Myocardial chip embedded with nanofiber and flexible circuit board and integrated forming method thereof

Abstract

The invention discloses an integral forming method of a myocardial chip embedded with nanofibers and a flexible circuit board, which adopts integral forming equipment, integrates a photocuring part and an electrostatic spinning part, and realizes collaborative operation through an upper computer and a driving device; the method comprises the following steps: firstly, forming a lower runner layer by adopting a hydrogel material through a photocuring component, and embedding a flexible circuit board into the top of the lower runner layer; forming a nanofiber bracket on the flexible circuit board by adopting a spinning material through the electrostatic spinning part; a hydrogel material is printed on the nanofiber support to form an upper flow channel layer, flow channel packaging is achieved, and efficient transmission of electrical stimulation and field potential signals is achieved through a flexible circuit board. According to the method, the problems of low alignment precision, strong manual dependence and insufficient automation degree in the traditional process are solved, the chip manufacturing efficiency and consistency are improved, and a detection platform with high integration level and high biocompatibility is provided for drug screening and heart disease research.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell detection, and particularly relates to a myocardial chip embedded with nanofibers and a flexible circuit board and an integrated molding method thereof. Background Art

[0002] In recent years, with the increasing demand for drug toxicity detection and disease mechanism analysis, myocardial chips have attracted much attention. By simulating the physiological environment of the human heart, myocardial chips can achieve efficient detection of drug toxicity and in-depth analysis of disease mechanisms. Such chips usually include four core components: a microfluidic chip, a tissue scaffold, a stimulating element, and a sensing element. The synergistic effect of these components endows myocardial chips with the characteristics of multi-materials, multi-components, and high integration, enabling precise simulation of the physiological functions of the heart and providing a powerful tool for drug screening and disease research.

[0003] Currently, the manufacturing process of myocardial chips mainly relies on the combination of multiple technologies. For example, the microfluidic chip part is manufactured through molding and soft lithography techniques, then the sensing element is manufactured through perfusion or direct writing techniques, and finally transferred to an electrospinning platform to manufacture the tissue scaffold. Most of the stimulation methods involve applying external platinum electrodes or carbon rods for stimulation during tissue culture. There are two problems with the above-mentioned chip manufacturing processes: First, the need to switch between multiple devices poses great challenges to the alignment accuracy of key components and the repeatability of the chips; second, the high dependence on manual operation and the low automation of the overall process. Manual operation is not only inefficient but also prone to introducing human errors, affecting the consistency and reliability of the chips. In addition, the low degree of automation of the overall process makes it difficult to achieve large-scale and high-efficiency production, limiting the wide application of myocardial chips in drug screening and disease research. Summary of the Invention

[0004] The purpose of the present invention is to develop an integrated molding method for a myocardial chip that can embed nanofibers and a flexible circuit board, including the design of equipment and the proposal of process methods, to achieve the integrated manufacturing of a highly integrated myocardial chip with four core components.

[0005] To achieve the above purpose, the technical solution of the present invention is as follows:

[0006] An integrated molding method for a myocardial chip embedded with nanofibers and a flexible circuit board, which uses an integrated molding device. The integrated molding device includes a main body and a host computer, and the main body is controlled by the host computer; the main body includes a working platform, a photocuring component, an electrospinning component, and a driving device, and the driving device is used to drive the photocuring component and the electrospinning component to move; the working platform integrates counter electrodes and a printing substrate; the integrated molding method includes the following steps:

[0007] 1) Move the photocuring component above the working platform and print a downstream channel layer on the printing substrate using a hydrogel material;

[0008] 2) Embed a flexible circuit board into the top of the downstream channel layer;

[0009] 3) Move the electrospinning component above the working platform and form a nanofiber scaffold on the flexible circuit board using a spinning material;

[0010] 4) Move the photocuring component above the working platform and print an upstream channel layer above the nanofiber scaffold using a hydrogel material.

[0011] Optionally, it further includes a pneumatic device which is controlled by the host computer; the photocuring component includes a hydrogel material discharging device, the electrospinning component includes a spinning material discharging device, and the pneumatic device is used to control the feeding of the hydrogel material discharging device and the spinning material discharging device.

