A method and system for sensing the excitation-contraction coupling of cardiomyocytes
By using multimodal microelectrode array devices and biosensing systems, the problem of simultaneously detecting electrical and mechanical signals of myocardial cells has been solved, achieving high-throughput, multi-site excitation-contraction coupling function, which is suitable for drug screening.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZJU HANGZHOU GLOBAL SCI & TECH INNOVATION CENT
- Filing Date
- 2022-05-09
- Publication Date
- 2026-05-12
AI Technical Summary
Existing technologies are difficult to simultaneously record electrical and mechanical signals of cardiomyocytes in a high-throughput, long-term, and multimodal manner. Furthermore, existing methods suffer from problems such as phototoxicity and operational complexity, and cannot accurately reflect the physiological information of the same research subject.
The system employs a multimodal microelectrode array device and a biosensing system, including a multimodal microelectrode array chip, a mechanical pulsation conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module, and a signal processing software module. The multimodal microelectrode array chip is used to detect the electrical and mechanical signals of cardiomyocytes.
It achieves high-throughput, multi-site recording of excitation-contraction coupling in cardiomyocytes, and can simultaneously detect electrical and mechanical signals, reflecting the excitation-contraction coupling state of cardiomyocytes, which is suitable for drug screening.
Smart Images
Figure CN115078466B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to sensing and detection technology, and in particular to a sensing and detection method and system for the excitation-contraction coupling of myocardial cells. Background Technology
[0002] Drug development is an inefficient and time-consuming process, with some candidate drugs failing to pass preclinical and early clinical evaluations due to cardiovascular safety concerns. Drug cardiotoxicity can cause cardiac dysfunction, and in severe cases, cardiac arrest. Furthermore, inefficient and ineffective drug screening tools increase the time and cost of drug development. Therefore, researchers have developed more preclinical cardiac safety models and technologies to improve drug screening. Among these, cardiomyocyte-based models are increasingly used to study the potential mechanisms or toxicities of drugs by measuring the electrical and mechanical properties of cardiomyocytes.
[0003] The detection of electrical activity at the cellular level is crucial for understanding cellular processes. Patch-clamp techniques, the gold standard for cellular electrophysiology for decades, measure high-quality action potentials in cardiomyocytes by creating a high seal and breaking the cell membrane. However, invasive patch-clamp techniques typically offer only a few hours of recording time, and the complexity of the procedure makes simultaneous recording of multiple cells difficult. On the other hand, the study of cardiomyocyte mechanical properties is used to assess the contractile state after the action potential. 2+ Transient recording is used to monitor intracellular calcium. 2+ While conventional strategies for reflecting the mechanical properties of cardiomyocytes have been developed, fluorescent labeling-based techniques suffer from phototoxicity and drug side effects affecting cell viability and limiting recording time. To meet the demand for high-throughput, sensitive, long-term, multimodal recording of cardiomyocytes, label-free and non-invasive cardiomyocyte-based biosensing technologies have been developed and applied to cardiomyocyte research. Microelectrode arrays are increasingly used to record the electrophysiology of cardiomyocytes over periods of several days or months. Simultaneously, impedance-based interdigital electrodes and hybrid cantilever arrays with integrated strain sensors can be used to measure the mechanical contraction of cardiomyocytes.
