A flexible bioelectronic device for detecting cardiac cell contraction function and its preparation method

Abstract

The present invention discloses a flexible bioelectronic device for detecting the contractile function of myocardial cells and a preparation method thereof. The bioelectronic device comprises, from bottom to top, a substrate, a first film layer, a second film layer, a strain sensing system, an insulating film layer, electrodes, and a cell culture device, successfully integrating a cell culture chamber, a biosensor, and a bioreactor into one system. In the above-mentioned bioelectronic device, the strain sensing system can measure the dynamic changes of the contractile force of myocardial cells in situ; there are air channels and hollow chambers between the substrate and the first film layer, which are used for pneumatic drive of the second film layer, thereby providing mechanical stimulation to the cells; the electrodes are arranged in pairs and in contact with the cell culture device, and are used to provide electrical stimulation to the cells. The above-mentioned flexible bioelectronic device can perform high-throughput contractility measurements on myocardial cells under specified microenvironmental conditions, and has good application prospects in high-throughput cardiac drug screening and in vitro cell culture.

Description

Technical Field

[0001] The present invention relates to the technical field of myocardial cell sensors, and in particular to a flexible bioelectronic device for detecting the contractile function of myocardial cells and a preparation method thereof. Background Art

[0002] Heart disease and heart failure are the leading causes of death worldwide, and healthcare expenditures are increasing dramatically. As the population continues to age, the incidence of heart disease is expected to rise dramatically. The development of in vitro cardiac models, heart-on-a-chip platforms, and biosensor technologies is crucial to accelerating cardiac drug discovery for treating heart disease. Traditional in vitro drug screening assays typically rely on neonatal animal cardiac cells or standard cell lines, such as HL-1 and H9c2. However, these cell sources exhibit differences in beating physiology, proteomics, and gene expression compared to human cardiomyocytes, resulting in poorly reliable reflection of human cardiac physiology, low accuracy in drug evaluation, off-target cardiotoxicity, and failure in late-stage clinical testing. Recent breakthroughs in somatic cell reprogramming and induced stem cell-derived cardiomyocytes (iPSC-CMs) have been applied to generate reliable induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) with a human proteome, ion channels, metabolism, and contractile phenotype, enabling the establishment of accurate in vitro cardiac models based on human cardiomyocytes. There is growing interest in using iPSC-CMs as more accurate models to understand cardiac physiology, investigate underlying disease mechanisms, and test human responses to potential drugs. Although iPSC-derived cardiomyocyte differentiation has been achieved and standardized, a major drawback of iPSC-derived cardiomyocytes is their immature phenotype, resembling human cardiomyocytes at the fetal or neonatal stage of development. This maturation defect in iPSC-CMs hinders accurate testing of adult cardiac responses to candidate drugs and also poses a risk of pro-arrhythmia during regenerative cell therapy. Therefore, the development of a platform technology to achieve in vitro microenvironment-induced maturation of iPSC-CMs, bringing their maturity close to that of native adult cardiomyocytes, is urgently needed.

[0003] Patients with congenital or acquired heart disease often experience contractile abnormalities, manifesting as weak contractility, fibrillation, and rhythm disturbances. Therefore, developing biosensing technologies to quantify contractility changes in in vitro cardiac models is crucial for analyzing cardiomyocyte contractile function and testing the therapeutic efficacy or potential cardiotoxicity of candidate drugs. To date, several biosensing technologies / platforms have been developed to measure contractility in single cardiomyocytes, 2D cell monolayers, and 3D in vitro cardiac tissue. Examples include microscopy-based analysis of dynamic mechanical motion of cardiomyocytes, atomic force microscopy (AFM), interdigitated electrode impedance changes induced by cardiomyocyte beating, traction force microscopy, flexible films, flexible cantilevers, or optical measurement of tissue wire (biowire) deflection. Piezoresistive strain sensors have also been integrated into flexible structures to enable continuous electrical readout of cardiomyocyte contraction. Despite significant progress, challenges remain, including improving device sensitivity to accurately capture weak contractile signals generated by in vitro cardiac models, increasing the capacity of high-throughput drug testing platforms, and integrating stimulation capabilities to investigate the causal relationship between cardiomyocyte development / maturation and microenvironmental factors. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a flexible bioelectronic device for detecting the contractile function of myocardial cells and a preparation method thereof. The present invention integrates a cell culture chamber, a biosensor and a bioreactor into an electronic device. The electronic device can provide controllable mechanical / electrical microenvironmental factors during the cell culture process, and at the same time realize continuous high-throughput measurement of the contractile function of monolayer myocardial cells in vitro, which can be used for in vitro myocardial cell culture and high-throughput cardiac drug screening.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A first aspect of the present invention provides a flexible bioelectronic device for detecting the contractile function of cardiac cells, the flexible bioelectronic device comprising:

