CN115078466A - Sensing detection method and system for exciting contraction coupling of myocardial cells - Google Patents
Sensing detection method and system for exciting contraction coupling of myocardial cells Download PDFInfo
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Abstract
The invention relates to a sensing detection technology, and aims to provide a sensing detection method and a sensing detection system for exciting and contracting coupling of myocardial cells. The system comprises a multi-mode microelectrode array device, a mechanical pulse conditioning module, an electrophysiological signal conditioning module and a high-speed parallel data acquisition module; the mechanical pulse conditioning module comprises an electrical impedance driving module and an electrical impedance amplifying module, wherein the electrical impedance driving module is electrically connected with a reference electrode of the multi-modal microelectrode array device, and the electrical impedance amplifying module is respectively electrically connected with the high-speed parallel data acquisition module and an electrode on the substrate of the multi-modal microelectrode array device; the electrophysiological signal conditioning module is electrically connected with the high-speed parallel data acquisition module, and the high-speed parallel data acquisition module is connected with the computer terminal through a signal wire. The invention can record the electric signal and the mechanical signal of single cells at multiple sites and high flux by detecting the integrated electro-mechanical signal by utilizing the multi-mode microelectrode array, and can realize the excitation-contraction coupling function of the cells.
Description
Technical Field
The invention relates to a sensing detection technology, in particular to a sensing detection method and a sensing detection system for exciting and contracting coupling of myocardial cells.
Background
The development of new drugs is an inefficient and time consuming process, typically requiring 12-15 years for the entire process, costing more than 10 billion dollars. In the united states, about 30% of drug candidates fail preclinical and early clinical stage assessments for cardiovascular safety reasons. Cardiotoxicity of drugs can cause cardiac dysfunction, and in severe cases, cardiac arrest. In addition, inefficient and inefficient drug screening tools increase the time and cost of drug development. Therefore, researchers have developed more preclinical cardiac safety models and techniques to improve drug screening. Among them, the cardiomyocyte-based model increasingly studies the potential mechanism or toxicity of the drug by measuring the electrical and mechanical properties of the cardiomyocytes.
The detection of electrical activity at the cellular level is key to understanding cellular processes. Patch clamp technology is the gold standard technique for studying cellular electrophysiology for decades and measures the high quality action potential of cardiomyocytes by forming a high seal and breaking the cell membrane. However, invasive patch clamp techniques typically record only a few hours and the complex procedure makes it difficult to record multiple cells simultaneously. On the other hand, studies of mechanical properties of cardiomyocytes are used to evaluate the state of contraction after an action potential. Ca 2+ Transient recording is monitoring intracellular Ca 2+ And conventional strategies reflecting the mechanical properties of cardiomyocytes, but fluorescence label-based techniques affect cell viability and limit recording time due to phototoxicity and drug side effects. In order to meet the requirements of high-throughput, sensitive and long-time multi-modal recording of the myocardial cells, a biosensing technology based on label-free and noninvasive myocardial cells is developed and applied to the research of the myocardial cells. Microelectrode arrays are increasingly used to record the electrophysiology of cardiomyocytes over a period of days or months. At the same time, an electrical impedance based interdigitated finger electrode and hybrid cantilever array with integrated strain sensors can be used to measure the mechanical contraction of the cardiomyocytes.
Most of the prior art monitors the electrical or mechanical signals of the cardiomyocytes separately and does not provide comprehensive correlation studies between the two properties. In order to simultaneously detect electrical and mechanical signals of cardiomyocytes, mechatronics recording has also been proposed by researchers to simultaneously monitor extracellular potential and mechanical pulsatile signals. However, the electrical signals and mechanical signals in mechatronic recording are rarely from the same cell or cell group due to the different positions of the electrical electrodes and the mechanical electrodes, and are difficult to accurately reflectSimultaneous physiological information of the same subject. In order to fully understand the electrical and mechanical signals of the same cell or cell group, Voltage Sensitive Dyes (VSDs) or Voltage Sensitive Fluorescent Proteins (VSFP) and Ca are used 2+ Sensitive dye binding for optical recording which tracks transmembrane voltage and Ca in a multi-site manner 2+ A change in transient signal. However, VSDs and Ca 2+ The disadvantages of sensitive dye phototoxicity and adverse drug reactions make the recording time only a few minutes, while VSFP is limited by the efficiency of the transgenic cell's optogenetic expression. Scanning probe microscopy probes have been reported to be used in conjunction with force-electrograms and force-controlled patch clamps for detecting electrical signals and mechanical contraction of cardiomyocytes. However, these methods cannot be widely used due to low throughput, complicated operation, and the like. Therefore, in the field of cardiology and pharmacology, there is still a high demand for the recording of high-throughput multimodal electrical and mechanical signals of single cells.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a sensing detection method and a sensing detection system for coupling excitation and contraction of myocardial cells.
