Suspended flexible microelectrode array device and manufacturing method and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a manufacturing method of a flexible suspended microelectrode array device, which comprises the following steps of: manufacturing a device frame with a culture tank, printing a suspended ordered carrier fiber array above the culture tank through a suspended melting electrospinning process, and carrying out metal layer patterning sputtering on an electrode on carrier fiber to obtain the flexible suspended microelectrode array device. Then forming a patterned insulating layer on the surface of the metal layer by using an electrostatic spraying mode, and finally integrating an electrostatic spinning ordered fiber scaffold as a cell culture scaffold. The prepared suspended and flexible microelectrode array device can ensure the attachment of the electrode and the cell for a long time in the cell in-vitro culture process, and realizes the long-term detection of a field potential signal. The manufacturing method does not need sacrifice materials, the preparation process is simple and efficient, the cost is reduced, and wide application prospects are achieved.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a suspended, flexible microelectrode array device and a manufacturing method and application thereof. Background Art

[0002] Drug development is a long process that consumes huge costs and has a high failure rate. Since animal models cannot fully simulate the correct response of the human body to drugs, about 92% of drugs that have been proven effective in preclinical trials cannot pass clinical trials. There are many cells in the human body that have electrophysiological properties, such as cardiomyocytes and nerve cells. Electrophysiological techniques based on in vitro cells can effectively test the effects of drugs on cells. Microelectrode arrays have very strong advantages in detecting in vitro field potential signals of cells due to their advantages such as high resolution and small size.

[0003] In the traditional detection of electrophysiological signals in engineered tissues, microelectrode arrays are usually manufactured on rigid substrates in combination with mature semiconductor technology. However, due to the large difference in modulus between the rigid substrate and the human tissue environment, it is difficult for cells cultured on the rigid substrate to form a good fit with the electrodes, resulting in mechanical mismatch, which makes it difficult for cells and electrodes to be in close contact for a long time and easy to fall off the electrode surface. In recent years, the trend of transition from rigid electrodes to flexible electrodes in microelectrode arrays can solve the problem of cell-electrode fit. By using materials with a Young's modulus of only a few kPa and a few MPa, such as hydrogels and PDMS, as substrates, field potential signal detection is performed on them, which is conducive to restoring the electrophysiological signals of cells in the body.

[0004] However, in terms of microstructure, these flexible substrates cannot provide good topographic induction and mechanical environment for some cells, such as muscle cells, resulting in disordered cell growth orientation, making it difficult to truly simulate the real structure and mechanical environment in vivo. Therefore, it is necessary to construct low-rigidity flexible electrodes in vitro to ensure good contact between cells and electrodes, while achieving three-dimensional culture and orderly growth of engineered tissues, and promoting high maturity of in vitro cell culture. This requires a new microelectrode array device. Summary of the invention

[0005] In view of the above difficulties, the purpose of the present invention is to construct a suspended, flexible microelectrode array device and its manufacturing method and application, which can not only provide a three-dimensional culture and orderly growth structure and mechanical environment for engineered tissues, but also can follow the contraction movement of cells through flexible electrodes, thereby solving the problem of long-term difficulty in detecting field potential signals of myocardial tissue.

[0006] The present invention is achieved through the following technical solutions:

[0007] A method for manufacturing a suspended, flexible microelectrode array device comprises the following steps:

[0008] Step 1: manufacturing a frame structure, wherein the frame structure has a culture tank;

[0009] Step 2: using a thermoplastic polymer material to manufacture a carrier fiber array through a melt electrospinning process, wherein the carrier fiber array is suspended and arranged on a culture tank;

[0010] Step 3: depositing a patterned metal layer by a sputtering process, wherein the metal layer is deposited on the surface of the carrier fiber and extends to the surface of the framework structure to form a conductive network;

[0011] Step 4: depositing a patterned insulating layer on the metal layer by a spray process, selectively exposing a portion of the metal layer on the surface of the carrier fiber as an electrode and a portion of the metal layer on the surface of the frame structure as a pad, to construct a microelectrode array;

[0012] Step 5: Fabricate ordered fiber scaffolds on the microelectrode array of the culture tank through electrospinning process.

[0013] Optionally, in step 1, the frame structure is manufactured by a melt deposition process, the thermoplastic polymer material is heated to a molten state, the inner diameter of the nozzle is 0.1 to 2 mm, the melting temperature is 100 to 300° C., the nozzle moving speed is 30 to 300 mm / s, and the extrusion pressure is 20 to 250 kPa. The thermoplastic polymer material for melt deposition includes but is not limited to one of polylactic acid (PLA), polycaprolactone (PCL), and thermoplastic polyurethane elastomer (TPU).