[0012] Optionally, the pneumatic device includes a gas cylinder and a pressure controller. The hydrogel material discharging device and the spinning material discharging device each include a syringe, and the syringe is connected to the gas cylinder through a pipeline. The pressure controller is arranged in the pipeline to regulate the air pressure.

[0013] Optionally, the electrospinning component further includes a positive high-voltage controller and a negative high-voltage controller. The spinning material discharging device includes a metal dispensing needle head. The positive high-voltage controller is used to apply a positive high voltage to the metal dispensing needle head, and the negative high-voltage controller is used to apply a negative high voltage to the counter electrode on the working platform.

[0014] Optionally, the upstream channel layer and the downstream channel layer enclose a channel up and down, and the flexible circuit board and the nanofiber scaffold are clamped in the channel, wherein the flexible circuit board is provided with a hollowed-out part.

[0015] Optionally, the flexible circuit board includes a stimulating electrode, a detecting and reference electrode, and an external electrode; the stimulating electrode and the detecting and reference electrode are located in the channel, and the external electrode is located outside.

[0016] Optionally, the driving device includes an X-axis driving unit, a Y-axis driving unit, and a Z-axis driving unit, and each driving unit respectively includes a driving circuit, a servo motor, and a feedback sensor.

[0017] 11. Optionally, the composition of the hydrogel material includes a photoinitiator, a monomer, deionized water, and lemon yellow, wherein the monomer is polyacrylamide (AAm) and polyethylene glycol diacrylate (PEGDA 700), and the photoinitiator is (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate).

[0018] Optionally, the photocuring component further includes a digital light processing device and a tensioning device. The tensioning device morphologically constrains and stores the hydrogel material droplets based on surface tension, drives the up-and-down movement through a driving device, and realizes 3D printing in combination with the digital light processing device.

[0019] A myocardial chip embedded with nanofibers and a flexible circuit board is prepared by the integral molding method of the myocardial chip embedded with nanofibers and a flexible circuit board as described above.

[0020] The beneficial effects of the present invention are as follows:

[0021] (1) Through the integral molding equipment integrating photocuring and electrospinning components, an automated process of downstream channel layer printing, flexible circuit board embedding, nanofiber scaffold preparation, and upstream channel layer encapsulation is realized, avoiding the component alignment errors caused by multi-device switching in the traditional process, significantly improving production efficiency and repeatability, and enhancing the consistency and reliability of chip manufacturing;

[0022] (2) The flexible circuit board is embedded in the channel layer, integrating stimulation electrodes, detection electrodes, and external electrodes. Combining with the two-dimensional ordered biomimetic structure of the nanofiber scaffold, it can not only simulate the natural microenvironment where the myocardial tissue is located, but also monitor electrophysiological signals in real time, enhancing the functionality of the chip in drug toxicity detection and disease mechanism research, and ensuring the stable acquisition and transmission of signals during long-term cultivation;

[0023] Other features and beneficial effects of the present invention will be described in the subsequent specification, and part of them will be obvious from the specification or understood by implementing the present invention. Description of the Drawings

[0024] Figure 1 Schematic diagram of the overall structure of the integral molding equipment for the embodiment;

[0025] Figure 2 Schematic diagram of the control principle of the driving device of the integral molding equipment for the embodiment;

[0026] Figure 3 Schematic diagram of the structure of the tensioning device (including the release film) of the integral molding equipment for the embodiment, a) overall structure diagram; b) sectional view;

[0027] Figure 4 Schematic diagram of the photocuring process based on surface tension constraint for the embodiment, a) schematic diagram of the printing step; b - e) actual photos of the printing process;

[0028] Figure 5 Schematic diagram of the structure of the working platform of the integral molding equipment for the embodiment;

[0029] Figure 6Integrated molding flowchart of the myocardial chip in the embodiment, a) Stereolithography based on surface tension constraint; b) Embedding a flexible circuit board; c) Embedding a nanofiber scaffold;

[0030] Figure 7 Schematic diagram of the overall structure of the myocardial chip embedding nanofibers and a flexible circuit board in the embodiment;