In order to solve the technical problem, the solution of the invention is as follows:
the multi-mode microelectrode array device comprises a reference electrode, a plastic cover, a glass ring, a multi-mode microelectrode array chip and a PCB adapter which are sequentially arranged from top to bottom, wherein the multi-mode microelectrode array chip is fixed at the central position of the surface of the PCB adapter; the glass ring is fixed on the multi-mode microelectrode array chip, and the radial size of the glass ring is matched with the multi-mode microelectrode array chip to cover the multi-mode microelectrode array chip; the plastic cover is covered on the glass ring, the reference electrode is fixed on the plastic cover, and the tail end of the reference electrode extends into the glass ring; the multi-mode microelectrode array chip takes a glass plate as a substrate, a plurality of electrodes extending from the centers of four circumferential directions are uniformly arranged on the substrate along the circumferential direction, and the electrodes are insulated from each other; the surface of the PCB adapter is provided with a plurality of microstrip lines extending from the periphery to the center, the number of the microstrip lines is the same as that of the chip electrodes, one end of each microstrip line is correspondingly connected with the chip electrodes one by one, and the other end of each microstrip line is correspondingly and electrically connected with the pins of the PCB adapter one by one; the reference electrode is electrically connected with the pins of the PCB adapter through the pin headers.
As a preferable scheme of the invention, the substrate of the multi-modal microelectrode array chip is a square or round glass plate.
As a preferable scheme of the invention, the area of the multi-mode microelectrode array chip is 324-576 mm 2 The diameter of the electrodes is 5-15 μm, and the number of the electrodes is 24-60; the diameter of the effective electrode area is 5-15 μm, and the distance between adjacent electrodes is 200-500 μm.
As a preferable scheme of the invention, the multi-mode microelectrode array chip is fixed on the surface of the PCB adapter through polydimethylsiloxane, and the glass ring is fixed on the multi-mode microelectrode array chip through polydimethylsiloxane.
The invention further provides a sensing detection system for exciting and contracting coupling of the myocardial cells, which comprises the multi-mode microelectrode array device, a mechanical pulse conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module and a signal processing software functional module; wherein the content of the first and second substances,
the high-speed parallel data acquisition module comprises an analog-to-digital converter (ADC) and a microcontroller based on a Field Programmable Gate Array (FPGA);
the mechanical pulse conditioning module comprises an electrical impedance driving module and an electrical impedance amplifying module, wherein the electrical impedance driving module is electrically connected with a reference electrode of the multi-modal microelectrode array device, and the electrical impedance amplifying module is respectively electrically connected with the high-speed parallel data acquisition module and an electrode on the substrate of the multi-modal microelectrode array device; the impedance change of the myocardial cell under the set detection frequency is measured, and a mechanical pulse signal of the myocardial cell is obtained;
the electrophysiological signal conditioning module is electrically connected with the high-speed parallel data acquisition module and is used for amplifying and filtering electrophysiological signals generated after spontaneous beating of the myocardial cells, and sampling the electrophysiological signals through the high-speed parallel data acquisition module;
the microcontroller is connected with the computer terminal through a signal wire, and the signal processing software functional module is arranged in the computer terminal and used for processing the mechanical pulsation signal and the electrophysiological signal and extracting the characteristic points.
