Study Of The Multiparameter Detection Method Of The Cellular Physiological Activity Based On The Union Of The Microelectrode Array And Atomic Force Microscope

The electrical activity and mechanical activity play important roles in the functions of excitable cells such as cardiomyocytes and skeletal muscle cells.In this paper,the detection method of the cellular electromechanical activity is studied by taking the cardiomyocytes as an example.In cardiomyocytes,the electrical activity and mechanical activity are closely related.During the action potential,the intracellular calcium concentration increases rapidly,and the calcium participates directly in the contractile activity of cardiomyocytes.In addition,the mechanical activity of cardiomyocytes has a feedback effect on their electrophysiological activities.The contraction of cardiomyocytes can affect the calcium sensitivity of the myofilaments and the state of the mechanically sensitive ion channels in the cell membrane,which will influence the electrical activity of cardiomyocytes.Simultaneous detection of electrical activity and mechanical activity of the cardiomyocytes can help to understand the cellular function under physiological and pathological conditions.On the one hand,the detection of the electromechanical activity of cardiomyocytes can investigate the cardiovascular drug effects in a comprehensive way and promote the efficiency of drug screening.The changes in the parameters of the electrical activity of cardiomyocytes,such as the firing rate and the action potential duration,have become the index for the evaluation of drug efficiency.At the same time,the mechanical parameters,such as the contractile force and diastolic period,are also used in the drug assessment.For some drugs,in a low concentration,the changes of the cellular electrophysiological activity were not remarkable,while the mechanical activity was obviously enhanced.It is helpful to improve the accuracy and efficiency of the drug evaluation by combining the electrical recording with the mechanical measurement.On the other hand,the mathematical model of cardiomyocytes provides a powerful tool for understanding the working mechanism of the cells,and has broad application prospects in the field of virtual teaching,evaluation of the disease treatment and so on.The early electrophysiological models of cardiomyocytes were mainly based on the cell membrane ion channel theory proposed by Hodgkin and Huxley.They described the generation of the action potential in cardiomyocytes.Additionally,the mathematical model describing the mechanical activity of cardiomyocytes was also established.The two types of models mentioned above are of great value in the study of cardiomyocytes.However,the simulation of only one of the electrical activity and mechanical activity can not comprehensively describe the physiological function of cardiomyocytes.Therefore,researchers combined the above two types of models to establish the electromechanical models.However,the validation and refinement of these models require a lot of experimental data which contain both the electrical and mechanical information of cardiomyocytes.The detection method of the electromechanical signals can provide support for the data acquisition.Due to the two reasons mentioned above,it is a very meaningful work to develop an efficient method for the detection of the cellular electomechanical signals.This paper proposed a method for the electromechanical signal detection,which was based on the combination of microelectrode array technique and atomic force microscopy.Moreover,the effectiveness of the method was verified.The main research contents are as follows:(1)The structural basis of cellular electrical and mechanical activities was described.The action potential generating mechanisms in the neuron and cardiomyocyte were represented.Additionally,the excitation-contraction coupling in cardiomyocytes was introduced.Finally,the significance and importance of the study on the detection method of cellular electromechanical activities were mentioned.(2)The existing cellular electrophysiological signal recording methods and the cell mechanical property measurement techniques were review.This paper introduced the development and application of the microelectrode array(MEA)as an extacellular electrical signal recording tool.Besides,the principle,development and biological applications of the force measurement tool,atomic force microscope(AFM)were introduced.(3)The olfactory bulb neurons were used as the research object.The electrical signal acquisition was carried out by the microelectrode array on the olfactory bulb neural network with the controllable neuron growth.First,a cell culture device with micro structures was fabricated and the surface of the device was modified.The olfactory bulb neurons were cultured on the device.The growth of the neurons could be controlled by the micro structures and the surface characteristics of the device,thereby forming a neural network with a fixed connection pattern.The olfactory bulb neurons in the network were identified by imnunocytochemistry.By combining the cell culture device with the MEA,the extacellular elecrtrical signals of neurons in the network were recorded.The experimental results and the existing problems were analyzed.