An enameled wire-based in-vitro myocardial flexible microelectrode array and a preparation method thereof

Abstract

The application discloses a kind of based on enameled wire's in-vitro myocardial flexible microelectrode array and preparation method thereof.The device includes PCB substrate, microelectrode array, cell culture ring and transparent observation window;The transparent observation window is embedded in the PCB substrate, and the top is provided with the cell culture ring;The PCB substrate is arranged with the microelectrode array;The microelectrode array is arranged by enameled wire, the enameled wire is composed of conductive inner core and insulating layer wrapped around the periphery of conductive inner core, and the enameled wire is exposed with conductive end face, and the conductive end face is located in the cell culture ring above the transparent observation window.The application can be used for real-time in-situ long-term monitoring in-vitro cultured myocardial cell field potential signal, with good biocompatibility, simple process, low cost, electrode size can be flexibly designed advantage, with wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to an in-vitro myocardial flexible microelectrode array based on enameled wire and a preparation method thereof. BACKGROUND

[0002] Myocardial cells have electrophysiological characteristics, and electrophysiological sensing technology can realize real-time and in-situ interpretation of the electrophysiological information of myocardial tissue, and is a key to the establishment of drug evaluation function. Traditional detection methods, such as voltage clamps, diaphragm clamps and fluorescence imaging, are not suitable for continuous high-throughput electrophysiological sensing due to invasiveness and complexity of operation. The microelectrode array (MEA) has the advantage of non-invasive measurement of extracellular field potential (FP), and can realize high-throughput detection of the heart tissue on the chip by capturing the electrical signal through a group of microelectrodes in close contact with the cells.

[0003] At present, the microelectrode array has gradually developed into a widely used electrophysiological sensing platform for in-vitro electrically active cells. By processing a metal electrode array matching the size of cells on a silicon-based, glass or PDMS flexible substrate, multi-channel detection of the electrophysiological signal of the extracellular field potential of the cells can be realized. However, since the myocardial cells and other cells with electrophysiological characteristics usually have a size of microns, the preparation of micron-level metal electrodes and their passivation layers is one of the biggest obstacles to reducing the manufacturing cost of the microelectrode array.

[0004] At present, the microelectrode array is widely manufactured by microprocessing technologies such as photolithography, magnetron sputtering and the like. Firstly, a metal layer is patterned on the surface of an insulating substrate, and then a passivation layer is further patterned on the surface of the metal layer to expose the metal electrode and realize insulation of the electrode lead. However, the above microprocessed microelectrode array has some limitations in design and manufacturing method. On the one hand, the metal electrode is fixed on the surface of the substrate and has high rigidity, which cannot conform to the contraction characteristics of the myocardial tissue. On the other hand, the microprocessing manufacturing technologies such as photolithography and magnetron sputtering have high manufacturing cost and high initial equipment investment, which limits the application of the technologies. SUMMARY

[0005] The application aims to overcome the deficiencies of the prior art, and provides an in-vitro myocardial flexible microelectrode array based on enameled wire and a preparation method thereof, which solves the problems in the above background technology.

[0006] One of the technical solutions adopted by the present application to solve its technical problems is: a flexible microelectrode array based on enameled wire is provided, which comprises a PCB substrate, a microelectrode array, a cell culture ring and a transparent observation window; the transparent observation window is arranged in the PCB substrate, and the cell culture ring is arranged above the transparent observation window; the microelectrode array is arranged on the PCB substrate; the microelectrode array is arranged by enameled wires, the enameled wire is composed of a conductive core and an insulating layer wrapped around the periphery of the conductive core, and the enameled wire is exposed with a conductive end face, and the conductive end face is located in the cell culture ring above the transparent observation window.

[0007] In a preferred embodiment of the present application, the conductive end face is perpendicular to the upper surface of the transparent observation window.

[0008] In a preferred embodiment of the present application, the conductive end face is a conductive core with a platinum black layer attached to the surface, and the material of the conductive core includes copper, platinum, gold, silver and tin.

[0009] In a preferred embodiment of the present application, the diameter of the conductive core is 25 μm to 1 mm; and the thickness of the insulating layer is 5 μm to 200 μm.

[0010] In a preferred embodiment of the present application, the transparent observation window is made of transparent material, including PDMS, quartz glass and PS.

[0011] In a preferred embodiment of the present application, the upper surface of the PCB substrate is provided with a positioning groove, the enameled wire is arranged along the positioning groove, and the end portion of the enameled wire without the conductive end face is welded on the solder pad of the PCB substrate and is connected with the interface through the lead wire in the PCB substrate.