The invention further provides a sensing detection method for coupling excitation and contraction of the cardiac muscle cells, which comprises the following steps:
(1) sterilizing the multi-mode microelectrode array device by using ethanol and irradiating by using ultraviolet, coating the surface with 10ng/mL fibrin solution, and putting the device in an incubator at 37 ℃ for 4 hours to promote cell adhesion;
(2) cleaning and cutting fresh animal ventricular muscle tissue, and digesting with 0.07% trypsin/0.05% type II collagenase at 37 deg.C for 2 hr to obtain cell suspension; terminating digestion with a medium containing 10% fetal bovine serum, centrifuging, filtering, and collecting cells again; then purified cells are obtained after differential wall sticking treatment;
(3) planting purified cells in the glass ring of a multi-modal microelectrode array device at 37 ℃ and 5% CO 2 Culturing in an incubator;
(4) after the myocardial cells generate spontaneous pulsation, performing signal secondary amplification and filtering processing on electrophysiological signals of the myocardial cells by using an electrophysiological signal conditioning module, and then sampling by using a high-speed parallel data acquisition module;
meanwhile, the electrical impedance driving module generates an alternating current signal and acts on a reference electrode of the multi-mode microelectrode array device, and after alternating current flows out of the electrode on the substrate of the multi-mode microelectrode array device, the alternating current is converted into an alternating current voltage signal through the electrical impedance amplifying module and secondary high-pass filtering is carried out; finally, the amplified signal is sampled by a high-speed parallel data acquisition module, and the mechanical pulsation signal of the myocardial cell is obtained by measuring the impedance change of the myocardial cell under the set detection frequency;
(5) in the high-speed parallel data acquisition module, after an analog-to-digital converter (ADC) converts a voltage signal into a digital signal, the digital signal is transmitted to a computer terminal by a microcontroller through a TCP/IP protocol, baseline removal and filtering processing are performed by a built-in signal processing software functional module, and after characteristic points are extracted, the frequency and amplitude of an electric signal and a mechanical signal are respectively calculated.
In the step (2), the rotation speed during centrifugation is 1000rpm, and the centrifugation time is 5 minutes; a 70-micron cell filter screen is used for filtering; differential adherent treatment was performed twice for 45 minutes each.
As a preferred embodiment of the present invention, in the step (3), the purified cells are planted at a density of 2.0X 10 5 Cells/cm 2 。
As a preferable scheme of the invention, in the step (4), the sampling rate of the electrophysiological signal is 20kHz, and the band-pass rate is 1 Hz-5 kHz; the sensitive detection frequency of the mechanical beating signal is 10 kHz.
Compared with the prior art, the invention has the beneficial effects that:
1. in the prior art, the detection of an electrical signal or a mechanical signal of a myocardial cell can be realized respectively, but cannot be realized simultaneously; therefore, it is difficult to accurately reflect the simultaneous physiological information of the same subject, and a comprehensive correlation study between the two characteristics cannot be provided. The invention can record the electric signal and the mechanical signal of single cells at multiple sites and high flux by detecting the integrated electro-mechanical signal by utilizing the multi-mode microelectrode array, and can realize the excitation-contraction coupling function of the cells.
2. The multi-mode microelectrode array device used in the invention has simple manufacturing process and is beneficial to large-scale processing; due to the adoption of the photoetching, magnetron sputtering and stripping technologies compatible with large-scale processes, complex and time-consuming processes such as electron beam exposure, ion beam etching and the like are avoided, and the commercial manufacturing with low cost is facilitated.
3. The invention uses the multi-mode microelectrode array device for high-flux multi-site electric signal and mechanical pulse signal integrated detection, and can realize the evaluation of ion channel drugs. The recorded mechatronics signal can reflect the excitation-contraction coupling state of the myocardial cells based on the biological sensing system of the multi-mode microelectrode array device. Through highly detailed recording, the influence of the ion channel blocking drug on the mechatronic signal of the myocardial cells can be checked, and drug screening is facilitated.
Drawings
FIG. 1 is an optical microscope view of a multi-modal microelectrode array chip;
FIG. 2 is a layout view of a multi-modal microelectrode array chip;
FIG. 3 is a block diagram of a multi-modal microelectrode array device;
FIG. 4 is an exploded view of a multi-modal microelectrode array device;
FIG. 5 is a block flow diagram of a multimodal microelectrode biosensing system;
FIG. 6 is a single cell electro-mechanical signal integration record for a multi-modal microelectrode array device;
FIG. 7 shows the mechatronics signals of cells of different channels;
figure 8 is a single cell electro-mechanical signal integrated record to evaluate ion channel drug efficacy.