[0031] Figure 8 Exploded view of the myocardial chip embedding nanofibers and a flexible circuit board in the embodiment;

[0032] Figure 9 Schematic diagram of the structure of the flexible circuit board of the myocardial chip in the embodiment;

[0033] Figure 10 Schematic diagram of the working principle of the myocardial chip integrating embedded nanofibers and FPC in the embodiment;

[0034] Figure 11 Experimental diagram of the myocardial tissue chip integrating an embedded nanofiber scaffold and a flexible circuit board in the embodiment, a) Preparation process of the myocardial chip; b) Morphology of the embedded nanofibers before swelling; c) Morphology of the embedded nanofibers after swelling; d) Myocardial chip before swelling; e) Myocardial chip after swelling; f) Optical characterization of cardiomyocytes on the nanofiber scaffold; g) Fluorescent staining map of apoptotic cardiomyocytes; h) Fluorescent staining map of surviving cardiomyocytes; i) Fluorescent staining map of cardiomyocyte nuclei; j) Mosaic result of the fluorescent staining map of surviving cardiomyocytes and the fluorescent staining map of nuclei;

[0035] Figure 12 Electrical property test of the myocardial chip before and after swelling in the embodiment, a) Impedance of the central recording electrode; b) Impedance of the outer recording electrode; c) Impedance of the reference / stimulation electrode; d) Signal acquisition and input test circuit; e) Noise of the measurement and control system; f) Noise spectrum of the measurement and control system; g) Input test of the stimulation signal before and after swelling of the hydrogel sample embedded with FPC; h) Recording of the simulated field potential signal before and after swelling of the hydrogel sample embedded with FPC. Detailed implementation mode

[0036] The following further explains the present invention in conjunction with the accompanying drawings and specific embodiments. The accompanying drawings of the present invention are only for illustration to facilitate understanding of the present invention, and its specific proportions can be adjusted according to design requirements.

[0037] Integrated molding method of the myocardial chip embedding nanofibers and a flexible circuit board in the embodiment, and the integrated molding equipment used is as Figure 1As shown in the figure, it mainly includes the prototype machine body 1, as well as the external pneumatic device 2 and the host computer 3. The body 1 includes a photocuring component 11, an electrospinning component 12, a working platform 13 and a driving device. The photocuring component 11 is used to realize the 3D printing function, the electrospinning component 12 is used to realize the electrospinning function, the working platform 13 integrates the printing platform and the electrospinning receiving platform, and the driving device is used to drive the photocuring component 11 and the electrospinning component 12 to move. The pneumatic device 2 is used for the discharge control in the 3D printing process and the electrospinning process. The prototype machine body 1 and the pneumatic device 2 are controlled by the host computer 3, and the above processes can be controlled through the host computer 3.

[0038] Specifically, the driving device is a three-axis driving device, and its function is to control the movement of the target feeding position of the photocuring function, the up and down movement during the photocuring process, and the movement of the target working position of the electrospinning function. The control schematic diagram of the three-axis driving device is as Figure 2 shown. It mainly consists of an X-axis driving unit, a Y-axis driving unit, a Z-axis driving unit, a control system, a motion control module and a closed-loop control module. Each driving unit respectively includes a driving circuit, a servo motor and a feedback sensor. First, the target position is input through the host computer, and the control system receives and processes the input signal of the host computer; then, the motion control module plans the motion path and drives the X / Y / Z-axis servo motors to move the working parts corresponding to the process. During the movement of the motor, the feedback sensor real-time feedbacks parameters such as position and speed to the closed-loop control module. The closed-loop control module adjusts the control instruction according to the feedback to make the working parts corresponding to the process move to the target position.

[0039] The photocuring component 11 is a photocuring component based on surface tension constraint, including a digital light processing (DLP) device 111, a tensioning device 112 and a hydrogel material discharging device 113. The hydrogel material discharging device 113 can be, for example, a syringe for photocuring and its fixture. The structure of the tensioning device 112 is as Figure 3 shown. It mainly consists of a tensioning device structural component and a release film 114. The digital light processing device 111 can realize high-precision 3D printing. The photocuring steps based on surface tension constraint are as Figure 4 shown. The main function of the tensioning device 112 is to use surface tension to perform morphological constraint and storage on the droplets discharged by the hydrogel material discharging device 113, and at the same time, combine the up and down movement of the Z-axis of the three-axis driving device and the digital light processing (DLP) device to complete the model printing.