(4)The atomic force microscope was used to detect the morphological changes of neurons induced by neural activity.A cell mechanical testing platform was set up.In order to verify the feasibility of this platform for the living cell recording,the experiment was focused on the plasticity of the rat cortical neurons.First,drawbacks of the neuron stimulation methods and morphological variation detection tools in previous researches were analyzed.Then the method with the combination of the laser uncaging and AFM imaging was proposed and used for the detection of neuronal activity induced micro structural changes of neurons.Under different conditions,the stimulus areas of the uncaged chemical molecules with different diffusion times were observed.The appropriate stimulation parameters were determined.The neurons of 4 groups were tested under different stimulation conditions.The advantages and disadvantages of the experimental platform as well as the problems in the experiment were discussed.(5)The MEA is combined with the mechanical measurement platform to detect the electromechanical activity of the cardiomyocytes.The research background of the electrical and mechanical signal detections in cardiomyocytes was introduced.After the existing cellular electromechanical signal detection methods were described,the shortage of these methods was analyzed.Then the experimental scheme of the combination of MEA and AFM was proposed.Cardiomyocytes were cultured on the MEA chip.The electrophysiological and mechanical activities of the cardiomyocytes were simultaneously recorded by the union method of MEA and AFM.The correlation between electrical signals and mechanical signals was analyzed.In order to verify the effectiveness of the experimental platform in drug screening,the electrical activity of cardiomyocytes was recorded before and after the drug administration.The drug effects were investigated by analyzing the inter firing interval and transverse force of the cardiomyocytes.The contractile force-stimulation frequency relationship curve was obtained.The experimental results and the shortages of the experiment were discussed.By means of the detection platform proposed in this paper,some fundamental problems in cell biology can be studied in depth.How the cardiomyocytes sense the mechanical and electrical stimuli in microenvironment and adjust their physiological activities is an interesting topic.The quantitative relationship between the magnitude of the mechanical stimuli and the rest membrane potential or the repolarization duration can be investigated by exerting forces of different intensities on catdiomyocytes while recording the electrophysiological signals.Imposing dynamic mechanical stimulations on cardiomyocytes by AFM can make the cell physiological activity synchronized with the stimulation frequency.In this process,researchers can examine the effects of stimulus amplitude and pattern on the cell physiological activity.If cardiomyocytes are cultured on the substrates with different elastic moduli,the effects of the mechanical environment alteration on the gene expression,protein synthesis and contraction-relaxation cycle can be revealed by a long-term electromechanical recording.Similarly,if the cardiomyocytes are subjected to a continuously changing electric field,the effects of the electric field on the physiological activities can be investigated.The molecular mechanism of the difference in the electrical activity(contraction frequency,beating force)between the cardiomyocyte cluster and cardiomyocyte-fibroblast co-culture system can also be analyzed by the method proposed in this paper.Previous studies have shown that the intermediate layer and inner layer of the cardiac muscle are different in physiological characteristics.If the cardiomyocytes from different parts of the cardiac muscle are accurately separated and the electromechanical properties of these cardiomyocytes are measured,a mathematical model of cardiac tissue with more reasonable parameters will be developed.The model may give a more accurate description of the cardiac activity in physiological or pathological conditions.By the platform proposed in this paper,researchers can combine the electrical property(cell membrane capacitance,resistance,etc.)testing and mechanical property(elastic modulus,etc.)testing to distinguish the tumor cells from the normal cells,providing a possible way for the cancer diagnosis and therapy evaluation.The experimental platform proposed is able to simultaneously implement mechanical,electrical and biochemical stimuli to a single cell.Recent studies indicate that the mechanical stimulation will change the cell microenvironment,induce stem cell differentiation,impact cell morphology and actin skeleton remodeling.In addition,electrical stimulation plays an important role in the development of myocardial conduction and contraction.The simultaneous application of electrical,mechanical,and chemical stimuli to cells is necessary for reconstructing the physiological microenvironment of muscle cells in vitro.In particular,for cardiomyocytes,the electrical and mechanical stimuli are tightly coupled in vivo.Multi-parameter cell stimulation and detection platform will provide a powerful tool for investigating the effect of one stimulus or synergies between stimulus on the cell development,differentiation and cell function,and may promote the development of tissue engineering and regenerative medicine.