[0004] Most existing technologies monitor the electrical or mechanical signals of cardiomyocytes separately, failing to provide a comprehensive correlation study between the two characteristics. To simultaneously detect both electrical and mechanical signals in cardiomyocytes, some researchers have proposed mechatronic recording to synchronously monitor extracellular potential and mechanical pulsation signals. However, the electrical and mechanical signals in mechatronic recordings rarely originate from the same cell or cell population due to the different positions of the electrical and mechanical electrodes, making it difficult to accurately reflect the simultaneous physiological information of the same research subject. To comprehensively understand the electrical and mechanical signals of the same cell or cell population, voltage-sensitive dyes (VSDs) or voltage-sensitive fluorescent proteins (VSFPs) are used in conjunction with Ca2+. 2+The use of sensitive dyes for optical recording allows for multi-site tracking of transmembrane voltage and Ca2+. 2+ Changes in transient signals. However, VSDs and Ca 2+ The phototoxicity of sensitive dyes and adverse drug reactions limit recording time to only a few minutes, while VSFP is limited by the optogenetic expression efficiency of transgenic cells. Scanning probe microscopy probes have been reported to be used in conjunction with force-electrocardiography and force-controlled patch clamp to detect electrical signals and mechanical contraction in cardiomyocytes. However, these methods are not widely applicable due to low throughput and operational complexity. Therefore, in cardiology and pharmacology, there remains a high demand for high-throughput, multimodal recording of electrical and mechanical signals from single cells. Summary of the Invention
[0005] The technical problem to be solved by the present invention is to overcome the shortcomings of the prior art and provide a sensing and detection method and system for excitation-contraction coupling of myocardial cells.
[0006] To solve the technical problem, the solution of the present invention is:
[0007] A multimodal microelectrode array device is provided, comprising, from top to bottom, a reference electrode, a plastic cover, a glass ring, a multimodal microelectrode array chip, and a PCB adapter. The multimodal microelectrode array chip is fixed at the center of the PCB adapter surface. The glass ring is fixed on the multimodal microelectrode array chip, and its radial dimension is adapted to the multimodal microelectrode array chip to cover it. The plastic cover covers the glass ring, and the reference electrode is fixed on the plastic cover with its end extending into the glass ring. The multimodal microelectrode array chip is based on a glass plate, on which several electrodes extending from the periphery to the center are uniformly arranged circumferentially, and each electrode is insulated from the others. Several microstrip lines are arranged on the surface of the PCB adapter extending from the periphery to the center, the number of which is the same as the number of chip electrodes. One end of each microstrip line is connected to a chip electrode, and the other end is electrically connected to a pin of the PCB adapter. The reference electrode is electrically connected to the pin of the PCB adapter through a pin header.
[0008] As a preferred embodiment of the present invention, the substrate of the multimodal microelectrode array chip is a square or circular glass plate.
[0009] As a preferred embodiment of the present invention, the area of the multimodal microelectrode array chip is between 324 and 576 mm². 2 Between these, the electrode diameter is 5–15 μm, and the number is 24–60; the diameter of the effective electrode area is 5–15 μm, and the spacing between adjacent electrodes is 200–500 μm.
[0010] As a preferred embodiment of the present invention, the multimodal microelectrode array chip is fixed to the surface of the PCB adapter by polydimethylsiloxane, and the glass ring is fixed to the multimodal microelectrode array chip by polydimethylsiloxane.
[0011] This invention further provides a sensing and detection system for the excitation-contraction coupling of cardiomyocytes, comprising the aforementioned multimodal microelectrode array device, as well as a mechanical pulsation conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module, and a signal processing software functional module; wherein,
[0012] The high-speed parallel data acquisition module includes an analog-to-digital converter (ADC) and a microcontroller based on a field-programmable gate array (FPGA);
[0013] The mechanical pulsation conditioning module includes an impedance drive module and an impedance amplification module. The former is electrically connected to the reference electrode of the multimodal microelectrode array device, and the latter is electrically connected to the high-speed parallel data acquisition module and the electrode on the substrate of the multimodal microelectrode array device, respectively. It is used to measure the impedance change of cardiomyocytes at a set detection frequency to obtain the mechanical pulsation signal of cardiomyocytes.
[0014] The electrophysiological signal conditioning module is electrically connected to the high-speed parallel data acquisition module. It is used to amplify and filter the electrophysiological signals generated after spontaneous beating of myocardial cells and to sample them through the high-speed parallel data acquisition module.
[0015] The microcontroller is connected to a computer terminal via a signal line. The signal processing software module is built into the computer terminal and is used to process mechanical pulsation signals and electrophysiological signals and extract feature points.
[0016] This invention further provides a sensing detection method for excitation-contraction coupling of cardiomyocytes, comprising the following steps:
[0017] (1) After sterilizing the multimodal microelectrode array device with ethanol and irradiating it with ultraviolet light, the surface was coated with 10 ng / mL fibrin solution and then placed in an incubator at 37°C for 4 hours to promote cell adhesion.