[0012] The second technical solution adopted by the present application to solve its technical problems is: a preparation method of the flexible microelectrode array based on enameled wire is provided, which comprises the following steps:

[0013] (1) exposing the conductive core of the enameled wire by mechanically cutting the enameled wire;

[0014] (2) arranging and fixing the cut enameled wire on the surface of the PCB substrate to form an electrode array;

[0015] (3) fixing the cell culture ring on the surface of the PCB substrate and locating the exposed conductive core in the cell culture ring;

[0016] (4) slotting the PCB substrate, the slotted area is located below the cell culture ring, a transparent observation window is made by using a flexible material to demold, and the transparent observation window is embedded in the slotted area of the PCB substrate and sealed.

[0017] (5) Replacing the exposed conductive inner core of the enameled wire in the cell culture ring with a potassium chloroplatinite solution to form a platinum black layer on the metal surface thereof.

[0018] In a preferred embodiment of the present application, in step (5), 50 μL to 1 mL of a potassium chloroplatinite solution with a concentration of 1 wt% to 8 wt% is added dropwise into the cell culture ring so that the potassium chloroplatinite solution completely immerses one end of the exposed conductive inner core of the enameled wire, and the reaction is carried out for 3 min to 1 h. The residual solution after the reaction is removed, 50 μL to 1 mL of pure water is added dropwise to clean the inner surface of the cell culture ring and the end surface of the enameled wire for 1 to 3 times, and the residual pure water is dried by heating.

[0019] In a preferred embodiment of the present application, in step (1), the enameled wire is vertically cut so that the cross section of the conductive inner core and the external insulating layer forms a concentric circle.

[0020] In a preferred embodiment of the present application, in step (2), the positioning grooves arranged at intervals are formed on the surface of the PCB substrate, the enameled wire is arranged along the positioning grooves, the axial position of the enameled wire is adjusted so that one end of the enameled wire with the exposed conductive inner core is located in the area to be slotted of the PCB substrate, the other end faces the pad of the PCB substrate, and the middle part of the enameled wire is fixed to the PCB substrate by glue.

[0021] Compared with the background art, the present technical solution has the following advantages:

[0022] 1. The microelectrode array of the present application is not directly anchored to a rigid substrate, but is carried by a flexible transparent observation window, has excellent bending deformation capability, can match the contraction characteristics of myocardial tissue, and can realize dynamic in-situ real-time monitoring of the field potential of myocardial tissue.

[0023] 2. The present application uses a pre-insulated enameled wire as a functional element of the microelectrode array, realizes low-cost rapid manufacturing of the microelectrode array, and solves the problems of complex preparation process and high manufacturing cost of the traditional microelectrode array patterned electrode and passivation layer.

[0024] 3. Compared with the traditional photolithography, sputtering and other processes for manufacturing planar electrodes attached to the surface of the substrate, the present application has a three-dimensional conductive end surface, which is more conducive to cell adhesion. Cells can wrap the conductive end surface in a spherical shape, which is better than the hemispherical wrapping of traditional planar microelectrode arrays, improves the sealing impedance between the metal-solution-cell, and further improves the signal amplitude and signal-to-noise ratio. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 Figure 1 is a structural diagram of an in-vitro myocardial flexible microelectrode array based on an enameled wire according to Embodiment 1 of the present application.

[0026] Figure 2 Figure 2 is an exploded view of an in-vitro myocardial flexible microelectrode array based on an enameled wire according to Embodiment 1 of the present application.

[0027] Figure 3 A schematic diagram of the cross-sectional structure of the enameled wire of Example 1;

[0028] Figure 4 A schematic diagram of the longitudinal cross-sectional structure of the enameled wire of Example 1 with a platinum black layer attached thereto;

[0029] Figure 5 A flow chart for preparing Example 2.

[0030] Wherein, 1-cell culture ring, 2-microelectrode array, 3-PCB substrate, 4-transparent observation window, 5-insulating layer, 6-conductive inner core, 7-platinum black layer. DETAILED DESCRIPTION

[0031] The technical solutions of the present application will be described clearly and completely below in combination with the drawings and examples.