[0007] substrate;

[0008] A first film layer is disposed above the substrate and bonded to the substrate, wherein one or more grooves are provided on a surface of the first film layer bonded to the substrate, each groove having one or more through holes extending through the first film layer, an air passage is formed between the grooves and the through holes and the substrate, and a hollow chamber is formed between the through holes and the substrate;

[0009] a second film layer, disposed above the first film layer and bonded to the first film layer;

[0010] a strain sensing system comprising one or more strain sensors fixedly disposed on a side of the second film layer away from the first film layer, wherein the strain sensors correspond one to one with the through holes and are located directly above the corresponding through holes;

[0011] an insulating film layer, disposed above the strain sensor and in contact with the second film layer;

[0012] Electrodes, including one or more pairs of paired electrodes, are fixedly arranged at intervals on a side of the insulating film layer away from the second film layer, the paired electrodes corresponding to the grooves one by one and located on both sides directly above the corresponding grooves;

[0013] The cell culture device comprises one or more cell culture containers, which are arranged above the paired electrodes and in contact with the paired electrodes. The cell culture containers correspond to the through holes one by one and are located directly above the corresponding through holes.

[0014] Furthermore, the substrate may be bonded to the first film layer after being treated with plasma.

[0015] Furthermore, the material of the substrate is preferably glass.

[0016] Furthermore, the material of the first film layer is preferably polydimethylsiloxane (PDMS).

[0017] Furthermore, the thickness of the first film layer is preferably 1 to 2 mm.

[0018] Furthermore, the depth of the groove is equal to or less than the thickness of the first film layer, and the width of the groove is preferably 200 μm to 800 μm, for example, 500 μm.

[0019] Furthermore, the diameter of the through hole is preferably 4 mm to 12 mm.

[0020] Furthermore, the material of the second membrane layer is preferably PDMS or silicone rubber.

[0021] Furthermore, the thickness of the second film layer is preferably 5 μm to 20 μm.

[0022] Furthermore, the strain sensor includes a flexible electrode with a serpentine structure, which is made from a composite of conductive nanoparticles and an elastic polymer material. The flexible electrode, made with conductive nanoparticles as the conductive filler, is more likely to disconnect when subjected to stress, resulting in a more sensitive resistance change.

[0023] Furthermore, the conductive nanoparticles are selected from one or more of silver nanoparticles, gold nanoparticles, and carbon black.

[0024] Furthermore, the conductive nanoparticles in the flexible electrode are more preferably carbon black, and the elastic substrate is more preferably PDMS with a curing ratio of 1:20, where the curing ratio is the mass ratio of the curing agent to the PDMS prepolymer; the mass proportion of carbon black in the flexible electrode is 15wt% to 30wt%.

[0025] Furthermore, the material of the insulating film layer is preferably polydimethylsiloxane or silicone rubber, such as Sylgard 527 silicone rubber, so as to provide a hardness environment similar to that of myocardial tissue in vivo.

[0026] Furthermore, the material of the second film layer and the insulating film layer is more preferably PDMS with a curing ratio of 1:20, and the elastic modulus of the obtained PDMS film is ˜624 kPa.

[0027] Furthermore, the insulating film layer is provided with one or more grooves at a position in contact with the cell culture container, wherein the grooves are annular or linear grooves and are used to guide the cells to be arranged circumferentially or in a single direction.

[0028] Furthermore, the groove is preferably an annular groove to simulate the circumferential arrangement of the myocardium, which helps to accumulate the circumferential contractile force of the myocardium.

[0029] Furthermore, the width of the groove is preferably 5 μm to 25 μm, and the depth is preferably 2 μm to 10 μm.

[0030] Furthermore, when the insulating film layer is provided with a plurality of grooves at a position in contact with the cell culture container, the spacing between adjacent grooves is 10 μm to 50 μm.

[0031] Furthermore, the electrode is preferably a carbon nanofiber bundle or a metal film, more preferably a carbon nanofiber bundle. Carbon fiber has low cost, a wide potential window and electrochemical inertness, and has better stability as an electrode.

[0032] Furthermore, the inner diameter of the cell culture container is larger than the distance between the two electrodes in the paired electrodes.

[0033] Furthermore, the flexible bioelectronic device has a multi-unit array structure, such as a bioarray electronic device with a 24-well (4×6) format.

[0034] Furthermore, the flexible bioelectronic device can be used for screening cardiac drugs.