[0018] (2) Fresh animal ventricular myocardial tissue was cleaned, chopped, and digested with 0.07% trypsin / 0.05% type II collagenase at 37°C for 2 hours to obtain a cell suspension; digestion was terminated with a culture medium containing 10% fetal bovine serum, and the cells were collected again by centrifugation and filtration; after differential adhesion treatment, purified cells were obtained.
[0019] (3) The purified cells were seeded in glass rings of a multimodal microelectrode array device and incubated at 37°C and 5% CO2. 2 Incubate in an incubator;
[0020] (4) After spontaneous beating of myocardial cells, the electrophysiological signals of myocardial cells are amplified and filtered twice by the electrophysiological signal conditioning module, and then sampled by the high-speed parallel data acquisition module.
[0021] Simultaneously, the impedance drive module generates an AC signal and applies it to the reference electrode of the multimodal microelectrode array device. After the AC current flows out from the electrode on the substrate of the multimodal microelectrode array device, it is converted into an AC voltage signal by the impedance amplification module and then subjected to secondary high-pass filtering. Finally, the high-speed parallel data acquisition module samples the amplified signal and obtains the mechanical pulsation signal of the myocardial cells by measuring the impedance change of the myocardial cells at the set detection frequency.
[0022] (5) In the high-speed parallel data acquisition module, the analog-to-digital converter (ADC) converts the voltage signal into a digital signal, which is then transmitted to the computer terminal by the microcontroller via the TCP / IP protocol. The built-in signal processing software module performs baseline removal and filtering, and calculates the frequency and amplitude of the electrical signal and mechanical signal after extracting feature points.
[0023] As a preferred embodiment of the present invention, in step (2), the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes; a 70µm cell filter is used for filtration; differential adhesion treatment is performed twice, each time for 45 minutes.
[0024] As a preferred embodiment of the present invention, in step (3), the seeding density of purified cells is 2.0 × 10⁻⁶. 5 cells / cm 2 .
[0025] As a preferred embodiment of the present invention, in step (4), the sampling rate of the electrophysiological signal is 20 kHz, the bandpass rate is 1 Hz to 5 kHz, and the sensitive detection frequency of the mechanical pulsation signal is 10 kHz.
[0026] Compared with the prior art, the beneficial effects of the present invention are:
[0027] 1. While existing technologies can detect electrical or mechanical signals in cardiomyocytes separately, they cannot do so simultaneously. Therefore, they struggle to accurately reflect the simultaneous physiological information of the same research subject and cannot provide a comprehensive correlation study between the two characteristics. This invention utilizes a multimodal microelectrode array to detect integrated electro-mechanical signals, enabling high-throughput recording of single-cell electrical and mechanical signals at multiple sites, thus realizing the excitation-contraction coupling function of cells.
[0028] 2. The multi-modal microelectrode array device used in this invention has a simple manufacturing process, which is conducive to large-scale processing. Since it can use photolithography, magnetron sputtering and stripping technology that are compatible with large-scale processes, it avoids complex and time-consuming processes such as electron beam exposure and ion beam etching, which is conducive to achieving low-cost commercial manufacturing.
[0029] 3. This invention utilizes a multimodal microelectrode array device for high-throughput, multi-site integrated detection of electrical and mechanical pulsation signals, enabling the evaluation of ion channel drugs. Based on the multimodal microelectrode array device-based biosensor system, the recorded electromechanical signals can reflect the excitation-contraction coupling state of cardiomyocytes. Through highly detailed recording, the effects of ion channel blocking drugs on the electromechanical signals of cardiomyocytes can be examined, facilitating drug screening. Attached Figure Description
[0030] Figure 1 An optical microscope image of a multimodal microelectrode array chip;
[0031] Figure 2 This is a layout diagram of a multimodal microelectrode array chip;
[0032] Figure 3 This is a structural diagram of a multimodal microelectrode array device;
[0033] Figure 4 This is an exploded view of a multimodal microelectrode array device.