[0032] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0033] Example 1

[0034] The present embodiment is an in-vitro myocardial flexible microelectrode array 2 based on enameled wire, which comprises a PCB substrate 3, a microelectrode array 2, a cell culture ring 1 and a transparent observation window 4; the transparent observation window 4 is embedded in the PCB substrate 3, and the cell culture ring 1 is arranged above the transparent observation window 4; the microelectrode array 2 is arranged on the PCB substrate 3; the microelectrode array 2 is arranged by enameled wire, the enameled wire is composed of a conductive inner core 6 and an insulating layer 5 wrapped around the outer periphery of the conductive inner core 6, and the enameled wire exposes a conductive end face, which is located in the cell culture ring 1 above the transparent observation window 4.

[0035] In the present embodiment, the transparent observation window 4 is made of PDMS material, and the cell culture ring 1 is a ring-shaped quartz glass which is bonded to the PCB substrate 3 by glue, thereby forming a cavity for culturing myocardial cells on the PCB substrate 3 with the embedded transparent observation window 4.

[0036] In the present embodiment, the material of the conductive inner core 6 of the enameled wire is copper, and the cross-sectional diameter is 80 μm; the material of the insulating layer 5 is polyurethane, and the thickness is 10 μm; the conductive end face is exposed by manually cutting the enameled wire perpendicular to the axis of the enameled wire by a cutter, and then a platinum black layer 7 is attached to the surface thereof by a displacement reaction.

[0037] In order to better fix the enameled wire, the PCB substrate 3 of the embodiment is provided with a customized structure with a groove, i.e., a positioning groove. The enameled wire is arranged in parallel on the upper surface of the PCB substrate 3, facilitating the leading-out of the other end of the enameled wire, so that the conductive end surface is perpendicular to the upper surface of the transparent observation window 4. In the embodiment, the enameled wire microelectrode is arranged in a symmetrical layout of 2x9, with a radial spacing of 1 mm. One of the 9 electrodes on one side is a pure platinum wire as a reference electrode. One end of the enameled wire is welded to the surface pad of the PCB substrate 3 through soldering, and is connected to the standard XH2.54 interface through the conductive wire on the PCB substrate 3.

[0038] The use method of the device of the embodiment is as follows: 300 μL of cell suspension containing 500,000 myocardial cells is inoculated into the cell culture ring 1, and is deposited, adhered and grown on the upper surface of the transparent observation window 4, gradually covering and coating the conductive end surface of the microelectrode array 2 located in the cell culture ring 1. When the myocardial cells generate electrophysiological activity, the extracellular field potential of the myocardial cells changes, and the microelectrode array 2 monitors the extracellular field potential through the electrical coupling of the electrode-solution-cell section. Since the conductive end surface is not closely attached to the transparent observation window 4 but is perpendicular to the upper surface of the transparent observation window 4, the myocardial cells can wrap the conductive end surface in a spherical shape during the myocardial cell culture process, improving the sealing impedance between the metal-solution-cell, and further improving the signal amplitude and signal-to-noise ratio.

[0039] Embodiment 2

[0040] Embodiment 2 provides a preparation method of the in-vitro myocardial flexible microelectrode array 2 based on the enameled wire of embodiment 1, including the following steps:

[0041] (1) A rigid PCB substrate 3 is designed using Altium Designer, and a 9x7 mm rectangular notch is designed in the center of the rigid substrate.

[0042] (2) 16 enameled wires and 2 platinum wires are manually cut using a cutter, and the interface metal core of the enameled wire is exposed;

[0043] (3) The enameled wires are arranged on the surface of the PCB substrate 3 and the spacing between the enameled wires is limited by the groove structure with a spacing of 1 mm. The axial position of the enameled wires is adjusted so that one end of the enameled wires is placed in the slotted area of the PCB substrate 3 and the other end is directed to the pad area of the PCB substrate 3. A dispensing needle is used to drop Krytox 705 silicone glue on the middle part of the enameled wire to avoid covering the two ends of the enameled wire with glue.

[0044] (4) The cell culture ring 1 is adhered to the surface of the rigid substrate using Krytox 705 silicone glue, and the silicone glue is cured for 8 h.

[0045] (5) Print a 9x7x1.6mm rectangular mold using a light-cured 3D printer, pour PDMS prepolymer (mass ratio: monomer: curing agent = 10:1), and place it in a constant temperature drying oven at 80°C for 3h to cure, demold to make a transparent observation window 4, embed it in the slotted area of the PCB rigid substrate, and seal the gap between the two with PDMS prepolymer, and then place it in a constant temperature drying oven at 80°C for 3h to cure again.