[0040] Refer to Figure 5, the surface of the working platform 13 is formed of an insulating material 133 such as Teflon, integrating a printing substrate 131 and a counter electrode 132. The material of the printing substrate 131 is stainless steel, and the material of the counter electrode 132 is brass. The counter electrode 132 is located outside the printing substrate 131. The above printing process can be realized on the printing substrate 131. The counter electrode 132 is used for the realization of the electrospinning process.

[0041] The electrospinning component 12 includes a spinning material discharging device 121, a positive high-voltage controller, a negative high-voltage controller, etc. The spinning material discharging device 121 is connected to the frame of the device through a connecting piece for easy movement. The spinning material discharging device 121 is, for example, a syringe for electrospinning and its fixture, which is provided with a metal dispensing needle head. The positive high-voltage controller is used to apply a positive high voltage to the metal dispensing needle head, and the negative high-voltage controller is used to apply a negative high voltage to the counter electrode 132 to form an electric field to drive the formation of nanofibers on the working platform 13.

[0042] The function of the pneumatic device 2 is to control the extrusion of the photocuring and electrospinning functional materials. The device is composed of a gas cylinder 21, a pressure controller 22, etc. The syringes of the hydrogel material discharging device 113 and the spinning material discharging device 121 are connected to the gas cylinder 21 through pipelines, and the pressure controller 22 is arranged in the pipeline to regulate the air pressure.

[0043] The upper computer 3 is responsible for the visual operation of photocuring, electrospinning and three-axis movement. The upper computer transmits data with the pneumatic device 2 and the prototype machine body 1 through the "Modbus485" communication protocol to realize air pressure control and process implementation.

[0044] Using the above integrated device to prepare a myocardial chip integrating embedded nanofibers and a flexible printed circuit board (FPC). The myocardial chip integrating embedded nanofibers and a flexible printed circuit board (FPC) integrates 4 core components, namely a microfluidic chip, a tissue scaffold in the form of nanofibers, and a stimulation and sensing component in the form of a flexible printed circuit board. The process flow chart of the preparation is as Figure 6 shown, and the structure of the obtained myocardial chip is as Figures 7 - 8 shown.

[0045] The one-piece molding process of the myocardial chip 4 integrating embedded nanofibers and a flexible printed circuit board (FPC) includes:

[0046] Lower channel layer 41: The three-axis drive device controls the movement of the hydrogel material discharging device 113 above the working platform 13, and drops an appropriate volume of hydrogel material; then, the light-curing components such as the digital light processing device 111 move above the working platform 13, and the curing program is output by the host computer 3 to complete the printing of the lower channel layer 41 of the myocardial chip, which has a flow channel groove 41a. The composition of the hydrogel material includes a photoinitiator, a monomer, deionized water, and lemon yellow, where the monomer is polyacrylamide (AAm) and polyethylene glycol diacrylate (PEGDA 700), and the photoinitiator is (lithium phenyl(2,4,6-trimethylbenzoyl)phosphate).

[0047] The hydrogel material is cured by UV light;

[0048] Functional layer: First, the flexible circuit board 42 is embedded on the top of the lower channel layer 41. Then, the spinning material discharging device 121 moves above the working platform 13, the positive high-voltage controller applies a positive high voltage (+12 kV) to the metal dispensing needle, and the negative high-voltage controller applies a negative high voltage (-2 kV) to the copper counter electrode 132 in the working platform; finally, the pneumatic device 2 controls the extrusion of the spinning material (TPU), and the nanofibers are received above the flexible circuit board 42 under the drive of the electric field to complete the embedded printing of the nanofiber scaffold 43;

[0049] Upper channel layer 44: The printing process is the same as that of the lower channel layer 41, and a similar flow channel groove is formed to complete the encapsulation of the myocardial chip.