The reference numbers in fig. 4 are: the device comprises a reference electrode 1, a plastic cover 2, a glass ring 3, a multi-mode microelectrode array chip 4 and a PCB adapter 5.
Detailed Description
The invention relates to a database technology, and is an application of a computer technology in the technical field of information security. In the implementation process of the invention, the application of a plurality of software functional modules is involved. The applicant believes that it is fully possible for one skilled in the art to utilize the software programming skills in his or her own practice to implement the invention, as well as to properly understand the principles and objectives of the invention, in conjunction with the prior art, after a perusal of this application. The aforementioned software functional modules include but are not limited to: the signal processing software functional modules and the like are all referred to in the present application, and the applicant does not enumerate one by one.
Those skilled in the art will appreciate that, in addition to implementing a portion of the system and its various devices, modules, units provided by the present invention as pure computer readable program code, the system and its various devices, modules, units provided by the present invention can be implemented with the same functionality in the form of logic gates, switches, application specific integrated circuits, programmable logic controllers, embedded microcontrollers and the like, simply by logically programming the method steps. Therefore, the system and various devices, modules and units thereof provided by the invention can be regarded as a hardware component, and the devices, modules and units included in the system for realizing various functions can also be regarded as structures in the hardware component; means, modules, units for performing the various functions may also be regarded as structures within both software modules and hardware components for performing the method.
It is further noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in the process, method, article, or apparatus that comprises the element.
The following describes an implementation of the present invention in detail with reference to the accompanying drawings.
As shown in fig. 1-4, the multi-modal microelectrode array device comprises a reference electrode 1, a plastic cover 2, a glass ring 3, a multi-modal microelectrode array chip 4 and a PCB adapter 5, which are sequentially arranged from top to bottom, wherein the multi-modal microelectrode array chip 4 is fixed at the central position of the surface of the PCB adapter 5 through Polydimethylsiloxane (PDMS); the glass ring 3 is also fixed on the multi-modal microelectrode array chip 4 through polydimethylsiloxane, and the radial dimension of the glass ring is matched with the multi-modal microelectrode array chip 4 to cover the multi-modal microelectrode array chip; a plastic cover 2 is placed over the glass ring 3 and a reference electrode 1 is fixed to the plastic cover 2 with its end extending into the glass ring 3.
The multi-mode microelectrode array chip 4 takes a glass plate as a substrate, a plurality of electrodes extending from the centers of four circumferential directions are uniformly arranged on the substrate along the circumferential direction, and the electrodes are insulated from each other; a plurality of microstrip lines are arranged on the surface of the PCB adapter 5 in an extending manner from the periphery to the center, the number of the microstrip lines is the same as that of the chip electrodes, one end of each microstrip line is correspondingly connected with the chip electrodes one by one, and the other end of each microstrip line is correspondingly and electrically connected with the pins of the PCB adapter 5 one by one; the reference electrode 1 is electrically connected to the pins of the PCB adapter 5 through the pin header.
The substrate of the multi-mode microelectrode array chip 4 can be a square or round glass plate. As an optional scheme, the area of the multi-mode microelectrode array chip 4 is 324-576 mm 2 The diameter of the electrodes is 5-15 μm, and the number of the electrodes is 24-60; the diameter of the effective electrode area is 5-15 μm, and the distance between adjacent electrodes is 200-500 μm.
The invention further utilizes the multi-mode microelectrode array device to build a sensing detection system for exciting, contracting and coupling the myocardial cells. As shown in figure 5, the system comprises the multi-modal microelectrode array device, a mechanical pulsation conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module and a signal processing software functional module.
