Physiological autophagy detection method based on myocardial cell sensing system

Abstract

The invention discloses a pathological autophagy detection method based on a myocardial cell sensing system. The pathological autophagy detection method comprises the following steps: constructing a pathological autophagy detection platform based on the myocardial cell sensing system, performing surface modification on a microelectrode array chip, culturing a cell autophagy sensitive element and performing in-vitro pathological autophagy detection. Through primary myocardial cell culture, rapamycin action and electrophysiological signal acquisition, time domain and frequency domain characteristics of myocardial cell extracellular local field potential signals are recorded and analyzed in real time, and the autophagy degree induced by rapamycin is judged. According to the method, the signal-to-noise ratio is improved through platinum black nanoparticle modification, it is ensured that the myocardial cell growth activity meets the detection requirement, the half inhibitory concentration of rapamycin is obtained through nonlinear regression analysis, and an effective means is provided for pathological autophagy research. The autophagy state can be obtained in real time without damaging the cell structure; any antigen or fluorescent molecular marker is not needed, so that cytotoxicity is avoided, and long-time monitoring is realized.

Description

Technical Field

[0001] The present invention relates to the technical field of micro-nano sensing and cell detection, and particularly to a method for detecting pathological autophagy based on a cardiomyocyte sensing system. Background Art

[0002] Autophagy is a biological mechanism for maintaining intracellular homeostasis. It helps cells adapt to various external stimuli by degrading proteins and polypeptides into small molecules such as amino acids and polysaccharides, thereby maintaining the dynamic balance of the body. Basic cellular autophagy is a key mechanism for the heart to maintain internal homeostasis, and can recycle and remove damaged proteins or organelles. However, excessive activation of autophagy will degrade normal organelles in the cell, thereby causing continuous damage to cardiomyocytes, leading to autophagic death, and ultimately resulting in heart failure. Therefore, rapid and real-time detection of the autophagy state of cardiomyocytes is particularly important for the discovery and treatment of related heart diseases, in order to avoid irreversible autophagic death of cardiomyocytes and provide reliable data support for the prognosis guidance of clinical patients.

[0003] Traditional autophagy detection methods, such as Western blotting to detect the expression levels of LC3 or p62 proteins, fluorescence protein labeling of the localization and expression of LC3 in cells, or transmission electron microscopy to observe the formation of autophagosomes in cells, etc., all require treating cells at a specific time point after autophagy induction, and then characterizing the autophagy level through various cell biology experiments. Moreover, the experimental period usually lasts from several hours to several days, and corresponding data cannot be obtained quickly in a timely manner. Similarly, the autophagy process or autophagy level cannot be monitored in real time. Therefore, developing an immediate, rapid and more intuitive method for characterizing cell autophagy will significantly improve the work efficiency of autophagy-related research.

[0004] The present invention uses cardiomyocytes as sensing elements, combines with a microelectrode array chip, and monitors the entire autophagy process in a real-time, continuous and non-invasive manner, providing a new method for detecting pathological cell autophagy. Summary of the Invention

[0005] The purpose of the present invention is to provide a method for detecting pathological autophagy based on a cardiomyocyte sensing system in view of the deficiencies of the prior art.

[0006] The purpose of the present invention is achieved by the following technical solutions:

[0007] A method for detecting pathological autophagy based on a cardiomyocyte sensing system, which is implemented on a platform for detecting pathological autophagy based on a cardiomyocyte sensing system. The platform includes a cell electrophysiological sensing device, a signal acquisition module, a signal amplification module, an upper computer and a cell incubator; wherein,

[0008] The cell electrophysiological sensing device includes a hollow glass culture chamber, a microelectrode array chip, and a PCB adapter; the signal acquisition module is connected to the signal amplification module through a long shielded cable; the signal amplification module is connected to the upper computer through a data connection line; after the cell electrophysiological sensing device is inserted into the signal acquisition module, it is placed in a cell culture incubator for collecting myocardial cell electrophysiological signals;

[0009] The method includes the following steps:

[0010] (1) Build a pathological autophagy detection platform based on the myocardial cell sensing system: Place the metal box with the cell electrophysiological sensing device and the signal acquisition module in the cell culture incubator, and place the metal box containing the signal amplification module outside the cell culture incubator; the two metal boxes are connected through a long shielded cable to achieve signal transmission;