[0050] The formed myocardial chip 4 includes, from bottom to top, a lower channel layer 41, a flexible circuit board 42, a nanofiber scaffold 43, and an upper channel layer 44. The flow channel grooves of the upper channel layer 44 and the lower channel layer 41 surround each other to form a flow channel, which is responsible for the delivery of the myocardial tissue culture solution. The upper channel layer 44 is provided with an inlet and outlet 441 of the flow channel. The flexible circuit board 42 and the nanofiber scaffold 43 are clamped in the flow channel, where the flexible circuit board 42 is provided with a hollow part 421 to realize the conduction of the flow channel, and the nanofiber scaffold provides tissue support for myocardial cells. Refer to Figure 9 , the flexible circuit board 42 integrates a microelectrode array, and its electrode potentials include a stimulating electrode 42a, a detecting and reference electrode 42b, and an external electrode 42c according to functions. The stimulating electrode 42a and the detecting and reference electrode 42b are located in the flow channel, and the external electrode 42c is located outside. Specifically, there are 12 electrode sites in the middle, among which 4 are stimulating electrodes 42a, providing the function of electrical stimulation for myocardial cells; the remaining 8 are detecting and reference electrodes 42b, used to monitor the field potential signals of myocardial tissue at different positions on the nanofiber scaffold. The 10 electrode sites symmetrically distributed on both sides outside are external electrodes 42c, used to connect with an external probe card to realize the external input of electrical stimulation signals and the output of field potential signals. Figure 10To integrate the embedded nanofibers, the FPC schematic diagram was presented, and the distribution of cardiomyocytes on the nanofiber scaffold, the transmission direction of the electrical stimulation signal, and the monitoring of the field potential signal were characterized.

[0051] Myocardial chip printing process, sample structure and biological experiments Figure 11 shown. Figure 11 a Characterizes the printing process of myocardial chip; Figure 11 bc characterized the structural integrity of the nanofibrous scaffolds before and after swelling; Figure 11 de shows that the myocardial chip with integrated embedded nanofibers and flexible circuit boards can maintain the integrity of the chip before and after swelling. After the chip was sterilized and coated with matrix gel, the myocardial cells were seeded on the nanofiber scaffold. After a period of incubation, immunofluorescence staining was performed to observe the survival rate and growth of myocardial cells. According to the immunofluorescence staining image ( Figure 11 fj), the survival rate of cardiomyocytes was extremely high, and their morphology was spindle-shaped along the fiber direction.

[0052] like Figure 12 As shown in Figures ac, the impedance change of the hydrogel sample embedded in FPC before and after swelling was tested. Due to the stable and excellent electrical properties of FPC, the impedance of the central and outer recording electrodes at a frequency of 1kHz before swelling was much less than 100kΩ, about 2.6~2.7kΩ. After swelling, the electrodes were affected by the deformation of the hydrogel, and the impedance value of the recording electrodes increased to 8.8~9.5kΩ. The impedance change of the reference / stimulation electrodes with a larger area before and after swelling was not obvious.

[0053] In order to simulate FPC as an electrical stimulation and electrophysiological sensing element, a signal generator was used to simulate the required generation / sampling signals ( Figure 12 d). Figure 12 ef, the amplitude range of the short-circuit noise at the end of the measurement and control system is between ±40μV. The small peaks of the typical myocardial cell field potential signal (usually a few hundred microvolts) will not be drowned by this noise. The noise is distributed relatively evenly in the spectrum. Although there is a certain amount of high-frequency noise in the spectrum, its amplitude is low and will not have a significant impact on the detection of myocardial cell field potential signals. Therefore, the measurement and control system can realize the acquisition of myocardial cell field potential signals.