[0014] Optionally, in step 2, the thermoplastic polymer material is one of polylactic acid, polycaprolactone, and thermoplastic polyurethane elastomer; the conditions of the melt electrospinning process are: the inner diameter of the nozzle is 0.1-1 mm, the melting temperature is 100-300°C, the nozzle moving speed is 150-300 mm / s, the extrusion pressure is 20-250 kPa, the melt electrospinning voltage is 1-8 kV, and the distance between the nozzle and the collecting plate is 1-5 mm.

[0015] Optionally, in the carrier fiber array, the diameter of the carrier fibers ranges from 5 to 100 μm, and the spacing between the carrier fibers ranges from 300 to 1000 μm.

[0016] Optionally, along the extension direction of the carrier fiber, the metal layer forms two spaced segments on each carrier fiber; the opposite ends of the two segments extend to the surface of the frame structure respectively, and the opposite ends are exposed under the insulating layer to form two electrodes on each carrier fiber.

[0017] Optionally, the metal layer is a stack of an adhesion promoting layer / conductive layer, the material of the adhesion promoting layer is Ti, and the material of the conductive layer is one of Pt and Au. The thickness of the adhesion promoting layer ranges from 3 to 8 nm, and the thickness of the conductive layer ranges from 200 to 300 nm.

[0018] Optionally, the insulating layer is made of a biocompatible insulating material, including one of ethyl cellulose (EC), polyimide (PI), and polyvinyl alcohol ether (PEG); the insulating layer material is dissolved in an organic solvent to form a spray solution, the spray process is an electrostatic spray process, the inner diameter of the needle is 0.11 mm, the receiving distance is 5 to 20 cm, the solution propulsion speed is 100 to 2000 μL / h, and the voltage at the nozzle is 5 to 30 kV.

[0019] The organic solvent of the spray solution includes but is not limited to one or a mixed solvent of anhydrous ethanol, dimethylformamide, and acetone; the content of the insulating layer material in the spray solution is 1 to 5 wt%.

[0020] Optionally, the material of the ordered fiber scaffold includes at least one of polycaprolactone (PCL), polylactic acid (PLA), thermoplastic polyurethane elastomer (TPU), and polyimide (PI); the ordered fiber scaffold material and an organic solvent are mixed to form an electrospinning solution, and the conditions of the electrospinning process are: the inner diameter of the needle is 0.25 mm, the voltage at the nozzle is 5 to 30 kV, the solution propulsion speed is 100 to 1000 μL / h, and the receiving distance is 10 to 30 cm.

[0021] The organic solvent of the electrospinning solution includes, but is not limited to, one or a mixed solvent of glacial acetic acid, chloroform, dimethylformamide, and acetone; the content of the ordered fiber scaffold material in the electrospinning solution is 15-25wt%.

[0022] Optionally, the metal layer and the insulating layer are patterned and deposited using corresponding masks. Microelectrodes of different stiffness and scale can be constructed by changing the electrode material and manufacturing process parameters.

[0023] The suspended, flexible microelectrode array device manufactured by the above-mentioned method for manufacturing a suspended, flexible microelectrode array device includes a frame structure having a culture tank in which a suspended, flexible microelectrode array is arranged, and an ordered fiber support is arranged on the microelectrode array.

[0024] The above-mentioned suspended, flexible microelectrode array device is used in the culture of myocardial cells, wherein the field potential signal of the myocardial cells is detected by the microelectrode array.

[0025] The beneficial effects of the present invention are:

[0026] The present invention provides a method for manufacturing a suspended, flexible microelectrode array device, which uses suspended melt electrospinning to manufacture flexible, micron-scale suspended fibers as electrode carriers; the metal layer and the insulating layer structure are patterned and manufactured on the carrier fiber array by sputtering and electrostatic spraying, respectively, to finally achieve selective exposure of electrodes and pads. The prepared suspended, flexible microelectrode array device can ensure the long-term adhesion of electrodes and cells during in vitro cell culture, realize long-term detection of field potential signals, and ensure that cells achieve three-dimensional and orderly growth during the culture process, and construct corresponding in vivo physiological structures and mechanical environments according to the growth requirements of different cells; solve the problem of difficulty in three-dimensional directional arrangement of in vitro cell culture and difficulty in long-term detection of field potential signals, and has very broad application prospects; the manufacturing method of the present invention does not require sacrificial materials, and the preparation process is simple and efficient. At the same time, a single mask plate can be used to manufacture electrodes with different size requirements, reducing the cost required to manufacture the mask plate, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is a flow chart of the manufacturing process of the suspended and flexible microelectrode array device of the embodiment;

[0028] Figure 2 The schematic diagram of the structure and the partially enlarged schematic diagram of the suspended and flexible microelectrode array device of the embodiment;

[0029] Figure 3 The actual picture and SEM image of the suspended and flexible microelectrode array device of the embodiment;

[0030] Figure 4 The optical morphology micrographs and immunofluorescence staining images of cardiomyocytes cultured for multiple days on a suspended, flexible microelectrode array device in the embodiment;

[0031] Figure 5 The present invention is a schematic diagram of multi-day field potential signal detection of cardiomyocytes on a suspended, flexible microelectrode array device. DETAILED DESCRIPTION

[0032] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments.

