Myocardial cell bionic taste and olfactory composite sensing system and detection method for evaluating tachycardia therapeutic drugs

Abstract

The present invention discloses a cardiomyocyte bionic taste-olfactory composite sensing system and a detection method for evaluating drugs for treating tachycardia. Cardiomyocytes that endogenously express taste receptors and olfactory receptors are used as taste-olfactory dual-sensing sensitive elements, and taste and olfactory receptors are used as drug targets. Microelectrode array potential sensors are used as secondary transducers. The system can detect the extracellular field potential signals of cardiomyocytes before and after the taste and olfactory substances act on an in vitro tachycardia model in a highly sensitive, real-time and rapid manner, and utilize time-domain and frequency-domain electrophysiological signal analysis methods to achieve the detection and quantitative evaluation of potential traditional Chinese medicines and odorants for treating tachycardia.

Description

Technical Field

[0001] The present invention relates to the field of bionic micro-nano sensing and drug evaluation, and in particular to a myocardial cell taste and smell composite sensing system and a detection method for evaluating tachycardia therapeutic drugs. Background Art

[0002] Taste receptors in oral taste buds and olfactory receptors in the nasal cavity act as peripheral chemoreceptors to mediate mammalian taste perception and olfactory perception, respectively. However, in recent years, combined with RT-PCR, gene sequencing, immunostaining, and spatial transcriptome techniques, studies have found that taste receptors, especially sweet, umami, and bitter G protein-coupled receptors and olfactory receptors, are also widely expressed in areas outside the taste and olfactory systems, including the respiratory system, gastrointestinal tract, urogenital system, cardiovascular system, thyroid, musculoskeletal system, immune system, and central nervous system. This is called "ectopic expression" of taste and olfactory receptors. Although receptor molecules and taste transduction cascades have similarities, taste and olfactory receptors play different chemical sensing functions in oral and nasal tissues depending on the location of expression, making them potential new drug targets for disease treatment. In the modern drug development process, drug screening based on taste receptors and olfactory receptors is expected to become a new approach to new drug development.

[0003] Arrhythmia is a heart disease that affects the heart rate or rhythm. Sudden cardiac death caused by arrhythmia can account for 25% of all deaths. Tachycardia is one of them. When the heart rate speeds up to more than 100 beats per minute, tachycardia occurs, which in turn affects the way the heart pumps blood. If not treated in time, it will destroy normal heart function and lead to serious complications such as heart failure, stroke, cardiac arrest or death. At present, antiarrhythmic drugs that can be used to treat tachycardia include beta-blockers such as acebutolol, metoprolol and other commonly used ion channel blockers such as amiodarone and sotalol. However, there are problems such as single receptor target, desensitization and even arrhythmic side effects. Therefore, it is urgent to develop new and safer tachycardia treatment drugs. Cardiomyocytes endogenously express taste and olfactory receptors. Bitter agonists and odorants that can activate taste and olfactory receptors can mediate the contractile response of cardiomyocytes and have an important dose-dependent negative inotropic effect on myocardial contraction. Therefore, the present invention utilizes the characteristics of myocardial cells endogenously expressing taste receptors and olfactory receptors, uses myocardial cells as dual-sensing sensitive elements for taste and smell, and uses taste and olfactory receptors as drug targets. Combined with microelectrode array chips, it records extracellular potential signals simply, quickly and efficiently, to provide a myocardial cell bionic taste and olfactory composite sensing system and detection method for evaluating tachycardia therapeutic drugs. Summary of the invention

[0004] The purpose of the present invention is to address the deficiencies of the prior art and provide a myocardial cell bionic taste and smell composite sensing system and a detection method for evaluating tachycardia therapeutic drugs.