[0034] Figure 5 A flowchart of a multimodal microelectrode biosensing system;
[0035] Figure 6 Integrated recording of single-cell electromechanical signals for multimodal microelectrode array devices;
[0036] Figure 7 These represent cellular mechatronic signals from different channels;
[0037] Figure 8 To evaluate the efficacy of ion channel drugs by integrating single-cell electromechanical signal recording.
[0038] Figure 4 The attached diagram is labeled as follows: 1. Reference electrode; 2. Plastic cover; 3. Glass ring; 4. Multimodal microelectrode array chip; 5. PCB adapter. Detailed Implementation
[0039] First, it should be noted that this invention relates to database technology, specifically an application of computer technology in the field of information security. The implementation of this invention involves the application of multiple software functional modules. The applicant believes that, after carefully reading the application documents and accurately understanding the implementation principles and objectives of this invention, and in conjunction with existing publicly known technologies, those skilled in the art can fully utilize their software programming skills to implement this invention. The aforementioned software functional modules include, but are not limited to, signal processing software functional modules, etc. All modules mentioned in this application fall within this scope, and the applicant will not list them all further.
[0040] Those skilled in the art will understand that, besides implementing a portion of the system and its various devices, modules, and units provided by this invention in the form of purely computer-readable program code, the same functions can be achieved entirely through logical programming of the method steps, enabling the system and its various devices, modules, and units to function in the form of logic gates, switches, application-specific integrated circuits, programmable logic controllers, and embedded microcontrollers. Therefore, the system and its various devices, modules, and units provided by this invention can be considered a hardware component, and the devices, modules, and units included therein for implementing various functions can also be considered structures within the hardware component; alternatively, the devices, modules, and units for implementing various functions can be considered both software modules implementing the method and structures within the hardware component.
[0041] It should also be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element.
[0042] The implementation of the present invention will now be described in detail with reference to the accompanying drawings.
[0043] like Figure 1-4As shown, the multimodal microelectrode array device includes a reference electrode 1, a plastic cover 2, a glass ring 3, a multimodal microelectrode array chip 4, and a PCB adapter 5 arranged sequentially from top to bottom. The multimodal microelectrode array chip 4 is fixed to the center of the surface of the PCB adapter 5 by polydimethylsiloxane (PDMS). The glass ring 3 is also fixed to the multimodal microelectrode array chip 4 by PDMS, and its radial dimension is adapted to the multimodal microelectrode array chip 4 to cover the latter. The plastic cover 2 covers the glass ring 3, and the reference electrode 1 is fixed to the plastic cover 2 with its end extending into the glass ring 3.
[0044] The multimodal microelectrode array chip 4 uses a glass plate as a substrate, on which several electrodes extending from the periphery to the center are evenly arranged in the circumferential direction, and each electrode is insulated from the others; several microstrip lines are arranged on the surface of the PCB adapter 5 extending from the periphery to the center, the number of which is the same as the number of chip electrodes, one end of each microstrip line is connected to a chip electrode in a one-to-one correspondence, and the other end is electrically connected to a pin of the PCB adapter 5 in a one-to-one correspondence; the reference electrode 1 is electrically connected to the pin of the PCB adapter 5 through a pin header.
[0045] The substrate for the multimodal microelectrode array chip 4 can be a square or circular glass plate. Alternatively, the area of the multimodal microelectrode array chip 4 can range from 324 to 576 mm². 2 Between these, the electrode diameter is 5–15 μm, and the number is 24–60; the diameter of the effective electrode area is 5–15 μm, and the spacing between adjacent electrodes is 200–500 μm.
[0046] This invention further utilizes a multimodal microelectrode array device to construct a sensing and detection system for the excitation-contraction coupling of cardiomyocytes. For example... Figure 5 As shown, the system includes the aforementioned multimodal microelectrode array device, as well as a mechanical pulsation conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module, and a signal processing software functional module.