[0054] like Figure 12g shows the results of the simulated electrostimulation experiments on the hydrogel samples embedded with FPC before and after swelling. From left to right, electrostimulation signals with frequencies of 0.2 Hz, 0.5 Hz, 1 Hz, 2 Hz, and 4 Hz, an amplitude of 1 V, and a stimulation duration of 1 ms were input through the stimulation electrodes. And the signal acquisition was carried out at the back end through the measurement and control system. Since the experiment was carried out in PBS buffer solution, the applied electrostimulation voltage value was slightly less than 1 V. However, there was no obvious influence on the frequency and stimulation duration of the electrostimulation signal before and after swelling. A field potential signal of 1 Hz was simulated using a signal generator and input for collection by the recording electrode ( Figure 12 h). The two key features of the field potential, namely spikes and small peaks, could be clearly observed at the electrodes before and after swelling. Thus, it can be seen that the swelling process did not cause significant influence on the performance of the embedded FPC electrodes and their leads, and the stability of the recording, reference / stimulation electrodes was effectively guaranteed.

[0055] The above embodiments are only used to further illustrate a myocardial chip embedded with nanofibers and a flexible circuit board and its one-piece molding method of the present invention. However, the present invention is not limited to the embodiments. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention all fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for integrally forming a myocardial chip embedded with nanofibers and a flexible circuit board, characterized in that: An integrated molding device is used, which includes a main body and a host computer, and the main body is controlled by the host computer; the main body includes a working platform, a light-curing component, an electrostatic spinning component and a driving device, and the driving device is used to drive the light-curing component and the electrostatic spinning component to move; the working platform integrates a counter electrode and a printing substrate; The one-piece molding method comprises the following steps: 1) moving the light-curing component to the top of the working platform, and printing a lower flow channel layer on the printing substrate using a hydrogel material; 2) embedding the flexible circuit board on top of the lower runner layer; 3) moving the electrospinning component above the working platform, and using the spinning material to form a nanofiber scaffold on the flexible circuit board; 4) The light-curing component is moved above the working platform, and a hydrogel material is used to print above the nanofiber support to form an upper flow channel layer.

2. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 1, characterized in that: It also includes a pneumatic device, which is controlled by the host computer; the light curing component includes a hydrogel material discharging device, and the electrostatic spinning component includes a spinning material discharging device, and the pneumatic device is used to control the feeding of the hydrogel material discharging device and the spinning material discharging device.

3. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 2, characterized in that: The pneumatic device includes a gas cylinder and an air pressure controller, the hydrogel material discharging device and the spinning material discharging device respectively include syringes, the syringes and the gas cylinders are connected by pipelines, and the air pressure controller is arranged in the pipeline to regulate the air pressure.

4. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 2, characterized in that: The electrospinning component also includes a positive high-voltage controller and a secondary high-voltage controller. The spinning material discharge device includes a metal dispensing needle. The positive high-voltage controller is used to apply positive high voltage to the metal dispensing needle, and the secondary high-voltage controller is used to apply negative high voltage to the electrodes on the working platform.

5. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 1, characterized in that: The upper flow channel layer and the lower flow channel layer are enclosed up and down to form a flow channel, and the flexible circuit board and the nanofiber support are clamped in the flow channel, wherein the flexible circuit board is provided with a hollow portion.

6. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 5, characterized in that: The flexible circuit board comprises stimulation electrodes, detection and reference electrodes and external electrodes; the stimulation electrodes, detection and reference electrodes are located in the flow channel, and the external electrodes are located outside.

7. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 1, characterized in that: The driving device comprises an X-axis driving unit, a Y-axis driving unit and a Z-axis driving unit, wherein each driving unit comprises a driving circuit, a servo motor and a feedback sensor.

8. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 1, characterized in that: The hydrogel material comprises a photoinitiator, a monomer, deionized water, and tartrazine, wherein the monomer is polyacrylamide (AAm) and polyethylene glycol diacrylate (PEGDA 700), and the photoinitiator is (phenyl (2,4,6-trimethylbenzoyl) lithium phosphate).

9. The method for integrally forming a myocardial chip with embedded nanofibers and a flexible circuit board according to claim 1, characterized in that: The photocuring component also includes a digital light processing device and a tensioning device. The tensioning device performs morphological constraints and storage on the hydrogel material droplets based on surface tension, and is driven to move up and down by a driving device and combined with the digital light processing device to achieve 3D printing.

10. A myocardial chip embedded with nanofibers and a flexible circuit board, characterized in that: The myocardial chip is prepared by the integrated molding method of the nanofiber embedded and flexible circuit board as described in any one of claims 1 to 9.

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