[0005] The object of the present invention is achieved through the following technical scheme: the system includes a microelectrode array chip design and processing module, a microelectrode array chip surface modification module, a taste and smell dual perception sensitive element cultivation module, a taste and smell dual perception sensitive element receptor expression identification module, an in vitro tachycardia model construction module and a drug detection and evaluation module;

[0006] The microelectrode array chip design and processing module is used to design the photolithography mask plate required for the microelectrode array chip. The photolithography mask plate includes two parts: a metal layer and an insulating layer. In the metal layer, the shape and spatial arrangement of the electrodes are designed according to the actual size; then, the insulating layer is designed according to the position of the electrodes. Except for the positions of the working electrode, the reference electrode and the gold wire transfer point, the remaining position areas are covered by the insulating layer; and the designed microelectrode array chip is processed by the MEMS manufacturing process;

[0007] The microelectrode array chip surface modification module is used to improve the signal-to-noise ratio of the microelectrode array chip detection signal by coating a gelatin / fibronectin solution and a Cell-Tak cell / tissue adhesive on the surface of the microelectrode array chip and enhancing the coupling degree between the microelectrode array chip and the cardiomyocytes;

[0008] The taste and smell dual-sensing sensitive element culture module is used to obtain primary cardiomyocytes or a cell line of cardiomyocytes by primary extraction, and configure them into a cardiomyocyte suspension; then the cardiomyocyte suspension is planted in a microelectrode array chip culture chamber, and the cardiomyocytes are evenly distributed on the electrodes of the microelectrode array chip or around the electrode points;

[0009] The taste and smell dual-sensory sensor receptor expression identification module is used to determine the gene expression of taste receptors and olfactory receptors in myocardial cells using real-time reverse transcription PCR (RT-PCR) technology;

[0010] The in vitro tachycardia model construction module is used to apply isoproterenol (ISO) of several concentration gradients to myocardial cells respectively, and then record the changes of the extracellular local field potential signals of myocardial cells on the microelectrode array chip in real time through a multi-channel in vitro electrophysiological signal acquisition system; and extract the time domain and frequency domain features of the detected potential signals and perform normalization to obtain the concentration-dependent response curves under the stimulation of different concentrations of ISO, and select the required concentration of ISO as the stimulus for the in vitro tachycardia model construction;

[0011] The drug detection and evaluation module is used to use taste and olfactory receptors as drug targets for tachycardia treatment. According to the gene expression obtained by the taste and olfactory dual perception sensitive element receptor expression identification module, stimulants that can activate taste receptors and olfactory receptors endogenously expressed in cardiomyocytes are selected, and act on the in vitro tachycardia model after ISO stimulation of the required concentration selected by the in vitro tachycardia model construction module to explore the changes in the extracellular local field potential signals of the cardiomyocytes caused by the stimulants of different concentrations, so as to obtain the evaluation effect of potential tachycardia treatment drugs.

[0012] Furthermore, the specific process of the MEMS process is to process the microelectrode array chip through photolithography, magnetron sputtering, lift-off stripping, and plasma enhanced chemical vapor deposition processing technology, and finally through the steps of scribing, bonding, and gold wire spot welding, the polydimethylsiloxane (PDMS) culture chamber is bonded to the surface of the microelectrode array chip.

[0013] Furthermore, the microelectrode array chip surface modification module can also perform surface modification of the microelectrode array chip by electroplating platinum black nanoparticles (PtNPs) on the electrode surface of the microelectrode array chip through the three-electrode system of the electrochemical workstation, thereby increasing the specific surface area of ​​the electrode, reducing the electrode impedance, thereby reducing noise and improving the signal-to-noise ratio of the microelectrode array chip detection signal.

[0014] Furthermore, after the sensitive element culture module plants cardiomyocytes in the microelectrode array chip, a live cell / dead cell staining method is required to determine the growth activity of the cardiomyocytes on the microelectrode array chip. If more than 80% of the cells are alive, the cell sensing detection requirements can be met, otherwise re-culture is required.

[0015] Furthermore, the process of the taste and olfactory dual perception sensitive element receptor expression identification module using real-time reverse transcription PCR (RT-PCR) technology to determine the gene expression of taste receptors and olfactory receptors in cardiomyocytes includes taste and olfactory receptor gene primer sequence design, cardiomyocyte RNA extraction, RNA reverse transcription synthesis of cDNA and RT-PCR amplification.