[0011] (2) Surface modification of the microelectrode array chip: Coating gelatin or fibronectin on the surface of the microelectrode array chip to improve the signal-to-noise ratio of the signals detected by the microelectrode array chip by enhancing the coupling degree between the microelectrode array chip and myocardial cells;

[0012] (3) Cultivation of cell autophagy sensitive elements: Obtain primary myocardial cells by primary cell extraction method and prepare a primary myocardial cell suspension; then plant the primary myocardial cell suspension in the culture chamber of the microelectrode array chip, and the myocardial cells are evenly distributed on or around the electrode points of the microelectrode array chip;

[0013] (4) In vitro pathological autophagy detection: After treating myocardial cells with rapamycin at any concentration in the range of 0.01 - 1 mM, use a multi-channel in vitro electrophysiological signal acquisition system to record the changes in the extracellular local field potential signals of myocardial cells on the microelectrode array chip in real time; extract the time-domain and frequency-domain characteristics of the detected potential signals and perform normalization processing to obtain the concentration-dependent response curve under the stimulation of this concentration of rapamycin, and judge the degree of autophagy induced by this concentration of rapamycin according to the potential signal attenuation trend shown by the curve.

[0014] Further, in the step (1), the signal acquisition module uses a multi-channel in vitro electrophysiological signal acquisition system for signal acquisition and recording. The extracellular electrophysiological signals of myocardial cells at different sites are synchronously recorded by multiple working electrodes. The signals are amplified and filtered by the signal amplification module, and then transmitted to the upper computer by a data acquisition card for offline analysis.

[0015] Further, in the step (2), the surface modification of the microelectrode array chip further includes: electroplating platinum black nanoparticles on the electrode surface of the microelectrode array chip through a three-electrode system of an 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 of the signals detected by the microelectrode array chip.

[0016] Further, in the step (2), the surface of the microelectrode array chip is coated with 1% gelatin, and then placed in an incubator for incubation for 2 - 4 h; the gelatin is completely aspirated before inoculating cells.

[0017] Further, in the step (3), the seeding density of the cardiomyocyte suspension in the culture chamber is 3.0×10 5 ~4.0×10 5 cells / cm 2 ; after seeding, ensure that the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip; change the culture medium every 24 h.

[0018] Further, in the step (3), after the cardiomyocytes are seeded in the microelectrode array chip, a live / dead cell staining method is used to determine the growth activity of the cardiomyocytes on the microelectrode array chip. If the survival rate reaches more than 80%, the requirements for cell sensing detection are met; otherwise, re - culture is required.

[0019] Further, in the step (4), the time - domain and frequency - domain features include: the amplitude and frequency of the electrophysiological signal; the half - maximal inhibitory concentration of rapamycin on the electrophysiological signal of cardiomyocytes is obtained through non - linear regression analysis.

[0020] The beneficial effects of the present invention are as follows: the detection method of the present invention can realize long - time continuous monitoring of cardiomyocytes in real - time, non - invasive, high - throughput, and high - sensitivity manner. The detection method of the present invention can obtain the autophagy state in real - time without destroying the cell structure; without any antigen or fluorescent molecule labeling, thus avoiding cytotoxicity to achieve long - time monitoring. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The present invention will be further described below with reference to the drawings and embodiments:

[0022] Figure 1 is a schematic diagram of the composition of the cardiomyocyte sensing platform of the present invention;

[0023] Figure 2 is a graph of the change in the electrophysiological signal of cardiomyocytes before and after adding rapamycin;

[0024] Figure 3 is a statistical chart of the characteristic parameters of the electrophysiological signal of cardiomyocytes;

[0025] Figure 4 is the IC 50 of rapamycin on the amplitude signal of cardiomyocytes;

[0026] Figure 5 is the IC 50 of rapamycin on the firing frequency of cardiomyocytes.

[0027] In the figure, there are a sensing device 1, a signal acquisition module 2, a signal amplification module 3, a host computer 4, and a cell incubator 5. Detailed implementation mode

[0028] In the present invention, the structure, preparation method and usage method of the cell electrophysiological sensing device are all based on the previous work results of the applicant. Specifically, reference can be made to the description of the "nano microelectrode array device" in the published patent application document "A sensing detection method and system for myocardial cell excitation-contraction coupling" (CN115078466A). Those skilled in the art can make detailed adjustments according to the specific experimental scheme design when reproducing the technical solution of the present invention, and the present invention will not be elaborated herein.