[0035] Furthermore, cardiac drugs are co-cultured with cardiomyocytes, and the dynamic changes in cardiomyocyte contractility are measured in situ by recording the changes in the resistance signal of the strain sensor to evaluate the efficacy of cardiac drugs.

[0036] The synchronous contraction of myocardial cells will cause the deflection of the suspension membrane (composed of a second membrane layer, a strain sensor, and an insulating membrane layer). The strain sensor embedded in the suspension membrane will deform, resulting in a change in resistance. By recording the dynamic changes in resistance, the contractile function of myocardial cells, including contraction rate, pulsation rate, and rhythm, can be evaluated.

[0037] Furthermore, the flexible bioelectronic device can be used for high-throughput cell culture.

[0038] Furthermore, the flexible bioelectronic device can promote the maturation of iPSC-CMs through controllable mechanical stimulation and electrical stimulation; use a pressure control system to provide pneumatic pressure to the air channel to expand the suspended membrane above the hollow chamber, providing mechanical stimulation to the cells in the cell culture container; and connect the electrodes to a multi-channel electrical stimulator to perform electrical pacing or long-term electrical stimulation on the cells.

[0039] The second aspect of the present invention provides a method for preparing the flexible bioelectronic device according to the first aspect, comprising the following steps:

[0040] (1) placing a film-forming liquid of the first film layer in a mold, peeling it off after curing to obtain a first film, transferring the first film to the upper surface of the substrate, and forming a first film layer bonded to the substrate after plasma treatment;

[0041] (2) spin coating and curing the film-forming liquid of the second film layer on the template to obtain a second film, transferring the second film to the upper surface of the first film layer, and forming a second film layer bonded to the first film layer after plasma treatment;

[0042] (3) preparing a conductive film having a preset pattern on the upper surface of the second film layer by spraying or printing a conductive ink;

[0043] (4) applying a film-forming liquid of the insulating film layer to a corresponding mold by spin coating and curing to obtain an insulating film, transferring the insulating film to the upper surface of the second film layer and covering the conductive film, and then subjecting the insulating film layer to plasma treatment to form an insulating film layer bonded to the second film layer;

[0044] (5) preparing electrodes on the upper surface of the insulating film layer according to a preset pattern;

[0045] (6) Place the customized cell culture container on the insulating film layer according to the preset position.

[0046] Furthermore, the membrane-forming liquid is prepared by mixing a prepolymer and a curing agent in a certain proportion.

[0047] In some preferred embodiments, the membrane-forming liquid of the first film layer is obtained by mixing PDMS prepolymer and curing agent in a mass ratio of 10:1; the membrane-forming liquid of the second film layer and the insulating film layer is obtained by mixing PDMS prepolymer and curing agent in a mass ratio of 20:1.

[0048] In some preferred embodiments, the conductive ink is obtained by mixing carbon black and PDMS prepolymer in a mass ratio of 1:3 and dispersing the mixture in isopropyl alcohol.

[0049] In some preferred embodiments, before cell culture, the surface of the insulating film layer is activated by oxygen plasma so that the cell culture container and the insulating film layer are closely attached.

[0050] Compared with the prior art, the present invention has the following beneficial effects:

[0051] 1. The present invention provides a flexible bioelectronic device that successfully integrates a cell culture chamber, a biosensor, and a bioreactor. By utilizing the resistance changes of a flexible strain sensor embedded in an ultrathin film, the device enables in situ, continuous, and highly sensitive measurement of cardiomyocyte contractility, which can be used for cardiac drug testing. Furthermore, the flexible bioelectronic device prepared by the present invention includes pneumatic air channels and electrodes distributed at both ends of the cell culture container. Through pneumatic and electrical stimulation, a controllable mechanical and electrical microenvironment can be achieved to promote the maturation of iPSC-CMs.

[0052] 2. The present invention uses a spray-deposited ink method to prepare a thin film sensing layer with a porous microstructure. This layer has high sensitivity and good stability, exhibits high repeatability in long-term sensing, and can record changes in the contractile ability of cardiomyocytes in real time, over a long period of time, and continuously. In addition, the present invention uses the high throughput of flexible bioelectronic devices to test different cardiac drugs. The test results show that the flexible bioelectronic device can measure the effects of drug types and dosages on the contractile performance of cardiomyocytes, thereby achieving the optimization of drug types and dosages. In addition, the present invention can promote the maturation of iPSC-CMs through controllable mechanical stimulation or electrical stimulation, and it has been found to be more effective under the combined action of mechanical and electrical stimulation.