[0016] Furthermore, the concentration gradients of isoproterenol selected in the in vitro tachycardia model construction module are 0.01, 0.1, 1, 10, 100 and 1000 μM respectively; the time domain and frequency domain characteristics of the local field potential signal analysis of myocardial cells include: amplitude, frequency, time course, release time interval and power spectral density of the electrophysiological signal.

[0017] Furthermore, the drug detection and evaluation module uses the stimulus as a traditional Chinese medicine and / or an odorant, obtains the concentration-dependent response curve of the stimulus through the change of the extracellular local field potential signal of the myocardial cells, and obtains the half maximal inhibitory concentration (IC) for inhibiting myocardial cell tachycardia through nonlinear regression analysis. 50 ).

[0018] The present invention also provides a detection method for evaluating tachycardia therapeutic drugs based on a myocardial cell bionic taste and smell composite sensing system, the specific steps of which are as follows:

[0019] (1) Design and processing of microelectrode array chip: designing the photolithography mask plate required for the microelectrode array chip, the photolithography mask plate includes two parts: a metal layer and an insulating layer. In the metal layer, the shape and spatial arrangement of the electrodes are designed according to the actual size; then, the insulating layer is designed according to the position of the electrodes. Except for the positions of the working electrode, the reference electrode and the gold wire transfer point, the remaining positions are covered by the insulating layer; and the designed microelectrode array chip is processed by MEMS manufacturing process;

[0020] (2) Surface modification of microelectrode array chips: by coating the surface of the microelectrode array chip with gelatin / fibronectin solution and Cell-Tak cell / tissue adhesive, the coupling degree between the microelectrode array chip and the cardiomyocytes is enhanced to improve the signal-to-noise ratio of the detection signal of the microelectrode array chip;

[0021] (3) Taste and smell dual-sensing sensitive element culture: primary cardiomyocytes or cardiomyocyte cell lines are obtained by primary extraction and prepared into a cardiomyocyte suspension; the cardiomyocyte suspension is then planted in the culture chamber of the microelectrode array chip, and the cardiomyocytes are evenly distributed on the electrodes of the microelectrode array chip or around the electrode points;

[0022] (4) Identification of taste and olfactory dual-sensory receptor expression: Real-time reverse transcription PCR (RT-PCR) was used to determine the gene expression of taste receptors and olfactory receptors in cardiomyocytes;

[0023] (5) Construction of an in vitro tachycardia model: After isoproterenol (ISO) of several concentration gradients was applied to cardiomyocytes, the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip were recorded in real time by a multi-channel in vitro electrophysiological signal acquisition system; the time domain and frequency domain features of the detected potential signals were extracted and normalized to obtain the concentration-dependent response curves under the stimulation of different concentrations of ISO, and the required concentration of ISO was selected as the stimulus for the construction of the in vitro tachycardia model;

[0024] (6) Drug testing and evaluation: Taste and olfactory receptors are used as targets for tachycardia treatment drugs. Based on the gene expression obtained in step (4), stimulants that can activate the taste receptors and olfactory receptors endogenously expressed in cardiomyocytes are selected to act on the in vitro tachycardia model after ISO stimulation at the required concentration selected in step (5). The changes in the extracellular local field potential signals of cardiomyocytes induced by the stimulation of different concentrations of stimulants are explored to obtain the evaluation effect of potential tachycardia treatment drugs.

[0025] Compared with the current technical shortcomings, the present invention has the following beneficial effects: The present invention provides a method for detecting and evaluating tachycardia therapeutic drugs based on a cardiomyocyte bionic taste and olfactory composite sensing system. This technology uses taste receptors and olfactory receptors endogenously expressed in cardiomyocytes as drug targets, combines microelectrode array chips, and utilizes bionic sensing technology to simply, quickly, and efficiently record extracellular potential signals after stimulation by taste and olfactory substances, thereby realizing the detection and evaluation of potential tachycardia therapeutic drugs, expanding the application scope of bionic taste and olfactory sensors, and opening up ideas for further research and development, detection and evaluation of tachycardia therapeutic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Schematic diagram of the bionic taste and smell composite sensing detection system based on cardiomyocytes of the present invention;