[0029] The following will describe the implementation mode of the present invention in detail with reference to the drawings, but it does not limit the present invention.

[0030] I. Experimental device of the present invention

[0031] As Figure 1 shown, a pathological autophagy detection platform based on a myocardial cell sensing system provided by the present invention includes a cell electrophysiological sensing device 1, a signal acquisition module 2, a signal amplification module 3, a host computer 4, and a cell incubator 5. Among them, the cell electrophysiological sensing device 1 is a technology recorded in existing published literature, including a hollow glass culture cavity, a microelectrode array chip, and a PCB adapter; the structure and processing method of this sensing device can be specifically referred to the patent application published document CN115078466A. The signal acquisition module 2 is connected to the signal amplification module 3 through a long shielded cable, and the signal amplification module 3 is connected to the host computer by a data connection line. After the cell electrophysiological sensing device 1 is inserted into the signal acquisition module 2, it can be placed in the cell incubator 5 for long-term acquisition of myocardial cell electrophysiological signals.

[0032] II. Detection method of the present invention

[0033] Based on the above experimental device, the present invention realizes a detection method for pathological autophagy, which specifically includes the following steps:

[0034] (1) Construction of the myocardial cell sensing platform

[0035] Place the metal box with the cell electrophysiological sensing device 1 and the signal acquisition module 2 in the cell incubator 5, and place the metal box containing the signal amplification module 3 outside the cell incubator 5; the two metal boxes are connected by a long shielded cable to achieve signal transmission. Among them, the signal acquisition module uses a multi-channel in vitro electrophysiological signal acquisition system to collect and record signals. The extracellular electrophysiological signals of cardiomyocytes at different sites are synchronously recorded by multiple working electrodes. The signals are amplified 500 times by the signal amplification module, filtered (<7.5 kHz), sampled at a frequency of 20 kHz, and then transmitted to the upper computer 4 by the data acquisition card for offline analysis.

[0036] (2) Surface modification of the microelectrode array chip

[0037] Coat the surface of the microelectrode array chip with gelatin or fibronectin to improve the signal-to-noise ratio of the signals detected by the microelectrode array chip by enhancing the coupling degree between the microelectrode array chip and cardiomyocytes.

[0038] As an example, coat the surface of the microelectrode array chip with 1% gelatin, and then place it in the incubator for 2 - 4 h; suck out the gelatin before inoculating cells.

[0039] Furthermore, when performing surface modification of the microelectrode array chip, platinum black nanoparticles (PtNPs) can also be electroplated on the electrode surface of the microelectrode array chip through a three-electrode system of an 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 of the signals detected by the microelectrode array chip.

[0040] (3) Cultivation of autophagy-sensitive elements in vitro

[0041] Obtain primary cardiomyocytes by primary cell extraction method and prepare a primary cardiomyocyte suspension; then plant the primary cardiomyocyte suspension in the culture chamber of the microelectrode array chip, and the cardiomyocytes are evenly distributed on or around the electrode points of the microelectrode array chip.

[0042] As an example, the seeding density of the cardiomyocyte suspension in the culture chamber is 3.0×10 5 ~4.0×10 5 cells / cm 2 ; after seeding, ensure that the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip; change the culture medium every 24 h.

[0043] Furthermore, after planting cardiomyocytes in the microelectrode array chip, a live / dead cell staining method needs to be used to determine the growth activity of cardiomyocytes on the microelectrode array chip. Those with a survival rate of more than 80% can meet the requirements of cell sensing detection, otherwise, they need to be cultured again.

[0044] (4) Detection of pathological autophagy in vitro

[0045] After treating cardiomyocytes with Rapamycin, the changes in the extracellular local field potential signals of cardiomyocytes on the microelectrode array chip were recorded in real time through a multi-channel in vitro electrophysiological signal acquisition system. As an example, the Rapamycin concentration range was 0.01 - 1 mM.