[0053] 3. The above-mentioned flexible electronic devices are easy to manufacture and can achieve high-throughput, multi-parameter screening and optimization. They have good application prospects in in vitro cardiomyocyte culture and high-throughput cardiac drug screening. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Schematic diagram of the preparation process of flexible bioelectronic devices (1a-1h), structural diagram (1i), and working principle diagram of the local structure (1j), where ① is the glass substrate, ② is the first film layer, ③ is the suspended membrane, ④ is the carbon fiber bundle electrode, ⑤ is the electric field, and ⑥ is the pneumatic pressure;

[0055] Figure 2 This is a scanning electron microscope image of the microgrooves on the surface of the insulating film layer;

[0056] Figure 3 is the stiffness of PDMS prepared by mixing PDMS prepolymer and curing agent at different mass ratios;

[0057] Figure 4 The scanning electron micrograph (4a) of the CB-PDMS composite material prepared by the spray deposition method, the scanning electron micrograph (4b) of the CB-PDMS composite material prepared by the screen printing method, and the uniaxial tensile test curves (4c) of the CB-PDMS composite material prepared by different methods, where ΔL / L0 is the initial length of the sample and ΔL is the difference between the length of the sample during stretching and the initial length;

[0058] Figure 5 The FEA simulation structure of the suspended membrane deformation driven by the starting pressure (5a) and the relationship between the applied pressure and the membrane area change calculated by finite element analysis (5b);

[0059] Figure 6 The resistance signal (ΔR / R0) change curve (6a) recorded by the strain sensor for a monolayer of iPSC-CM cultured in a flexible bioelectronic device cell culture incubator from day 1 to day 7 of culture, the ΔR / R0 amplitude (6b), the beating time interval (6c), and the beating frequency (6d) as a function of culture time;

[0060] Figure 7 Live (green) / dead (red) staining images of a monolayer of iPSC-CMs cultured next to carbon fiber electrodes (7a), green: AM calcein, red: BOBO-3 iodide, scale bar 200 μm; resistance signals generated by spontaneous beating of a monolayer of iPSC-CMs or under 1 Hz and 2 Hz electrical pacing on day 7 of cell culture (7b);

[0061] Figure 8 Bright-field optical images and confocal immunofluorescence images of a monolayer of iPSC-CMs cultured on a circular microgrooved membrane surface. Blue: DAPI, green: α-actinin, red: connexin-43. Scale bar: 100 μm.

[0062] Figure 9Figure 2 shows the dose-dependent responses of monolayer iPSC-CMs to cardiac drug candidates: (9a) isoproterenol, (9b) omecamtiv mecarbil, (9c) blebbistatin, and (9d) flecainide. (i) shows the curve of cell contractility changing with drug dose, (ii) shows the curve of cell volatility changing with drug dose, and (iii) shows the contraction curve of cells at different drug concentrations.

[0063] Figure 10 Figure 10a shows immunostaining images of monolayer iPSC-CMs under control, mechanical stimulation, electrical stimulation, and combined electromechanical stimulation conditions. Cells were stained with a combination of two antibodies: α-actin (green) and connexin-43 (red) or cardiac troponin-T (CTNT, cyan) and myosin light chain-2 (MLC2V, magenta); quantification of contractile force (10b), sarcomere length (10c), connexin-43 expression (10d), and MLC2V expression (10e) under different conditions, where the fluorescence intensity was normalized to the nuclear fluorescence intensity to calculate the expression levels of connexin-43 and MLC2V; measured peak contractile force: n = 8 for each condition, measured sarcomeres: n = 30 for each condition, and measured connexin-43 and MLC2V expression intensities: n = 6 for each condition. Horizontal bars between groups indicate statistical significance (p < 0.05). DETAILED DESCRIPTION

[0064] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification of the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0065] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0066] Example 1

[0067] This embodiment relates to the preparation of a flexible bioelectronic device, the preparation process is as follows Figure 1 As shown, the details are as follows:

[0068] (1) PDMS prepolymer and curing agent were prepared in a mass ratio of 10:1 to obtain a first PDMS film-forming solution, and the first PDMS film-forming solution was placed in an aluminum mold ( Figure 1a), after curing, the PDMS base film was peeled off and transferred to a glass substrate. The PDMS base film was treated with plasma (PDC-001, Harrick plasma) for 1 minute to bond the PDMS base film to the glass substrate to obtain a 1 mm thick first film layer ( Figure 1 b); Six grooves are provided on one side of the first film layer that is bonded to the substrate, and four through holes are opened at equal intervals on each groove to penetrate the first film layer. Air channels are formed between the grooves and the through holes and the substrate, and a hollow chamber is formed between the through holes and the substrate. The width of the grooves is 500μm and the depth is 500μm; the diameter of the through holes is 6.35mm.