[0028] Figure 2 It is a schematic diagram of the processing flow of the microelectrode array chip of the present invention;

[0029] Figure 3 The expression of taste receptors and olfactory receptors in myocardial cells determined by RT-PCR technology in the present invention;

[0030] Figure 4 It is the extracellular local field potential signal of the myocardial cell collected by the multi-channel electrophysiological signal collection system of the present invention;

[0031] Figure 5 The in vitro tachycardia model constructed by the present invention using isoproterenol stimulation;

[0032] Figure 6 It is the response signal of the bionic taste and smell composite sensor of the present invention to the potential tachycardia therapeutic drug;

[0033] Figure 7 It is a response signal of inhibiting bitter taste signal transduction pathway detected by the bionic taste and smell composite sensor of the present invention. DETAILED DESCRIPTION

[0034] The present invention is described in detail below with reference to the accompanying drawings and specific embodiments, but the present invention is not limited thereto.

[0035] The present invention provides a bionic taste and smell composite sensing detection system based on cardiomyocytes for use in tachycardia model construction, bitter Chinese medicine and / or odorant detection, and tachycardia therapeutic drug evaluation methods, such as Figure 1 As shown in the figure, the system uses cardiomyocytes as sensitive elements and microelectrode array chips as secondary transducers to record the electrophysiological response signals of cardiomyocytes to taste and olfactory substances; extracts the characteristics of the change of extracellular local field potential signals, studies the changes in the potential emission frequency after bitter Chinese medicine and odorants act on the tachycardia model, and realizes the quantitative detection and evaluation of potential tachycardia therapeutic drugs. The construction method of the system includes the following steps:

[0036] (1) Microelectrode array chip design: The photolithography mask required for microelectrode array chip processing is designed by AutoCAD software, which mainly includes two parts: the metal layer and the insulating layer. In the metal layer, the shape and spatial arrangement of the electrode are drawn in AutoCAD software according to the actual size. The microelectrode array sensor used has a glass substrate chip with a 12mm square structure. 60 working electrode points with a diameter of 30μm and a spacing of 200μm are arranged in an 8×8 array in the middle area. There are 4 reference electrodes with a size of 1 / 4 circle around the working electrode. The periphery is connected to the PCB through a gold wire transfer point, and different electrodes are connected through wires (line width 10μm). After completing the design of the electrode structure of each chip unit, 37 chip units are arranged on a mask plate (5 inches). After completing the metal layer design of the microelectrode array chip, the insulating layer is designed according to the position of the electrode. Except for the working electrode, reference electrode and the location of the gold wire transfer point, the remaining area is covered by the insulating layer. In the same way, the insulating layer mask of the 64-channel microelectrode array chip can be designed.

[0037] (2) Microelectrode array chip MEMS processing: It is manufactured using MEMS technology. The manufacturing process is as follows Figure 2As shown. The specific operation process is as follows: a four-inch quartz glass sheet (Pyrex Corning 7740 glass, 0.5mm) is cleaned and used as a substrate. It is light-transmissive, which is convenient for observing the growth of cells on the microelectrode array chip with an inverted microscope in the experiment; spin-coat positive photoresist on the glass substrate; cover the metal layer mask plate that determines the electrode layout on the substrate, and expose and develop it under ultraviolet light; use magnetron sputtering technology to deposit an adhesion layer (Cr, 20nm) and a conductive layer (Au, 300nm) on the glass substrate in turn; use the lift-off stripping process to wash off the photoresist and the Cr and Au covering it to form a preset metal layer pattern; deposit an insulating layer (Si3N4, 500nm) by plasma enhanced chemical vapor deposition (Plasma Enhanced Chemical Vapor Deposition, PECVD); spin-coat photoresist on the Si3N4 layer; expose and develop the coated photoresist under the insulating layer mask plate under ultraviolet light; use reactive ion etching (Reactive-Ion Etching (RIE) is used to expose the electrode points and metal transfer points, and the remaining Si3N4 covering the Au layer is etched out with a KOH solution; the photoresist is removed, and the prepared microelectrode array chip is supported by a printed circuit board (PCB), and the electrode points are connected to the PCB metal transfer points with gold wires; finally, after steps such as scribing, chip mounting, and gold wire spot welding, a polydimethylsiloxane (PDMS) culture chamber is bonded to the surface of the microelectrode array chip.