[0046] Extract the time-domain and frequency-domain characteristics of the detected potential signals, namely amplitude and firing frequency, and perform normalization processing. That is, the electrical signal value collected before Rapamycin treatment is denoted as X 0 , and the electrical signal value collected at time t after treatment is denoted as X t , and after normalization calculation, the electrical signal value X nor = X t / X 0 , so as to eliminate the differences in electrical signals between different channels or different batches of cells, and thus obtain the concentration-dependent response curve under the stimulation of this concentration of Rapamycin. According to the decay trend of the potential signal shown in the curve, judge the degree of autophagy induced by this concentration of Rapamycin.

[0047] Furthermore, the time-domain and frequency-domain characteristics include: the amplitude and frequency of the electrophysiological signal; the half maximal inhibitory concentration (IC 50 ) of Rapamycin on the electrophysiological signal of cardiomyocytes obtained through non-linear regression analysis.

[0048] Example

[0049] 1. Fabrication of the cell electrophysiological sensing device

[0050] Spin-coat photoresist onto a 16 square-inch quartz glass substrate, and then bake it at 120 °C for 2 min. Before depositing Au / Ti (100 / 10 nm) on the substrate, remove the photoresist using acetone. After lithography, an insulating SU-8 layer is formed, post-baked at 95 °C, and developed using propylene glycol monomethyl ether acetate. Divide the glass substrate into 25 device units, each unit having a size of 2 × 2 cm 2 , where each unit houses 32 microelectrodes. The components of the device include a printed circuit board (PCB) adapter, a cell culture glass ring, and a sensor chip. Fix a culture ring with a diameter of 1.4 cm and a height of 1 cm to the center of the sensor chip using polydimethylsiloxane (PDMS). Then bond the electrode array chip to the PCB adapter with PDMS, and ensure the electrical connection between all electrode pads and their corresponding PCB pads through a combination of silver conductive adhesive and gold bonding wires. Finally, solder the pin headers to the PCB to adapt to the interface of the electrophysiological recording system.

[0051] 2. Primary cardiomyocyte culture

[0052] Cardiomyocytes were isolated from 1-day-old Sprague Dawley rats. The ventricular tissues were dissected rapidly and rinsed three times in cold Dulbecco's Modified Eagle Medium (DMEM). The cardiac tissues were pre-cut into fragments of approximately 1 mm 3 in cold Hank's Balanced Salt Solution (HBSS), and then digested in HBSS containing 0.05% type II collagenase and 0.07% trypsin, with the tissue digestion enzymes being replaced every 8 minutes. After digestion, the dissociated cells in the loose tissue were collected by centrifugation at 1000 rpm for 5 min. Subsequently, the cell suspension was filtered through a 70-μm cell strainer. Cardiomyocytes were further purified by differential adhesion for two 45-minute periods to remove other cells. The purified cardiomyocytes were seeded onto a microelectrode array (MEA) device at a density of 3.5×10 5 cells / cm 2 . The culture medium used was high-glucose DMEM containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin. The instrument was placed in an incubator at 37 °C and 5% CO 2 for long-term monitoring to ensure an appropriate culture environment. The culture medium was renewed every 24 hours.

[0053] 3. Detection of pathological autophagy in cardiomyocytes

[0054] When the cardiomyocytes had grown on the microelectrode array chip for 2 - 3 days, rapamycin was added to the culture medium to mimic the occurrence of autophagy. Electrophysiological signals were recorded through the cardiomyocyte sensing platform to evaluate the degree of autophagy in the cells. The specific steps were as follows:

[0055] 3.1 Record the electrophysiological signals of normal cardiomyocytes for 1 min as a control.

[0056] 3.2 Add rapamycin at final concentrations of 0.01 mM, 0.1 mM, and 1 mM to the cell culture chambers respectively to induce autophagy.

[0057] 3.3 Refer to Figure 2 and Figure 3 , and record the electrophysiological signals of cardiomyocytes at different rapamycin concentrations every 5 min ( Figure 2 ), and extract the characteristic parameters of the electrophysiological signals ( Figure 3 ) for analyzing the effects of different autophagy degrees on the electrophysiological activity of cardiomyocytes.

[0058] 3.4 Refer to Figure 4 and Figure 5, according to the changes in the amplitude and firing frequency of cardiomyocytes at different time points, the IC of rapamycin at the corresponding time points was calculated 50 , indicating that this platform can quantitatively analyze the autophagy level of cardiomyocytes ( Figure 4 and Figure 5 ).