[0069] (2) The PDMS prepolymer and the curing agent were prepared in a mass ratio of 20:1 to obtain a second PDMS film-forming solution. The first PDMS film-forming solution was spin-coated on the silanized PDMS plate at a speed of 5000 rpm for 1 min, and then placed at 80°C for overnight curing to form a base film. The base film was transferred to the first film layer and bonded to the first film layer after plasma treatment for 1 min to obtain a 10 μm thick second film layer ( Figure 1 c);

[0070] (3) Carbon black (CB) and PDMS prepolymer were mixed in a mass ratio of 1:3, and isopropyl alcohol was added. The mixture was dispersed in an ultrasonic bath for 5 minutes to obtain a CB-PDMS ink. The CB-PDMS film was prepared on the second film layer covered with the existing rigid shadow mask by spray deposition. After drying, a 5 μm thick conductive film with a serpentine structure was obtained ( Figure 1 d);

[0071] (4) The second PDMS film-forming liquid was spin-coated on the silanized PDMS plate with the microgrooved stamp structure at a speed of 5000 rpm for 1 min ( Figure 1 e), and cured overnight at 80°C to form a top film, which was transferred to the upper surface of the second film layer and covered with a conductive film. The surface layer was plasma treated for 1 min and then bonded to the second film layer to obtain a 10 μm thick insulating film layer ( Figure 1 f), there are circular microgrooves on the insulating film layer just above each through hole (such as Figure 2 As shown), the circular microgrooves have a depth of 5 μm, a width of 10 μm, and a spacing of 20 μm;

[0072] (5) Figure 1 As shown in g, carbon fiber bundles (1000 wires per bundle, each wire diameter 7 μm) are placed in parallel and spaced on both sides directly above each groove, and PDMS first film-forming liquid is dripped on both ends and heated and cured, and then fixed on the insulating film layer. The interval between each pair of electrodes is 7 mm;

[0073] (6) 24 custom-made glass cylinders (8 mm inner diameter, 10 mm outer diameter, 10 mm height) were connected to the insulating film layer just above the corresponding through-holes and contacted with the carbon fiber electrodes ( Figure 1 h).

[0074] like Figure 1 As shown in Figures 1i and 1j, in the flexible bioelectronic device, the second membrane layer, the conductive film, and the insulating membrane layer constitute a suspended membrane. The CB-PDMS flexible strain sensor embedded in the suspended membrane is used to measure the contractile force of the cells. The air channel formed by the grooves and through holes of the first membrane layer and the substrate is used to pneumatically drive the suspended membrane. The carbon fiber bundle electrode is used for electrical pacing; and the glass cylinder is used to culture cells.

[0075] 1. The present invention reduces the rigidity of the suspension membrane by increasing the mass ratio of PDMS prepolymer to curing agent, thereby improving the sensitivity of flexible bioelectronic devices to myocardial cell contractility testing.

[0076] Figure 3 The suspended films with a total thickness of 25 μm were prepared using different curing ratios. As can be seen from the figure, when the mass ratio of PDMS prepolymer to curing agent increased from 10:1 to 20:1, the Young's modulus of the suspended film decreased from 1.27 MPa to 624 kPa (n = 6 indentation points).

[0077] 2. During the above-mentioned conductive film preparation process, the present invention explored the effects of different methods of preparing the conductive film on strain performance. The specific operations are as follows:

[0078] The above-mentioned CB-PDMS ink was spray deposited and screen-printed on a 10mm×5mm×50μm PDMS substrate to prepare conductive films of the same shape to obtain CB-PDMS composite materials. The samples prepared by different methods were subjected to uniaxial tensile testing (cyclic loading of 0.3% strain using a micro-tensile testing platform to simulate the low strain generated by the contraction of a monolayer of cardiomyocytes). The conductive film was connected to a precision multimeter, and the relative resistance change under cyclic stretching (ΔR / R0, R0 is the initial resistance of the sample, and ΔR is the difference between the resistance of the sample during stretching and the initial resistance) was recorded to evaluate the piezoresistive strain sensing performance.

[0079] The scanning electron microscope images of the conductive films prepared by different methods are shown in Figure 2. Figure 4 As shown in Figure 4a (spray deposition) and 4b (screen printing), it can be seen that the surface morphology of the conductive film prepared by spray deposition is rougher and has a porous microstructure; while the conductive film prepared by screen printing has a smoother and denser structure. Figure 4As shown in Figure c, the ΔR / R0 values ​​of the samples prepared by the two different methods show a highly linear relationship with the tensile strain size, and the hysteresis is small. Among them, the slope of the sample prepared by spray deposition is significantly greater than that of the sample prepared by screen printing, which also shows that the sample prepared by spray deposition has higher sensitivity.