[0033] The schematic diagram of the manufacturing method of the suspended, flexible microelectrode array device of the embodiment is as follows Figure 1 The structural diagram and partial enlarged diagram of the fabricated suspended and flexible microelectrode array device are shown in Figure 2 As shown, combined Figure 1 and Figure 2 The embodiments are described in detail.

[0034] See also Figure 1Step I, adding thermoplastic polylactic acid (PLA) material into the heating barrel of the 3D printer, heating it to a molten state at 190°C, and using a fused deposition process to print a frame structure 1 with a culture tank 11. The fused deposition process conditions are: the inner diameter of the nozzle is 400 μm, the nozzle moving speed is 50 mm / s, the extrusion pressure is 100 kPa, and the melting temperature is 190°C.

[0035] See also Figure 1 Step II, add thermoplastic polyurethane elastomer (TPU) into the barrel of the 3D printer, heat it to a molten state at 250°C, and use the suspended melt electrospinning process to manufacture suspended, flexible, ordered micro-nanoscale fibers to form a carrier fiber array. The carrier fiber array is arranged in order in the culture tank 11. The suspended melt electrospinning process conditions: the inner diameter of the nozzle is 200μm, the nozzle movement speed is 250mm / s, the melting temperature is 250°C, the voltage at the nozzle is 2.4kV, the receiving distance is 3mm, and the extrusion pressure is 50kPa. The carrier fibers 2 in the array are arranged in parallel and spaced, and the two ends are overlapped on the surface of the frame structure 1 at the edge of the culture tank 11; the diameter of the carrier fiber 2 is 10μm and the spacing is 900μm.

[0036] See also Figure 1 In step III and step IV, a stainless steel mask is used to selectively expose the metal layer area that needs to be sputtered, and a magnetron sputtering machine is used to deposit 5nm of Ti and 250nm of Pt in succession to construct the conductive network of the device. The Ti / Pt metal layer 3 is deposited on the surface of the carrier fiber 2 and extends to the surface of the framework structure 1. Specifically, along the extension direction of the carrier fiber 2, the metal layer 3 forms two interval sections 3a and 3b on each carrier fiber 2, and the interval area (i.e., the area where the metal is not deposited) is preferably located in the middle of the carrier fiber 2, and the length is, for example, 500μm; the opposite ends of the two sections 3a and 3b extend to the surface of the framework structure 1, and the distance between the opposite ends is the length of the interval area.

[0037] Weigh 3 g of ethyl cellulose powder into 97 g of anhydrous ethanol solution to make a 3% (w / w) ethyl cellulose solution, and stir on a magnetic stirrer until the solution is clear. Figure 1 In step V and step VI, a stainless steel mask is used to selectively expose the area that needs to be covered with the insulating layer, and an electrostatic spray equipment is used to electrostatically spray and deposit the insulating layer 4. The electrostatic spray process conditions are: the inner diameter of the needle is 0.18 mm, the receiving distance is 10 cm, the solution propulsion speed is 1200 μL / h, and the voltage at the nozzle is 8 kV. The insulating layer 4 covers the metal layer 3, so that the opposite ends of the sections 3a and 3b are exposed (i.e., not covered with insulating material) to form electrodes, and the end of the metal layer located on the surface of the frame structure is exposed as a pad to construct a microelectrode array, so that two electrodes are formed on each carrier fiber 2.

[0038] Polycaprolactone (PCL) and anhydrous ethanol were mixed in a mass ratio of 2:8 to form an electrospinning solution. Figure 1 Step VII, construct an ordered fiber scaffold 5 on the microelectrode array by electrospinning process, and the deposition conditions are: the inner diameter of the needle is 0.25mm, the voltage at the nozzle is 10kV, the solution propulsion speed is 500μL / h, and the receiving distance is 10cm. The ordered fiber scaffold 5 is parallel and spaced fibers, with a fiber diameter of 1.19±0.20μm, a fiber density of 250pcs / mm, and a fiber extension direction perpendicular to the extension direction of the carrier fiber 2. The engineered tissue of the ordered fiber scaffold 5 provides a three-dimensional culture, orderly growth structure and mechanical environment, and the ordered fiber scaffold 5 is directly formed on the suspended, flexible microelectrode array surface, which can achieve good connection and synchronous action.