[0047] The mechanical pulsation conditioning module includes an impedance drive module and an impedance amplification module. The former is electrically connected to the reference electrode of the multimodal microelectrode array device, while the latter is electrically connected to both the high-speed parallel data acquisition module and the electrodes on the substrate of the multimodal microelectrode array device. This module measures the impedance changes of cardiomyocytes at a set detection frequency to obtain the mechanical pulsation signal of the cardiomyocytes. The electrophysiological signal conditioning module is electrically connected to the high-speed parallel data acquisition module. It amplifies and filters the electrophysiological signals generated after spontaneous pulsation of cardiomyocytes and samples them through the high-speed parallel data acquisition module.
[0048] The high-speed parallel data acquisition module includes an analog-to-digital converter (ADC) and a microcontroller based on a field-programmable gate array (FPGA). The ADC converts the voltage signal into a digital signal, which is then transmitted to the microcontroller for subsequent signal processing. Finally, the signal is transmitted to the signal processing software module in the computer terminal via the TCP / IP protocol to process the mechanical pulsation signal and electrophysiological signal and extract feature points.
[0049] Based on the above-mentioned sensing and detection system, the sensing and detection method for excitation-contraction coupling of myocardial cells provided by the present invention includes the following steps:
[0050] (1) After sterilizing the multimodal microelectrode array device with ethanol and irradiating it with ultraviolet light, the surface was coated with 10 ng / mL fibrin solution and then placed in an incubator at 37°C for 4 hours to promote cell adhesion.
[0051] (2) Fresh animal ventricular myocardial tissue was cleaned, chopped, and digested with 0.07% trypsin / 0.05% type II collagenase at 37°C for 2 hours to obtain a cell suspension. The digestion was terminated with a culture medium containing 10% fetal bovine serum. The cells were collected again by centrifugation and filtration. After differential adhesion treatment, purified cells were obtained. The centrifugation speed was 1000 rpm and the centrifugation time was 5 minutes. A 70µm cell filter was used for filtration. Differential adhesion treatment was performed twice, each time for 45 minutes.
[0052] (3) The purified cells were seeded in glass rings of a multimodal microelectrode array device and incubated at 37°C and 5% CO2. 2 The cells were cultured in an incubator; the seeding density of the purified cells was 2.0 × 10⁶. 5 cells / cm 2 .
[0053] (4) After spontaneous pulsation of cardiomyocytes, the electrophysiological signals of cardiomyocytes are amplified and filtered twice using an electrophysiological signal conditioning module, and then sampled by a high-speed parallel data acquisition module. At the same time, the mechanical pulsation signal of cardiomyocytes is obtained by measuring the impedance change of cardiomyocytes at a set detection frequency using a mechanical pulsation conditioning module, and sampled by a high-speed parallel data acquisition module; wherein, the sampling rate of the electrophysiological signal is 20 kHz, the bandpass rate is 1 Hz~5 kHz, and the sensitive detection frequency of the mechanical pulsation signal is 10 kHz.
[0054] (5) In the high-speed parallel data acquisition module, the analog-to-digital converter (ADC) converts the voltage signal into a digital signal, which is then transmitted to the computer terminal by the microcontroller via the TCP / IP protocol. The built-in signal processing software module performs baseline removal and filtering, and calculates the frequency and amplitude of the electrical signal and mechanical signal after extracting feature points.
[0055] More detailed specific implementation examples:
[0056] Step 1:
[0057] The multimodal microelectrode array chip is fabricated using common photolithography, magnetron sputtering, and lift-off techniques.
[0058] A pattern of 32 electrodes with a diameter of 10 μm was fabricated on a 20 mm × 20 mm × 1 mm glass substrate. Positive photoresist RZJ-390PG-30 was spin-coated at 3000 rpm / min, exposed at the i-line (365 nm) with a dose of 300 mJ / cm², and then the microelectrode pattern was formed on the substrate using RZX3038 developer. After defining the microelectrode array pattern, a 10 nm Ti / 100 nm Au layer was deposited by magnetron sputtering, and the photoresist was stripped with acetone and ethanol to prepare a metal layer. Next, the electrode leads were insulated, and an effective electrode region was defined. A 2 μm-think SU-8 2002 photoresist was spin-coated onto the sample at 3000 rpm / min, exposed using a photolithography machine with a dose of 120 mJ / cm², developed in propylene glycol methyl ether acetate for 1 minute, and rinsed with isopropanol. Finally, the multimodal microelectrode array chip was fabricated by hard baking at 150℃ for 30 min with N2.