[0080] 3. The present invention further studies the relationship between air pressure and the change in the area of ​​the suspended membrane. The specific operation is as follows:

[0081] A pressure control system is used to provide pneumatic pressure into the microchannel to expand the suspended membrane. The change in membrane area under different air pressures is calculated by finite element analysis (COMSOL Multiphysics). Each suspended membrane unit is simulated as a two-dimensional axisymmetric structure ( Figure 5 a).

[0082] The results are as follows Figure 5 As shown in b, under the action of 1.7 kPa air pressure, the membrane area of ​​the suspension membrane increased by 15%, which could produce mechanical stimulation to the monolayer of cardiomyocytes attached to the top of the suspension membrane.

[0083] 4. The present invention further explores the effects of electrical stimulation on cells, and the specific operations are as follows:

[0084] The flexible bioelectronic devices were sterilized by gamma ray. Before cell culture, the PDMS membrane surface was activated by oxygen plasma for 1 minute. Then, cell culture medium was added to each culture chamber and placed in a 37°C incubator overnight. Cells were thawed and dispersed in cell culture medium at a density of 1.5×10 5 cells / cm 2 Cells were seeded at a density of 100 μg / ml to form a two-dimensional monolayer. The monolayer cells were cultured at 37°C in an atmosphere of 5% CO2, and the culture medium was changed every two days.

[0085] Over the course of culture days, iPSC-CMs gradually established intracellular connections, generated spontaneous cell beating, and exerted compressive stress to deflect the suspended membrane. Figure 6 a is the dynamic change of the resistance signal of the flexible bioelectronic device with the increase of culture days. As can be seen from the figure, iPSC-CM began to beat spontaneously on the second day. From the second day to the seventh day, the cell contraction force gradually increased with the increase of culture days, as shown in Figure 1. Figure 6 b. Statistical analysis showed that the time interval between two adjacent signal peaks changed from the dispersed pattern on the second day to a uniform value ( Figure 6 c), indicating that the cell beating rhythm gradually shifted from an irregular state to a stable state. In addition, the myocardial cell beating rate tended to decrease from the second to the fourth day, and then remained stable on the fifth and seventh days ( Figure 6 d).

[0086] The carbon fiber electrodes in the flexible bioelectronic device were connected to a multi-channel electrical stimulator to perform electrical pacing on the cell monolayer. After 7 days, the activity of iPSC-CMs around the carbon fiber electrodes was measured using a kit (R37601, Thermo Fisher Scientific). Live cells were stained with Calcein-AM (green) and dead cells were stained with BOBO-3 iodide (red). The results are shown in Figure 2. Figure 7 As shown in a, the cell viability is greater than 90%.

[0087] The resistance signals generated by spontaneous beating of monolayer iPSC-CMs and electrical pacing at different frequencies (1 Hz, 2 Hz) are shown in Figure 2. Figure 7 As shown in b, the monolayer of iPSC-CMs successfully adjusted to different beating frequencies under different electrical stimulations.

[0088] 5. The present invention further observes the distribution of cells in flexible bioelectronic devices, and the specific operations are as follows:

[0089] The monolayer of iPSC-CMs cultured for 7 days was immunostained to analyze cell distribution, sarcomere structure, and intracellular connections between adjacent iPSC-CMs by immunostaining of nuclei (blue), α-actin (green), and gap junction protein (connexin-43, red).

[0090] Imaging results such as Figure 8 As shown in the figure, cardiomyocytes are evenly distributed on the surface of the microgrooved membrane and arranged along the circular microgrooves. Cyclic contraction of cardiomyocytes is generated by the dynamic sliding of actin and myosin filaments between each sarcomere unit. Thin actin filaments are anchored to the z-line by α-actinin. α-actin immunostaining results show that the sarcomeres are striped and arranged along the circular microgrooves, effectively mimicking the circumferential arrangement of the myocardium and facilitating the accumulation of circumferential contractile force. Furthermore, clear expression of the connexin-43 protein indicates that iPSC-CMs have established effective intercellular connections for communication and synchronized cell beating.