The mechanical pulse conditioning module comprises an electrical impedance driving module and an electrical impedance amplifying module, wherein the electrical impedance driving module is electrically connected with a reference electrode of the multi-modal microelectrode array device, and the electrical impedance amplifying module is respectively electrically connected with the high-speed parallel data acquisition module and an electrode on the substrate of the multi-modal microelectrode array device; the method is used for measuring the impedance change of the myocardial cell at a set detection frequency to obtain a mechanical pulse signal of the myocardial cell. The electrophysiological signal conditioning module is electrically connected with the high-speed parallel data acquisition module and is used for amplifying and filtering electrophysiological signals generated after spontaneous beating of the myocardial cells, and sampling the electrophysiological signals through the high-speed parallel data acquisition module;
the high-speed parallel data acquisition module comprises an analog-to-digital converter (ADC) and a microcontroller based on a Field Programmable Gate Array (FPGA); an analog-to-digital converter (ADC) converts the voltage signal to a digital signal, which is then transmitted to a microcontroller for subsequent signal processing. And finally, transmitting the signals to a signal processing software functional module in the computer terminal through a TCP/IP protocol, processing the mechanical pulse signals and the electrophysiological signals and extracting characteristic points.
Based on the sensing detection system, the sensing detection method for coupling excitation and contraction of the myocardial cells provided by the invention comprises the following steps of:
(1) sterilizing the multi-mode microelectrode array device by using ethanol and irradiating by using ultraviolet, coating the surface with 10ng/mL fibrin solution, and putting the device in an incubator at 37 ℃ for 4 hours to promote cell adhesion;
(2) cleaning and cutting fresh animal ventricular muscle tissue, and digesting with 0.07% trypsin/0.05% type II collagenase at 37 deg.C for 2 hr to obtain cell suspension; terminating digestion with a medium containing 10% fetal bovine serum, centrifuging, filtering, and collecting cells again; then purified cells are obtained after differential wall sticking treatment; the rotating speed during centrifugation is 1000rpm, and the centrifugation time is 5 minutes; a 70-micron cell filter screen is used for filtering; differential adherent treatment was performed twice for 45 minutes each.
(3) Planting purified cells in the glass ring of a multi-modal microelectrode array device at 37 ℃ and 5% CO 2 Culturing in an incubator; the planting density of the purified cells is 2.0 multiplied by 10 5 Cells/cm 2 。
(4) After the myocardial cells generate spontaneous pulsation, the electrophysiological signals of the myocardial cells are subjected to signal secondary amplification and filtering processing by the electrophysiological signal conditioning module, and then are sampled by the high-speed parallel data acquisition module. Meanwhile, the mechanical pulsation conditioning module is used for measuring the impedance change of the myocardial cells under the set detection frequency to obtain mechanical pulsation signals of the myocardial cells, and the high-speed parallel data acquisition module is used for sampling; wherein the sampling rate of the electrophysiological signals is 20kHz, and the band-pass rate is 1 Hz-5 kHz; the sensitive detection frequency of the mechanical beating signal is 10 kHz.
(5) In the high-speed parallel data acquisition module, after an analog-to-digital converter (ADC) converts a voltage signal into a digital signal, the digital signal is transmitted to a computer terminal by a microcontroller through a TCP/IP protocol, a built-in signal processing software functional module performs baseline removal and filtering processing, and after characteristic points are extracted, the frequency and amplitude of an electric signal and a mechanical signal are respectively calculated.
More detailed specific examples:
step 1:
the multi-mode microelectrode array chip is prepared by general photoetching, magnetron sputtering and stripping technologies.
An array pattern of 32 electrodes 10 μm in diameter was prepared on a 20mm by 1mm glass plate as a substrate. A positive photoresist RZJ-390PG-30 was spin coated at 3000rpm/min, exposed at a dose of 300mJ/cm2 at i-line (365nm), and then microelectrode patterned on the substrate with RZX3038 developer. After defining the microelectrode array pattern, the metal layer was prepared by magnetron sputtering deposition of 10nm Ti/100nm Au, acetone and ethanol stripping of the photoresist. The electrode leads were then insulated and defined an effective electrode area, 2 μm-think SU-82002 was spin coated onto the sample at 3000rpm/min on the electrode layer, exposed to light using a photolithography machine at a dose of 120mJ/cm2, developed in propylene glycol methyl ether acetate for 1 minute, and rinsed with isopropanol. Finally, by N 2 And hard baking at 150 ℃ for 30min to finish the preparation of the multi-mode microelectrode array chip.
As an example, the size of a single multi-modal microelectrode array chip is 20mm multiplied by 20mm, 32 electrodes are provided, the diameter of an electrode effective area is 10 μm, and the distance between adjacent electrodes is 300 μm.
Step 2:
and assembling the prepared multi-modal microelectrode array into a device.