[0038] (3) Surface modification of microelectrode array chips: In order to improve the signal-to-noise ratio of the detection signal of the microelectrode array chip, on the one hand, platinum black nanoparticles (PtNPs) can be electroplated on the surface of the microelectrode array chip electrode through the three-electrode system of the electrochemical workstation to increase the specific surface area of ​​the electrode, reduce the electrode impedance, thereby reducing noise and improving the signal-to-noise ratio; on the other hand, gelatin / fibronectin solution or Cell-Tak cell / tissue adhesive can be coated on the surface of the microelectrode array chip to enhance the coupling between the microelectrode array chip and the cells.

[0039] (4) In vitro culture of sensitive element cardiomyocytes: primary cardiomyocytes or cardiomyocyte cell lines are obtained by primary extraction and configured into cardiomyocyte suspension. Then, 1 mL of cell suspension and cell culture medium is added to the cell culture chamber of the microelectrode array chip to ensure that the number of cells is about 50,000 per well. Cardiomyocytes are planted in the culture chamber of the microelectrode array chip. Cardiomyocytes are evenly distributed on the microelectrode array electrodes or around the electrode points, forming a cell network for potential conduction, ensuring that the electrodes can effectively record the electrophysiological signals of cardiomyocytes. The live cell / dead cell staining method is used to determine the growth activity of cells on the microelectrode array chip. Calcein-AM (0.3 mg / L) and propidium iodide (PI, 0.5 mg / L) fluorescent dyes are used to stain live cells and dead cells respectively. For more than 80% of the cells, it is shown that the microelectrode array chip has good biocompatibility and can meet the requirements of cell sensing detection.

[0040] (5) RT-PCR in sensitive cardiomyocytes to measure the expression of taste receptors and olfactory receptors: Real-time reverse transcription PCR (RT-PCR) was used to measure the gene expression of taste receptors and olfactory receptors in the studied cardiomyocytes. The specific steps included design of primer sequences for taste receptor and olfactory receptor genes, extraction of cardiomyocyte RNA, reverse transcription of RNA to synthesize cDNA, and RT-PCR amplification. Amplification data were collected from three independent experiments using 2 -△△Ct The internal reference gene β-Actin was selected as a reference to quantify the relative expression level of the target gene. Figure 3 As shown in the figure, among the 35 bitter taste receptors of mice, the expression levels of bitter taste receptors such as TAS2R108, TAS2R122, TAS2R126, TAS2R129, TAS2R135, TAS2R136, TAS2R137 and TAS2R143 are relatively higher than other receptors. In addition, the expression levels of Olfr544 and Olfr558 olfactory receptors related to the perception of medium- and short-chain fatty acid odors in cardiomyocytes are relatively high, but Olfr78 receptors are almost not expressed. On the basis of the established culture system, the functionality of taste and olfactory receptors is further explored by combining calcium imaging technology.

[0041] (6) Recording of electrophysiological signals of cardiomyocytes: When cardiomyocytes spontaneously or stimulatedly generate action potentials, transient transmembrane potentials and ionic currents are generated. The electrode is polarized by reconstructing the charge distribution at the electrode-electrolyte cell interface, causing changes in the electrode potential. A multi-channel in vitro electrophysiological signal acquisition system (MEA2100-Systems) is used for signal acquisition and recording. Extracellular electrophysiological signals of cardiomyocytes at different sites are recorded synchronously by multiple working electrodes ( Figure 4), the signal is amplified 1200 times by a preamplifier and filtered (<500Hz), the sampling frequency is 1kHz, and then transmitted to a computer by a data acquisition card for offline analysis.

