high-throughput assay for detection of ion channel marine toxins is developed. Three typical ion channel marine toxins (STX, TTX, PbTx-2) are selected to be analyzed. The recording signal parameters, cell index (CI), beating amplitude (BA), beating rate (BR) extracted from cell impedance signals of the impedance biosensor, are analyzed to quantitatively evaluate toxicological risk of STX, TTX and PbTx-2.. The results show that BR is a sensitive parameter to STX with the detection limit of 0.087 ng/ml and the detection range of 0.25～20 nM and 0.18～14.6μM after 24 h treatment. Also BR is a sensitive parameter to TTX with the detection limit of 89 ng/ml and the detection range of 0.4～20μM after 24 h treatment. But the impednce biosensor presents poor sensitivity to PbTx-2 and shows no response to PbTx-2 of high concentration (4μM,3.6 ppm). Baesd on the important advantages of high sensitivity and good repeatability, cardiomyocyte-based impedance biosensor will be a novel and high-throughput platform for detection of ion channel marine toxins.4. A novel and rapid assay for detection of saxitoxin (STX), tetrodotoxin (TTX) and brevetoxin 2 (PbTx-2) by cardiomyocytes-based potential biosensor.Based on the method of cardiomyocyte-based impedance biosensor, high-performance cardiomyocyte-based potential biosensor is used to study the toxicity of ion channel marine toxins, and a novel and rapid assay for detection of ion channel marine toxins is developed. Three typical ion channel marine toxins (STX, TTX, PbTx-2) are selected to be analyzed. The recording signal parameters, spike amplitude (SA), firing rate (FR) and 50% of spike potential duration (SPD50) extracted from extracelluar field potential (EFP) signals of the potential biosensor .are analyzed to quantitatively evaluate toxicological risk of STX, TTX and PbTx-2. The results show that FR is a sensitive parameter to STX and TTX with the detection limit of 0.35 ng/ml and 83 ng/mL within 5 min, respectively. And the detection range of STX and TTX is 1.56～400 nM_and 400～3200 nM, respectively. SPD50 is a sensitive parameter to PbTx-2 with the detection limit of 1.55 ng/ml within 5 min, and the detection range of PbTx-2 is 1.56～400 nM. Baesd on the important advantages of high sensitivity and good repeatability, cardiomyocyte-based potential biosensor will be a novel and rapid platform for detection of ion channel marine toxins.
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