The multi-modal micro-electrode array was fixed to a custom Printed Circuit Board (PCB) adapter with Polydimethylsiloxane (PDMS), and 32 electrode pads were bonded to the PCB pads with conductive silver paste. Next, a 1.4 cm diameter wide by 1 cm height glass ring was mounted in the center of the chip for cell culture, and the reference electrode was mounted on a plastic cover. And finally, the pins are welded on the PCB to adapt to an interface of the multi-mode microelectrode biological sensing system, so that the assembly of the multi-mode microelectrode array device is completed. The device consists of a glass ring, a multi-mode microelectrode array chip and a customized PCB. The glass ring is fixed above the electrode through PDMS for cell culture, and the multi-mode microelectrode array chip is communicated with the PCB to be accessed into an instrument to adapt to multi-mode cell electro-mechanical signal integrated detection.
And step 3:
establishment of a multi-modal biosensing system:
the hardware module of the multi-modal biological sensing system comprises an electrophysiological signal conditioning module, a mechanical pulse conditioning module and a high-speed parallel data acquisition module.
The high-speed parallel data acquisition module comprises an analog-to-digital converter (ADC) and a microcontroller based on a Field Programmable Gate Array (FPGA). The mechanical pulse conditioning module comprises an electrical impedance driving module and an electrical impedance amplifying module, wherein the electrical impedance driving module is electrically connected with a reference electrode of the multi-modal microelectrode array device, and the electrical impedance amplifying module is respectively electrically connected with the high-speed parallel data acquisition module and an electrode on the substrate of the multi-modal microelectrode array device; the method is used for measuring the impedance change of the myocardial cell at a set detection frequency to obtain a mechanical pulse signal of the myocardial cell. The electrophysiological signal conditioning module is electrically connected with the high-speed parallel data acquisition module, and is used for amplifying and filtering electrophysiological signals generated after spontaneous beating of the cardiac myocytes, and sampling the electrophysiological signals through the high-speed parallel data acquisition module. The microcontroller is connected with the computer terminal through a signal wire, and the signal processing software functional module is arranged in the computer terminal and used for processing the mechanical pulsation signal and the electrophysiological signal and extracting the characteristic points.
And 4, step 4:
culturing the myocardial cells on the multi-modal microelectrode array device. Before cell culture, the multi-mode microelectrode array device is sterilized by 75% ethanol, placed in a biological safety cabinet for 2 hours of ultraviolet irradiation, then 10ng/mL fibrin solution is covered on the microelectrode array device, and placed in an incubator at 37 ℃ for 4 hours to promote cell adhesion. Newborn SD rats 1-3 days old were sterilized, centrifuged from their hearts to ventricular muscle tissue, and washed in ice-cold medium to remove blood. Subsequently, the tissue was minced to about 1mm with scissors in ice cold balanced salt solution 3 The debris of (4) was digested with 0.07% trypsin/0.05% collagenase type II at 37 ℃ for 2 hours to form a cell suspension. Followed by a solution containing 10% fetal calf serumThe medium was digested, centrifuged at 1000rpm for 5 minutes, filtered through a 70 μm cell strainer, and the cells were collected again. After two differential adherents for 45 minutes, purified cells were obtained and planted in a multi-modal microelectrode array device at a density of 2.0X 105 cells/cm 2 and cultured in a 5% CO2 incubator at 37 ℃.
And 5:
recording of electrical signals and mechanical impulses was performed based on the prepared multi-modal microelectrode array devices and cultured cardiomyocytes. After the myocardial cells generate spontaneous pulsation (usually 2-3 days after culture), the multi-mode microelectrode biosensing system is used for carrying out the integrated detection of the electric-mechanical signals. The sampling rate of the electrophysiological signals is 20kHz, and the band-pass rate is 1 Hz-5 kHz. The sensitive detection frequency of the mechanical beating signal is 10 kHz.
After the myocardial cells generate spontaneous pulsation, the electrophysiological signals of the myocardial cells are subjected to signal secondary amplification and filtering processing by the electrophysiological signal conditioning module, and then are sampled by the high-speed parallel data acquisition module.