[0042] (7) Construction of myocardial cell tachycardia model: Isoprenaline (ISO), as a non-selective β-adrenergic receptor agonist, can activate β1-AR receptors on myocardial cells. It is a widely used drug in clinical practice and is often used to treat bradycardia or heart block. Therefore, ISO was selected as the stimulating drug for the myocardial tachycardia model. Figure 4 As shown, after ISO with concentrations of 0.01, 0.1, 1, 10, 100 and 1000 μM acted on cardiomyocytes, the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip were recorded in real time by a multi-channel in vitro electrophysiological signal acquisition system. Figure 5 Figure A shows the typical extracellular field potential (EFP) signal of HL-1 cardiomyocytes in response to different concentrations of ISO stimulation. The firing rate is an important parameter for measuring the electrophysiological activity of cardiomyocytes. Raster graphs are usually used to represent the time domain distribution of action potential signals. In addition, according to the statistical results, Figure 5 As can be seen in B, 0.0-100 μM ISO stimulation significantly increased the potential firing frequency of cardiomyocytes. After t-test, the signal firing frequency was significantly different from that of PBS (control group). However, 1000 μM ISO caused a significant decrease in the firing frequency and amplitude of cardiomyocytes, which is related to the toxic side effects of high-concentration ISO stimulation on cardiomyocytes. To further quantify the difference between signals, the firing frequency of the detected cardiomyocyte extracellular field potential signal was extracted and normalized to obtain the ISO concentration-dependent response curve, which is obtained by Figure 5 As shown in C, the results show that the normalized firing frequency increases with the increase of ISO concentration. The normalized firing frequency of the control group is 1.0, while the normalized firing frequency induced by 100μM ISO stimulation is 1.68±0.20. The EC50 of ISO is 1.36μM after nonlinear fitting, which also shows the high sensitivity of the system. Therefore, in subsequent experiments, ISO with a concentration of 100μM was selected as the stimulating drug for constructing the tachycardia model.

[0043] (8) Finally, the cardiomyocyte bionic taste-olfactory composite sensing system was used in combination with electrophysiological signal decoding and analysis to realize the detection and evaluation of bitter Chinese medicine and odorants for the treatment of tachycardia: After first constructing an in vitro tachycardia model using isoproterenol, bitter Chinese medicines that can activate the taste receptors endogenously expressed in cardiomyocytes and odorants that activate the olfactory receptors were selected, and then the extracellular electrical signals induced by different concentrations of taste and olfactory substances were explored. 0.001-1000 μM salicin, 1-400 μM artemisinin, 1-250 μM Zanthoxylum bungeanum and 0.001-100 μM azelaic acid were added to the in vitro tachycardia model, and the extracellular electrical signals induced by different concentrations of the three bitter substances were analyzed. The action potential firing frequency of myocardial cells was used as the output parameter of the sensor. The relationship between the firing frequency and the stimulus concentration was established based on the difference in firing frequency. The concentration-dependent response curves of the three bitter Chinese medicines and azelaic acid were obtained by statistical analysis of the potential firing frequency and its normalization. The half maximal inhibitory concentration (IC) of salicin, artemisinin, Zanthoxylum bungeanum and azelaic acid for inhibiting myocardial tachycardia was obtained by nonlinear regression analysis. 50 ) values ​​were 3.6nM, 309.8μM, 14.68μM and 0.102nM, respectively. Figure 6 shown.

[0044] Furthermore, the specific detection performance of the cardiomyocyte bionic taste and olfactory composite sensing system was verified by using the PLCβ2 inhibitor U73122 to inhibit the Gβγ-PLCβ2 signal transduction pathway activated by taste receptors. 1μM U73122 can significantly inhibit the reduction of the potential firing frequency of cardiomyocytes caused by salicin, Zanthoxylum bungeanum and artemisinin. Figure 7 The data were presented using artemisinin as an example. The experimental results showed that the changes in the contraction frequency of myocardial cells after stimulation by bitter Chinese medicine were indeed related to the activation of bitter receptors.