[0046] (6) Solder one end of the enameled wire to the PCB substrate 3 pad, drop 50μL-1mL of 1wt%-8wt% potassium chloroplatinite solution into the culture ring, so that it completely immerses the end of the enameled wire exposed to the conductive cross section, react for 3min-1h, absorb the residual solution after reaction, drop 50μL-1mL of pure water into the culture ring 1-3 times to clean the inner surface of the culture ring and the end surface of the enameled wire, and heat and dry the residual pure water.

[0047] The above examples are only used to illustrate the technical solutions of the present application, and not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent substitutions for part or all of the technical features; and these modifications or substitutions do not make the essence of the corresponding technical solution deviate from the scope of the technical solutions of the embodiments of the present application.

Claims

1. A flexible microelectrode array based on enameled wire for extracorporeal myocardium, characterized by: The application relates to a microelectrode array culture chip, which comprises a PCB substrate, a microelectrode array, a cell culture ring and a transparent observation window; the transparent observation window is arranged in the PCB substrate and is provided with the cell culture ring above; the microelectrode array is arranged on the PCB substrate; the microelectrode array is arranged by enameled wires, the enameled wire is composed of a conductive inner core and an insulating layer wrapped around the outer periphery of the conductive inner core, and the enameled wire is exposed with a conductive end face, which is located in the cell culture ring above the transparent observation window.

2. The flexible microelectrode array based on enameled wire for extracorporeal myocardium according to claim 1, characterized in that: The conductive end face is perpendicular to the upper surface of the transparent observation window.

3. The flexible microelectrode array based on enameled wire for extracorporeal cardiac muscle according to claim 1, characterized in that: The conductive end face is a conductive inner core with a platinum black layer attached to the surface, and the material of the conductive inner core comprises copper, platinum, gold, silver and tin.

4. The flexible microelectrode array based on enameled wire for extracorporeal cardiac muscle according to claim 1, characterized in that: The diameter of the conductive inner core is 25-1mm; and the thickness of the insulating layer is 5-200mu.

5. The flexible microelectrode array based on enameled wire for extracorporeal cardiac muscle according to claim 1, characterized in that: The upper surface of the PCB substrate is provided with a positioning groove, and the enameled wire is arranged along the positioning groove.

6. The flexible microelectrode array based on enameled wire for extracorporeal cardiac muscle according to claim 1, characterized in that: The end of the enameled wire is welded on the pad of the PCB substrate and is in conduction with the interface through the lead wire in the PCB substrate.

7. A method for fabricating a flexible microelectrode array based on enameled wire for extracorporeal myocardium, characterized by: The application further discloses a manufacturing method of the microelectrode array culture chip. (1) exposing the conductive inner core of the enameled wire by mechanically cutting the enameled wire; (2) arranging and fixing the cut enameled wire on the surface of the PCB substrate to form an electrode array; (3) fixing the cell culture ring on the surface of the PCB substrate and locating the exposed conductive inner core in the cell culture ring; (4) slotting the PCB substrate, locating the slotted area below the cell culture ring, manufacturing the transparent observation window by using a flexible material, embedding the transparent observation window in the slotted area of the PCB substrate and sealing the transparent observation window; (5) replacing the exposed conductive inner core of the enameled wire in the cell culture ring with potassium chloroplatinite solution to form a platinum black layer on the metal surface of the conductive inner core.

8. The method for fabricating an in vitro flexible myocardial microelectrode array based on enameled wire according to claim 7, characterized in that: In step (5), 50-1000muL of potassium chloroplatinite solution with a concentration of 1-8wt% is added dropwise in the cell culture ring, the potassium chloroplatinite solution completely immerses one end of the exposed conductive inner core of the enameled wire, the reaction lasts for 3min-1h, the residual solution after the reaction is absorbed, 50-1000muL of pure water is added dropwise to clean the inner surface of the cell culture ring and the end face of the enameled wire for 1-3 times, and the residual pure water is heated and dried.

9. The method for fabricating an in vitro flexible myocardial microelectrode array based on enameled wire according to claim 7, characterized in that: In step (1), the enameled wire is vertically cut, and the cross section of the conductive inner core and the external insulating layer forms a concentric circle.

10. The method for fabricating an in vitro flexible myocardial microelectrode array based on enameled wire according to claim 7, characterized in that: In step (2), the positioning grooves are arranged on the surface of the PCB substrate, the enameled wire is arranged along the positioning grooves, the axial position of the enameled wire is adjusted, one end of the enameled wire with the exposed conductive inner core is located in the slotted area of the PCB substrate, the other end faces the pad of the PCB substrate, and the middle part of the enameled wire is fixed to the PCB substrate by glue.

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