The mechanical beating signal of the cardiomyocyte is obtained by measuring the impedance change of the cell electrode at a sensitive detection frequency. Generating an alternating current signal through an electrical impedance driving module in the mechanical pulsation conditioning module and acting on a reference electrode of the multi-mode microelectrode array device; after alternating current flows out from an electrode on the substrate of the multi-mode microelectrode array device, the alternating current is converted into an alternating voltage signal through the electrical impedance amplification module, and secondary high-pass filtering is performed; and finally, sampling the amplified signals by a high-speed parallel data acquisition module. An analog-to-digital converter (ADC) in the high-speed parallel data acquisition module package converts the voltage signal into a digital signal, then transmits the digital signal to a microcontroller for subsequent signal processing, transmits the signal to a signal processing software function module arranged in a computer for baseline removal and filtering processing through a TCP/IP protocol, and respectively calculates the frequency and amplitude of an electric signal and a mechanical signal after extracting characteristic points.
Figure 6 shows the mechatronic signal recorded from day 2 to day 4. The electrophysiological signal and the mechanical pulse signal appear on almost the same day (day 3) according to the excitation-contraction coupling of the cardiomyocytes. The amplitude of the electrophysiological signal gradually increases, and the discharge frequency tends to be rhythmic and stable. The amplitude and pulse rate of the mechanical pulse signal appeared large and stable on days 3 and 4. The cardiomyocyte mechatronic model at day 4 is suitable for obtaining high-quality mechatronic signals.
Based on the multi-modality device configuration, each channel is capable of simultaneously recording electrophysiological and mechanical pulsatile signals from the same cardiomyocyte. The high throughput and consistent nature of the electromechanically integrated signals recorded by the multimodal devices allows continuous recording on a multi-site single unit. FIG. 7 shows a typical simultaneous mechatronic recording using three different channels under the same culture conditions.
Step 6:
the multi-modal microelectrode biosensor system evaluates the influence of ion channel blocking drugs on the mechatronic signals of the myocardial cells: flucalix (a Na + channel blocker) is selected as a tool drug to test the multi-modal microelectrode biosensing system. Frakaempde is a typical Na + channel blocker and can reduce the frequency and amplitude of action potential of cardiomyocytes during rapid depolarization, thereby significantly reducing contractility of cardiomyocytes during mechanical beating. The control group recorded the electromechanical signal in the absence of the drug. The addition of different concentrations of drug as tested in the experiment, the peak-to-peak separation of the electromechanical signal under the drug action was extended and a dose-dependent response was shown (as shown in figure 8).
Claims (9)
1. A multi-mode microelectrode array device is characterized by comprising a reference electrode, a plastic cover, a glass ring, a multi-mode microelectrode array chip and a PCB adapter which are sequentially arranged from top to bottom, wherein the multi-mode microelectrode array chip is fixed at the central position of the surface of the PCB adapter; the glass ring is fixed on the multi-mode microelectrode array chip, and the radial size of the glass ring is matched with the multi-mode microelectrode array chip to cover the multi-mode microelectrode array chip; the plastic cover is covered on the glass ring, the reference electrode is fixed on the plastic cover, and the tail end of the reference electrode extends into the glass ring;
the multi-mode microelectrode array chip takes a glass plate as a substrate, a plurality of electrodes extending from the centers of four circumferential directions are uniformly arranged on the substrate along the circumferential direction, and the electrodes are insulated from each other; the surface of the PCB adapter is provided with a plurality of microstrip lines extending from the periphery to the center, the number of the microstrip lines is the same as that of the chip electrodes, one end of each microstrip line is correspondingly connected with the chip electrodes one by one, and the other end of each microstrip line is correspondingly and electrically connected with the pins of the PCB adapter one by one; the reference electrode is electrically connected with the pins of the PCB adapter through the pin headers.
2. The multi-modal microelectrode array device of claim 1, wherein the substrate of the multi-modal microelectrode array chip is a square or circular glass plate.
3. The multi-modal microelectrode array device of claim 1, wherein the multi-modal microelectrode array chip has an area of 324-576 mm 2 The diameter of the electrodes is 5-15 μm, and the number of the electrodes is 24-60; the diameter of the effective electrode area is 5-15 μm, and the distance between adjacent electrodes is 200-500 μm.