[0045] The above results indicate that the cardiomyocyte-based bionic taste and olfactory composite sensor designed in the present invention can effectively and quantitatively evaluate the inhibitory effects of bitter Chinese medicine and odorants on tachycardia models, can be used to assist in drug evaluation, and has potential application value for the development of new drugs around new mechanisms and new targets.

[0046] The above embodiments are used to explain the present invention, rather than to limit the present invention. Any modification and change made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia therapeutic drugs, characterized in that: The system includes a microelectrode array chip design and processing module, a microelectrode array chip surface modification module, a taste and smell dual perception sensitive element cultivation module, a taste and smell dual perception sensitive element receptor expression identification module, an in vitro tachycardia model construction module and a drug detection and evaluation module; The microelectrode array chip design and processing module is used to design the photolithography mask plate required for the microelectrode array chip. The photolithography mask plate includes two parts: a metal layer and an insulating layer. In the metal layer, the shape and spatial arrangement of the electrodes are designed according to the actual size; then, the insulating layer is designed according to the position of the electrodes. Except for the positions of the working electrode, the reference electrode and the gold wire transfer point, the remaining position areas are covered by the insulating layer; and the designed microelectrode array chip is processed by the MEMS manufacturing process; The microelectrode array chip surface modification module is used to improve the signal-to-noise ratio of the microelectrode array chip detection signal by coating a gelatin / fibronectin solution and a Cell-Tak cell / tissue adhesive on the surface of the microelectrode array chip and enhancing the coupling degree between the microelectrode array chip and the cardiomyocytes; The taste and smell dual-sensing sensitive element culture module is used to obtain primary cardiomyocytes or a cell line of cardiomyocytes by primary extraction, and configure them into a cardiomyocyte suspension; then the cardiomyocyte suspension is planted in a microelectrode array chip culture chamber, and the cardiomyocytes are evenly distributed on the electrodes of the microelectrode array chip or around the electrode points; The taste and smell dual-sensing sensitive element receptor expression identification module is used to determine the gene expression of taste receptors and olfactory receptors in myocardial cells using real-time reverse transcription PCR technology; The in vitro tachycardia model construction module is used to apply several concentration gradients of isoproterenol ISO to myocardial cells respectively, and then record the changes of the extracellular local field potential signals of myocardial cells on the microelectrode array chip in real time through a multi-channel in vitro electrophysiological signal acquisition system; and extract the time domain and frequency domain features of the detected potential signal and perform normalization to obtain the concentration-dependent response curve under the stimulation of different concentrations of ISO, and select the required concentration of ISO as the stimulus for the construction of the in vitro tachycardia model; The drug detection and evaluation module is used to use taste and olfactory receptors as drug targets for tachycardia treatment. According to the gene expression obtained by the taste and olfactory dual perception sensitive element receptor expression identification module, salicin, artemisinin, bitter Chinese medicine of Zanthoxylum bungeanum toxin and azelaic acid odorant that can activate taste receptors and olfactory receptors endogenously expressed in cardiomyocytes are selected as stimulants, and act on the in vitro tachycardia model after ISO stimulation of the required concentration selected by the in vitro tachycardia model construction module. Through the changes in the extracellular local field potential signals of the cardiomyocytes, the concentration-dependent response curve of the stimulant is established according to the difference in the action potential firing frequency, and the half-inhibitory concentration for inhibiting the tachycardia of the cardiomyocytes is obtained through nonlinear regression analysis. The changes in the extracellular local field potential signals of the cardiomyocytes caused by the stimulants of different concentrations are explored to obtain the evaluation effect of potential tachycardia treatment drugs.

2. The myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: The specific process of the MEMS manufacturing process is to process the microelectrode array chip through photolithography, magnetron sputtering, lift-off stripping, and plasma enhanced chemical vapor deposition processing technology, and finally through the steps of scribing, bonding, and gold wire spot welding, the polydimethylsiloxane culture chamber is bonded to the surface of the microelectrode array chip.