[0091] Example 2

[0092] The contraction of cardiomyocytes involves a series of processes, including changes in membrane potential, ion channel activity, calcium dynamics, myofilament sliding, and intercellular communication through gap junctions or membrane current conduction. Therapeutic cardiac drugs or cardiotoxic molecules can regulate the contractile function of cardiomyocytes by regulating the above processes. This example verifies the effectiveness of the above-mentioned flexible bioelectronic devices in high-throughput drug testing by evaluating the effects of four representative anticardiac compounds (isoproterenol, omecamtiv mecarbil, blebbistatin, and flecainide). The specific operations are as follows:

[0093] Drug experiments were performed on days 5-7 of iPSC-CM culture. Before the drug experiments, the culture medium was replaced with 250 μL of fresh maintenance medium and incubated for 2 hours to avoid the effects of nutrients and temperature on beating behavior. Then, 2.5 μL of drug stock solution was added every 10 minutes. In the incubator environment, the resistance signal was continuously recorded. The dose-dependent response of contractility and beating rate was extracted from the resistance signal and fitted into an S-shaped curve to obtain the effect of drug dose on cell contractility, beating frequency and rhythm. The test results are shown in Figure 2. Figure 9 As shown in a-9d.

[0094] Isoproterenol is an agonist that can be used to treat bradycardia. This example tests the drug response of isoproterenol at the following doses: 1 nM, 50 nM, 100 nM, 250 nM, 500 nM, 1 μM, 2 μM, and 5 μM. Figure 9 As shown in a, the contraction force and beating rate increased in a dose-dependent manner. The contraction force EC 50 The value is 348nM, and the beating frequency EC 50 When the effective dose of isoproterenol is 250 nM, the contractility of monolayer cardiomyocytes increases by 15% and the beating rate increases from 0.7 Hz to 1.12 Hz.

[0095] Omecamtiv mecarbil (OM) is a cardiac-specific myosin activator that effectively enhances the formation and duration of connections between myofiber filaments. Figure 9 As shown in b, the test results of different drug concentrations (1nM, 10nM, 50nM, 100nM, 200nM, 500nM, 1μM, 2μM and 5μM) showed that the contractility of cells gradually increased with the increase of OM dose and tended to be stable at around 1μM. 50 The value was 973 nM. Due to potential side effects, the effect of OM on improving contractility was weakened when the dose reached 5 μM. In contrast to the changes in contractility, OM administration had little effect on cell beating rate.

[0096] In addition, myofilament inhibitors and ion channel blockers were also tested to verify the effectiveness of the flexible bioelectronic device of the present invention in measuring the pharmacological effects of inhibiting cardiomyocytes and potential cardiotoxicity. Compared with OM, blebbistatin is a myosin filament inhibitor that can block the bridge between myosin and actin filaments by affecting the ATPase activity of myosin heavy chain. Figure 9 As shown in c, the Blebbistatin detection results (10nM, 100nM, 250nM, 500nM, 1μM, 2μM, 5μM and 10μM) showed that the cell contractility decreased with the increase of drug concentration, IC 50=584nM. Furthermore, when the drug dose was increased to 1μM, the beating suddenly stopped. This phenomenon is attributed to the electromechanical decoupling mechanism of blebbistatin, which only inhibits the sliding of contractile filaments but has no effect on the regularity of membrane potential.

[0097] Flecainide is a multi-channel blocker that mainly acts on sodium channels and hERG potassium channels. This example tests the effects of different doses of Flecainide (1nM, 50nM, 100nM, 200nM, 500nM, 1μM, 2μM, 5μM, 10μM) on cell contractility and beating rate. Figure 9 As shown in Figure d, the contractility and beating rate gradually decreased with the increase of Flecainide concentration. 50 Tested at 2.48 μM, beat rate IC 50 The value was 1.09 μM. When the concentration of Flecainide was greater than 10 μM, the cells stopped beating. These results show that the flexible bioelectronic array of the present invention can measure the effects of drug types and dosages on the contractile properties of cardiomyocytes.

[0098] Example 3

[0099] In this example, mechanical stimulation and / or electrical stimulation were applied to the monolayer of iPSC-CMs to observe cellular changes.

[0100] The specific operations are as follows:

[0101] After seeding, iPSC-CMs were cultured for 48 hours to allow for the formation of focal adhesions, intercellular connections, and synchronized beating. Starting on day 2, mechanical stimulation was applied by membrane expansion deformation, applying a 15% static strain. Simultaneously, electrical stimulation was applied by applying periodic rectangular pulses (2 ms duration, 1 Hz, 2.5 V amplitude, 3.57 V / cm) via parallel carbon fiber electrodes. At the end of each culture day, mechanical and electrical stimulation were suspended for 1 hour to allow for medium replacement. Contractility of the iPSC-CM monolayer was measured and immunostained at day 7.