4. The multi-modal microelectrode array device of claim 1, wherein the multi-modal microelectrode array chip is attached to the surface of the PCB adapter by polydimethylsiloxane, and the glass ring is attached to the multi-modal microelectrode array chip by polydimethylsiloxane.
5. A sensing detection system coupled with excitation and contraction of myocardial cells is characterized by comprising the multi-mode microelectrode array device as claimed in claim 1, a mechanical pulse conditioning module, an electrophysiological signal conditioning module, a high-speed parallel data acquisition module and a signal processing software function module; wherein the content of the first and second substances,
the high-speed parallel data acquisition module comprises an analog-to-digital converter (ADC) and a microcontroller based on a Field Programmable Gate Array (FPGA);
the mechanical pulse conditioning module comprises an electrical impedance driving module and an electrical impedance amplifying module, wherein the electrical impedance driving module is electrically connected with a reference electrode of the multi-modal microelectrode array device, and the electrical impedance amplifying module is respectively electrically connected with the high-speed parallel data acquisition module and an electrode on the substrate of the multi-modal microelectrode array device; the impedance change of the myocardial cell under the set detection frequency is measured, and a mechanical pulse signal of the myocardial cell is obtained;
the electrophysiological signal conditioning module is electrically connected with the high-speed parallel data acquisition module and is used for amplifying and filtering electrophysiological signals generated after spontaneous beating of the myocardial cells, and sampling the electrophysiological signals through the high-speed parallel data acquisition module;
the microcontroller is connected with the computer terminal through a signal wire, and the signal processing software functional module is arranged in the computer terminal and used for processing the mechanical pulsation signal and the electrophysiological signal and extracting the characteristic points.
6. A sensing detection method for coupling excitation and contraction of myocardial cells is characterized by comprising the following steps:
(1) sterilizing the multi-mode microelectrode array device by using ethanol and irradiating by using ultraviolet, coating the surface with 10ng/mL fibrin solution, and putting the device in an incubator at 37 ℃ for 4 hours to promote cell adhesion;
(2) cleaning and cutting fresh animal ventricular muscle tissue, and digesting with 0.07% trypsin/0.05% collagenase II at 37 deg.C for 2 hr to obtain cell suspension; terminating digestion with a medium containing 10% fetal bovine serum, centrifuging, filtering, and collecting cells again; then purified cells are obtained after differential wall sticking treatment;
(3) planting purified cells in the glass ring of a multi-modal microelectrode array device at 37 ℃ and 5% CO 2 Culturing in an incubator;
(4) after the myocardial cells generate spontaneous pulsation, performing signal secondary amplification and filtering processing on electrophysiological signals of the myocardial cells by using an electrophysiological signal conditioning module, and then sampling by using a high-speed parallel data acquisition module;
meanwhile, the electrical impedance driving module generates an alternating current signal and acts on a reference electrode of the multi-mode microelectrode array device, and after alternating current flows out of the electrode on the substrate of the multi-mode microelectrode array device, the alternating current is converted into an alternating current voltage signal through the electrical impedance amplifying module and secondary high-pass filtering is carried out; finally, the amplified signal is sampled by a high-speed parallel data acquisition module, and the mechanical pulsation signal of the myocardial cell is obtained by measuring the impedance change of the myocardial cell under the set detection frequency;
(5) in the high-speed parallel data acquisition module, after an analog-to-digital converter (ADC) converts a voltage signal into a digital signal, the digital signal is transmitted to a computer terminal by a microcontroller through a transmission control protocol/internet protocol (TCP/IP), a built-in signal processing software function module performs baseline removal and filtering processing, and after characteristic points are extracted, the frequency and amplitude of an electric signal and a mechanical signal are respectively calculated.
7. The method according to claim 6, wherein in the step (2), the rotation speed at the time of centrifugation is 1000rpm, and the centrifugation time is 5 minutes; a 70-micron cell filter screen is used for filtering; differential adherent treatment was performed twice for 45 minutes each.
8. The method according to claim 6, wherein in the step (3), the purified cells are planted at a density of 2.0X 10 5 Cells/cm 2 。
9. The method according to claim 6, wherein in step (4), the electrophysiological signal has a sampling rate of 20kHz, a bandpass rate of 1Hz to 5 kHz; the sensitive detection frequency of the mechanical beating signal is 10 kHz.
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