3. The myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: The microelectrode array chip surface modification module can also perform surface modification of the microelectrode array chip by electroplating platinum black nanoparticles on the electrode surface of the microelectrode array chip through the three-electrode system of the electrochemical workstation, thereby increasing the specific surface area of ​​the electrode, reducing the electrode impedance, thereby reducing noise and improving the signal-to-noise ratio of the microelectrode array chip detection signal.

4. The myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: After the sensitive element culture module plants cardiomyocytes in the microelectrode array chip, a live cell / dead cell staining method is required to determine the growth activity of the cardiomyocytes on the microelectrode array chip. If more than 80% of the cells are alive, the cell sensing detection requirements can be met, otherwise re-culture is required.

5. The myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: The process of using real-time reverse transcription-PCR technology to determine the gene expression of taste receptors and olfactory receptors in cardiomyocytes includes designing primer sequences of taste and olfactory receptor genes, extracting RNA from cardiomyocytes, synthesizing cDNA through reverse transcription of RNA, and RT-PCR amplification.

6. The myocardial cell bionic taste and smell composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: The time domain and frequency domain characteristics analyzed by the in vitro tachycardia model building module include: the amplitude, frequency, time course, firing time interval and power spectral density of the extracellular field potential signal of the myocardial cells.

7. The myocardial cell bionic taste and olfactory composite sensing system for evaluating tachycardia treatment drugs according to claim 1, characterized in that: The concentration gradient of isoproterenol (ISO) was 0.01, 0.1, 1, 10, 100 and 1000 μM.

8. A detection method for evaluating tachycardia therapeutic drugs based on the myocardial cell bionic taste and olfactory composite sensing system according to any one of claims 1 to 7, characterized in that: The specific steps are as follows: (1) Design and processing of microelectrode array chips: Design the photolithography mask required for the microelectrode array chip. The photolithography mask includes a metal layer and an insulating layer. In the metal layer, the shape and spatial arrangement of the electrodes are designed according to the actual size. Then, the insulating layer is designed according to the position of the electrodes. Except for the working electrode, reference electrode and the gold wire transfer point, the remaining position area is covered by the insulating layer. The designed microelectrode array chip is then processed through the MEMS manufacturing process. (2) Surface modification of microelectrode array chips: by coating the surface of the microelectrode array chip with gelatin / fibronectin solution and Cell-Tak cell / tissue adhesive, the coupling degree between the microelectrode array chip and the cardiomyocytes is enhanced to improve the signal-to-noise ratio of the microelectrode array chip detection signal; (3) Culture of taste and smell dual-sensing sensitive elements: Primary cardiomyocytes or cardiomyocyte cell lines are obtained by primary extraction and prepared into a cardiomyocyte suspension; the cardiomyocyte suspension is then planted in the culture chamber of the microelectrode array chip, and the cardiomyocytes are evenly distributed on the electrodes of the microelectrode array chip or around the electrode points; (4) Identification of taste and olfactory dual-sensory receptor expression: Real-time reverse transcription PCR was used to determine the gene expression of taste receptors and olfactory receptors in cardiomyocytes; (5) Construction of an in vitro tachycardia model: After isoproterenol (ISO) with several concentration gradients was applied to cardiomyocytes, the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip were recorded in real time by a multi-channel in vitro electrophysiological signal acquisition system. The time domain and frequency domain features of the detected potential signals were extracted and normalized to obtain the concentration-dependent response curves under the stimulation of different concentrations of ISO. The required concentration of ISO was selected as the stimulus for the construction of an in vitro tachycardia model. (6) Drug testing and evaluation: Taste and olfactory receptors are used as targets for tachycardia treatment drugs. Based on the gene expression obtained in step (4), stimulants that can activate the taste receptors and olfactory receptors endogenously expressed in cardiomyocytes are selected and applied to the in vitro tachycardia model after ISO stimulation at the required concentration selected in step (5). The changes in the extracellular local field potential signals of cardiomyocytes induced by the action of stimulants of different concentrations are explored to obtain the evaluation effect of potential tachycardia treatment drugs.

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