[0059] As an example, a single multimodal microelectrode array chip has a size of 20 mm × 20 mm, 32 electrodes, an effective electrode area diameter of 10 μm, and a spacing of 300 μm between adjacent electrodes.
[0060] Step 2:
[0061] The prepared multimodal microelectrode array is assembled into a device.
[0062] The multimodal microelectrode array was fixed to a custom printed circuit board (PCB) adapter using polydimethylsiloxane (PDMS), and 32 electrode pads were bonded to the PCB pads using conductive silver paste. Next, a glass ring 1.4 cm in diameter and 1 cm in height was fixed to the center of the chip for cell culture, and a reference electrode was fixed to a plastic cap. Finally, pins were soldered to the PCB to accommodate the interface of the multimodal microelectrode biosensing system, completing the assembly of the multimodal microelectrode array device. The device consists of a glass ring, a multimodal microelectrode array chip, and a custom PCB. The glass ring is fixed above the electrodes using PDMS for cell culture, and the multimodal microelectrode array chip is connected to the PCB for instrument access, enabling integrated detection of multimodal cell electromechanical signals.
[0063] Step 3:
[0064] Establishment of a multimodal biosensing system:
[0065] The hardware modules of the multimodal biosensor system include an electrophysiological signal conditioning module, a mechanical pulsation conditioning module, and a high-speed parallel data acquisition module.
[0066] The high-speed parallel data acquisition module includes an analog-to-digital converter (ADC) and a microcontroller based on a field-programmable gate array (FPGA). The mechanical pulsation conditioning module includes a impedance drive module and an impedance amplification module. The former is electrically connected to the reference electrode of the multimodal microelectrode array device, while the latter is electrically connected to both the high-speed parallel data acquisition module and the electrodes on the substrate of the multimodal microelectrode array device; it is used to measure the impedance changes of cardiomyocytes at a set detection frequency to obtain the mechanical pulsation signal of the cardiomyocytes. The electrophysiological signal conditioning module is electrically connected to the high-speed parallel data acquisition module and is used to amplify and filter the electrophysiological signals generated after spontaneous pulsation of cardiomyocytes, and then sample them through the high-speed parallel data acquisition module. The microcontroller is connected to a computer terminal via signal lines, and the signal processing software module is built into the computer terminal to process the mechanical pulsation signal and electrophysiological signal and extract feature points.
[0067] Step 4:
[0068] Cardiomyocytes were cultured on a multimodal microelectrode array device. Prior to cell culture, the multimodal microelectrode array device was sterilized with 75% ethanol and irradiated with UV light in a biosafety cabinet for 2 hours. Then, a 10 ng / mL fibrin solution was applied to the microelectrode array device, and it was incubated at 37°C for 4 hours to promote cell adhesion. Ventricular myocardial tissue was isolated from the hearts of 1-3 day old neonatal SD rats after sterilization and washed with ice-cold culture medium to remove blood. Subsequently, the tissue was minced into approximately 1 mm pieces using scissors in ice-cold balanced salt solution. 3 Cell fragments were digested with 0.07% trypsin / 0.05% type II collagenase at 37°C for 2 hours to form a cell suspension. Digestion was then terminated with culture medium containing 10% fetal bovine serum. Cells were centrifuged at 1000 rpm for 5 minutes, filtered through a 70 µm cell filter, and collected again. After two 45-minute differential adhesion cycles, purified cells were obtained and seeded at a density of 2.0 × 10⁵ cells / cm² in a multimodal microelectrode array device and cultured at 37°C in a 5% CO₂ incubator.