[0059] The above embodiments are used to explain the present invention, rather than limiting the present invention. Any modifications and changes made to the present invention within the spirit and scope of the claims of the present invention fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent shall be subject to the appended claims.

Claims

1. A method for detecting pathological autophagy based on a cardiomyocyte sensing system, characterized in that: The method is implemented on a pathological autophagy detection platform based on a cardiomyocyte sensing system, wherein the platform includes a cell electrophysiological sensing device, a signal acquisition module, a signal amplification module, a host computer and a cell culture box; wherein, The cell electrophysiological sensing device comprises a hollow glass culture chamber, a microelectrode array chip and a PCB adapter; the signal acquisition module is connected to the signal amplification module through a long shielded cable; the signal amplification module is connected to a host computer through a data connection line; after the cell electrophysiological sensing device is inserted into the signal acquisition module, it is placed in a cell culture box to collect electrophysiological signals of myocardial cells; The method comprises the following steps: (1) Build a pathological autophagy detection platform based on the cardiomyocyte sensing system: place a metal box with a cell electrophysiological sensing device and a signal acquisition module in a cell culture incubator, and place a metal box containing a signal amplification module outside the cell culture incubator; the two metal boxes are connected by a long shielded cable to achieve signal transmission; (2) Surface modification of microelectrode array chips: coating the surface of microelectrode array chips with gelatin or fibronectin to enhance the coupling between microelectrode array chips and cardiomyocytes so as to improve the signal-to-noise ratio of the detection signal of the microelectrode array chips; (3) Cultivation of cell autophagy sensitive elements: primary cardiomyocytes are obtained by primary cell extraction to prepare a primary cardiomyocyte suspension; the primary cardiomyocyte suspension is then planted in a microelectrode array chip culture chamber, and the cardiomyocytes are evenly distributed on or around the electrode points of the microelectrode array chip; (4) In vitro pathological autophagy detection: After the cardiomyocytes are treated with any concentration of rapamycin in the concentration range of 0.01 to 1 mM, the changes in the extracellular local field potential signals of the cardiomyocytes on the microelectrode array chip are recorded in real time by a multi-channel in vitro electrophysiological signal acquisition system; the time domain and frequency domain characteristics of the detected potential signal are extracted and normalized to obtain the concentration-dependent response curve under the stimulation of the concentration of rapamycin, and the degree of autophagy induced by the concentration of rapamycin is determined based on the attenuation trend of the potential signal shown in the curve.

2. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In the step (1), the signal acquisition module uses a multi-channel in vitro electrophysiological signal acquisition system to collect and record signals. The extracellular electrophysiological signals of cardiomyocytes at different sites are synchronously recorded by multiple working electrodes. The signals are amplified and filtered by the signal amplification module and then transmitted to the host computer by the data acquisition card for offline analysis.

3. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In the step (2), the surface modification of the microelectrode array chip also includes: electroplating platinum black nanoparticles on the electrode surface of the microelectrode array chip through an electrochemical workstation three-electrode system to increase the specific surface area of ​​the electrode, reduce the electrode impedance, thereby reducing noise and improving the signal-to-noise ratio of the microelectrode array chip detection signal.

4. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In the step (2), the surface of the microelectrode array chip is coated with 1% gelatin and then placed in an incubator for incubation for 2-4 hours; the gelatin is completely absorbed before inoculating cells.

5. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In step (3), the planting density of the cardiomyocyte suspension in the culture chamber is 3.0×10 5 ~4.0×10 5 cells / cm 2 ; After implantation, ensure that the cardiomyocytes are evenly distributed on the surface of the microelectrode array chip; replace the culture medium every 24 hours.

6. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In the step (3), after the cardiomyocytes are planted in the microelectrode array chip, a live cell / dead cell staining method is used to determine the growth activity of the cardiomyocytes on the microelectrode array chip. If the survival rate reaches more than 80%, the cell sensing detection requirement is met, otherwise the cells are re-cultured.

7. The method for detecting pathological autophagy based on a cardiomyocyte sensing system according to claim 1, characterized in that: In the step (4), the time domain and frequency domain characteristics include: the amplitude and frequency of the electrophysiological signal; and the half-maximal inhibitory concentration of rapamycin on the electrophysiological signal of myocardial cells is obtained by nonlinear regression analysis.

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