[0102] like Figure 10 As shown in a, under the four culture conditions, a good monolayer of iPSC-CM was formed on the top surface of the suspension membrane. Troponin-t (ctnt) and α-actin are both biomarkers expressed in the early differentiation stage of cardiomyocytes. Immunofluorescence analysis showed that ctnt and α-actin filaments in the monolayer of iPSC-CM were clearly striped. In addition, under mechanical, electrical and combined stimulation conditions, the fluorescence intensity of connexin-43 and myosin light chain-2 (mlc2v) increased. The changes in the contractile ability of cardiomyocytes under four different culture conditions are shown in Figure 4. Figure 10As shown in b-10e, compared with the control group, the contraction amplitude increased by 35.1% in the mechanical stimulation group, 30% in the electrical stimulation group, and 62.3% in the combined electromechanical stimulation group, which also indicates that electrical or mechanical stimulation can promote the maturation of iPSC-CMs.

[0103] The above-described embodiments are merely preferred examples for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A flexible bioelectronic device for detecting the contractile function of cardiac cells, characterized in that: The flexible bioelectronic device comprises: substrate; A first film layer is disposed above the substrate and bonded to the substrate, wherein one or more grooves are provided on a surface of the first film layer bonded to the substrate, each groove having one or more through holes extending through the first film layer, an air passage is formed between the grooves and the through holes and the substrate, and a hollow chamber is formed between the through holes and the substrate; a second film layer, disposed above the first film layer and bonded to the first film layer; The strain sensing system includes one or more strain sensors fixedly disposed on a side of the second film layer away from the first film layer, the strain sensors corresponding one to each of the through holes and located directly above the corresponding through holes; the strain sensors include flexible electrodes having a serpentine structure, the flexible electrodes being composited from conductive nanoparticles and an elastic polymer material; an insulating film layer, disposed above the strain sensor and in contact with the second film layer; Electrodes, including one or more pairs of paired electrodes, are fixedly arranged at intervals on a side of the insulating film layer away from the second film layer, the paired electrodes corresponding to the grooves one by one and located on both sides directly above the corresponding grooves; a cell culture device comprising one or more cell culture containers, disposed above and in contact with the paired electrodes, wherein the cell culture containers correspond one-to-one to the through holes and are located directly above the corresponding through holes; The insulating film layer is provided with one or more grooves at a position in contact with the cell culture container, and the grooves are annular grooves.

2. The flexible bioelectronic device according to claim 1, characterized in that The thickness of the first film layer is 1 mm to 2 mm, and the material of the first film layer is polydimethylsiloxane.

3. The flexible bioelectronic device according to claim 1, characterized in that The width of the groove is 200 μm to 800 μm; the diameter of the through hole is 4 mm to 12 mm.

4. The flexible bioelectronic device according to claim 1, characterized in that The thickness of the second film layer is 5 μm to 20 μm, and the material of the second film layer is polydimethylsiloxane or silicone rubber.

5. The flexible bioelectronic device according to claim 1, characterized in that The conductive nanoparticles are selected from one or more of silver nanoparticles, gold nanoparticles, and carbon black.

6. The flexible bioelectronic device according to claim 1, characterized in that The thickness of the insulating film layer is 5 μm to 20 μm, and the material of the insulating film layer is polydimethylsiloxane or silicone rubber.

7. The flexible bioelectronic device according to claim 6, characterized in that: The width of the groove is 5 μm to 25 μm, and the depth is 2 μm to 10 μm. When the insulating film layer is provided with multiple grooves at the position in contact with the cell culture container, the spacing between adjacent grooves is 10 μm to 50 μm.

8. The flexible bioelectronic device according to claim 1, characterized in that: The inner diameter of the cell culture container is greater than the distance between two electrodes in the paired electrodes; and the electrodes are carbon nanofiber bundles or metal films.

9. A method for preparing a flexible bioelectronic device according to any one of claims 1 to 8, characterized in that: The following steps are involved: (1) placing a film-forming liquid of the first film layer in a mold, peeling it off after curing to obtain a first film, transferring the first film to the upper surface of the substrate, and forming a first film layer bonded to the substrate after plasma treatment; (2) Spin coating and curing the film-forming liquid of the second film layer on the template to obtain a second film, transferring the second film to the upper surface of the first film layer, and forming a second film layer bonded to the first film layer after plasma treatment; (3) preparing a conductive film having a preset pattern on the upper surface of the second film layer by spraying or printing a conductive ink; (4) Spin coating and curing the film-forming liquid of the insulating film layer on the corresponding mold to obtain an insulating film, transferring the insulating film to the upper surface of the second film layer and covering the conductive film, and forming an insulating film layer bonded to the second film layer after plasma treatment; (5) Preparing electrodes on the upper surface of the insulating film layer according to a preset pattern; (6) Place the customized cell culture container on the insulating film layer according to the preset position.

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