[0069] Step 5:
[0070] Electrophysiological signals and mechanical pulsations were recorded using a fabricated multimodal microelectrode array device and cultured cardiomyocytes. After spontaneous pulsation of the cardiomyocytes (usually 2-3 days after culture), integrated electro-mechanical signal detection was performed using a multimodal microelectrode biosensor system. The sampling rate for electrophysiological signals was 20 kHz, and the bandpass rate was 1 Hz–5 kHz. The sensitive detection frequency for mechanical pulsation signals was 10 kHz.
[0071] After spontaneous beating of myocardial cells, the electrophysiological signals of myocardial cells are amplified and filtered twice by the electrophysiological signal conditioning module, and then sampled by the high-speed parallel data acquisition module.
[0072] The mechanical pulsation signal of cardiomyocytes is obtained by measuring the impedance change of the cell electrodes at a sensitive detection frequency. After passing through the impedance drive module in the mechanical pulsation conditioning module, an AC signal is generated and applied to the reference electrode of the multimodal microelectrode array device. The AC current flows out from the electrodes on the substrate of the multimodal microelectrode array device, and is converted into an AC voltage signal by the impedance amplification module, followed by secondary high-pass filtering. Finally, the amplified signal is sampled by the high-speed parallel data acquisition module. The analog-to-digital converter (ADC) in the high-speed parallel data acquisition module converts the voltage signal into a digital signal, which is then transmitted to the microcontroller for further signal processing. The signal is then transmitted via TCP / IP protocol to the computer's built-in signal processing software module for baseline removal and filtering. After extracting feature points, the frequency and amplitude of the electrical and mechanical signals are calculated separately.
[0073] Figure 6 The electromechanical integration signals recorded from day 2 to day 4 are shown. Based on the excitation-contraction coupling of cardiomyocytes, electrophysiological and mechanical pulsation signals appear almost simultaneously on day 3. The amplitude of the electrophysiological signal gradually increases, and the firing frequency tends to be rhythmic and stable. The amplitude and pulsation rate of the mechanical pulsation signal are relatively large and stable on days 3 and 4. The cardiomyocyte electromechanical integration model on day 4 is suitable for obtaining high-quality electromechanical integration signals.
[0074] Based on the multimodal device configuration, each channel can simultaneously record electrophysiological and mechanical pulsation signals from the same cardiomyocyte. The high throughput and consistency of the electromechanical integrated signals recorded by the multimodal device allow for continuous recording on a single cell at multiple sites. Figure 7 This shows a typical synchronous electromechanical integration record using three different channels under the same culture conditions.
[0075] Step 6:
[0076] Evaluation of the Effects of Ion Channel Blocking Drugs on Mechatronic Signals in Cardiomyocytes using a Multimodal Microelectrode Biosensing System: Flucainide (a Na+ channel blocker) was used as the tool drug to test the multimodal microelectrode biosensing system. Flucainide is a typical Na+ channel blocker that reduces the frequency and amplitude of action potentials in cardiomyocytes during rapid depolarization, thereby significantly reducing the contractility of cardiomyocytes during mechanical pulsation. A control group recorded electromechanical signals without the drug. Different concentrations of the drug were added for testing; under drug influence, the interpeak intervals of the electromechanical signals were prolonged, and a dose-dependent response was observed (e.g., ...). Figure 8 (As shown).
Claims
1. A sensing and detection system for excitation-contraction coupling of myocardial cells, characterized in that, It includes a multimodal microelectrode array device, as well as a mechanical pulsation conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module, and a signal processing software functional module; among which, The multimodal microelectrode array device includes, from top to bottom, a reference electrode, a plastic cover, a glass ring, a multimodal microelectrode array chip, and a PCB adapter. The multimodal microelectrode array chip is fixed to the center of the PCB adapter surface using polydimethylsiloxane. The glass ring is fixed to the multimodal microelectrode array chip using polydimethylsiloxane, and its radial dimensions are adapted to cover the multimodal microelectrode array chip. The plastic cover covers the glass ring, and the reference electrode is fixed to the plastic cover with its end extending into the glass ring. The multimodal microelectrode array chip uses a circular glass plate as a substrate, on which several electrodes extending from the periphery to the center are evenly arranged circumferentially, and each electrode is insulated from the others. Several microstrip lines are arranged on the surface of the PCB adapter extending from the periphery to the center, the number of which is the same as the number of chip electrodes. One end of each microstrip line is connected to a chip electrode, and the other end is electrically connected to a pin of the PCB adapter. The reference electrode is electrically connected to the pin of the PCB adapter via a pin header. The high-speed parallel data acquisition module includes an analog-to-digital converter (ADC) and a microcontroller based on a field-programmable gate array (FPGA). The mechanical pulsation conditioning module includes an impedance driving module and an impedance amplification module. The former is electrically connected to the reference electrode of the multimodal microelectrode array device, and the latter is electrically connected to the high-speed parallel data acquisition module and the electrode on the substrate of the multimodal microelectrode array device, respectively. It is used to measure the impedance change of cardiomyocytes at a set detection frequency to obtain the mechanical pulsation signal of cardiomyocytes. The electrophysiological signal conditioning module is electrically connected to the high-speed parallel data acquisition module. It is used to amplify and filter the electrophysiological signals generated after spontaneous beating of myocardial cells and to sample them through the high-speed parallel data acquisition module. The microcontroller is connected to a computer terminal via a signal line. The signal processing software module is built into the computer terminal and is used to process mechanical pulsation signals and electrophysiological signals and extract feature points.
2. The sensing and detection system according to claim 1, characterized in that, The area of the multimodal microelectrode array chip is between 324 and 576 mm². 2 Between these, the electrode diameter is 5–15 μm, and the number is 24–60; the diameter of the effective electrode area is 5–15 μm, and the spacing between adjacent electrodes is 200–500 μm.
3. A method for sensing and detecting the excitation-contraction coupling of myocardial cells using the sensing and detection system described in claim 1 or 2, characterized in that, Includes the following steps: (1) After sterilizing the multimodal microelectrode array device with ethanol and irradiating it with ultraviolet light, the surface was coated with 10 ng / mL fibrin solution and then placed in an incubator at 37°C for 4 hours to promote cell adhesion. (2) Fresh animal ventricular myocardial tissue was cleaned, chopped, and digested with 0.07% trypsin / 0.05% type II collagenase at 37°C for 2 hours to obtain a cell suspension; digestion was terminated with a culture medium containing 10% fetal bovine serum, and the cells were collected again by centrifugation and filtration; after differential adhesion treatment, purified cells were obtained. (3) The purified cells were seeded in glass rings of the multimodal microelectrode array device and cultured in an incubator at 37°C and 5% CO2. (4) After spontaneous beating of myocardial cells, the electrophysiological signals of myocardial cells are amplified and filtered twice by the electrophysiological signal conditioning module, and then sampled by the high-speed parallel data acquisition module. Simultaneously, the impedance driving module generates an AC signal and applies it to the reference electrode of the multimodal microelectrode array device. After the AC current flows out from the electrode on the substrate of the multimodal microelectrode array device, it is converted into an AC voltage signal by the impedance amplification module and then subjected to secondary high-pass filtering. Finally, the high-speed parallel data acquisition module samples the amplified signal and obtains the mechanical pulsation signal of the myocardial cells by measuring the impedance change of the myocardial cells at a set detection frequency. The sampling rate of the electrophysiological signal is 20 kHz, and the bandpass rate is 1 Hz to 5 kHz. The sensitive detection frequency of the mechanical pulsation signal is 10 kHz. (5) In the high-speed parallel data acquisition module, the analog-to-digital converter (ADC) converts the voltage signal into a digital signal, which is then transmitted to the computer terminal by the microcontroller via the TCP / IP protocol. The built-in signal processing software module performs baseline removal and filtering, and calculates the frequency and amplitude of the electrical and mechanical signals after extracting feature points.
4. The method according to claim 3, characterized in that, In step (2), the centrifugation speed is 1000 rpm and the centrifugation time is 5 minutes; a 70µm cell filter is used for filtration; differential adhesion treatment is performed twice, each time for 45 minutes.
5. The method according to claim 3, characterized in that, In step (3), the seeding density of purified cells is 2.0 × 10⁻⁶. 5 cells / cm 2 .