[0039] A suspended, flexible microelectrode array device is manufactured through the above steps. Figure 3 See Figure 1 Step VIII: To facilitate in vitro culture of myocardial tissue, a culture ring is attached to the device surface using Kraft glue to confine the culture of myocardial cells within the culture ring. Myocardial cells derived from pluripotent stem cells obtained by in vitro culture and differentiation of human induced pluripotent stem cells are inoculated on the device surface. After inoculation, culture medium is added for culture. The growth of myocardial cells within 10 days and immunofluorescence staining are shown in Figure 4. Figure 4 As shown, the cells showed good activity and grew in an orderly manner.

[0040] The sections 3a and 3b of the metal layer 3 extend to the pads on the surface of the frame structure 1 to connect to the field potential signal detection system. The field potential signal of the myocardial cells on the device is detected for up to ten days through two electrodes. The field potential signal results are as follows: Figure 5 As shown, from left to right are the electrophysiological signals of myocardial tissue monitored by the microelectrode on the 2nd day, the 6th day and the 10th day. The field potential signal of the myocardial tissue increases with the increase of culture days, and the electrophysiological maturity of the cells increases accordingly. At the same time, the device shows good long-term culture ability.

[0041] The above embodiments are only used to further illustrate a suspended, flexible microelectrode array device, a manufacturing method and an application of the present invention, but the present invention is not limited to the embodiments. Any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention fall within the protection scope of the technical solution of the present invention.

Claims

1. A method for manufacturing a suspended, flexible microelectrode array device, characterized in that: The steps include: Step 1: manufacturing a frame structure, wherein the frame structure has a culture tank; Step 2: using a thermoplastic polymer material to manufacture a carrier fiber array through a melt electrospinning process, wherein the carrier fiber array is suspended and arranged on a culture tank; Step 3: depositing a patterned metal layer by a sputtering process, wherein the metal layer is deposited on the surface of the carrier fiber and extends to the surface of the framework structure to form a conductive network; Step 4: depositing a patterned insulating layer on the metal layer by a spray process, selectively exposing a portion of the metal layer on the surface of the carrier fiber as an electrode and a portion of the metal layer on the surface of the frame structure as a pad, to construct a microelectrode array; Step 5: Fabricate ordered fiber scaffolds on the microelectrode array of the culture tank through electrospinning process.

2. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: In step 1, the frame structure is manufactured by a molten deposition process, the thermoplastic polymer material is heated to a molten state, the inner diameter of the nozzle is 0.1 to 2 mm, the melting temperature is 100 to 300° C., the nozzle moving speed is 30 to 300 mm / s, and the extrusion pressure is 20 to 250 kPa.

3. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: In step 2, the thermoplastic polymer material is one of polylactic acid, polycaprolactone, and thermoplastic polyurethane elastomer; the conditions of the melt electrospinning process are: the inner diameter of the nozzle is 0.1-1 mm, the melting temperature is 100-300°C, the nozzle moving speed is 150-300 mm / s, the extrusion pressure is 20-250 kPa, the melt electrospinning voltage is 1-8 kV, and the distance between the nozzle and the collecting plate is 1-5 mm.

4. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: In the carrier fiber array, the diameter of the carrier fibers ranges from 5 to 100 μm, and the spacing between the carrier fibers ranges from 300 to 1000 μm.

5. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: Along the extension direction of the carrier fiber, the metal layer forms two spaced sections on each carrier fiber; the opposite ends of the two sections extend to the surface of the frame structure respectively, and the opposite ends are exposed under the insulating layer to form two electrodes on each carrier fiber.

6. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: The metal layer is a stack of an adhesion-promoting layer and a conductive layer. The material of the adhesion-promoting layer is Ti, and the material of the conductive layer is one of Pt and Au.

7. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: The material of the insulating layer is one of ethyl cellulose, polyimide, and polyvinyl alcohol ether; the insulating layer material is dissolved in an organic solvent to form a spray solution, the spray process is an electrostatic spray process, the inner diameter of the needle is 0.11mm, the receiving distance is 5-20cm, the solution propulsion speed is 100-2000μL / h, and the voltage at the nozzle is 5-30kV.

8. The method for manufacturing a suspended, flexible microelectrode array device according to claim 1, characterized in that: The material of the ordered fiber scaffold includes at least one of polycaprolactone, polylactic acid, thermoplastic polyurethane elastomer, and polyimide; the ordered fiber scaffold material and an organic solvent are mixed to form an electrospinning solution, and the conditions of the electrospinning process are: the inner diameter of the needle is 0.25 mm, the voltage at the nozzle is 5 to 30 kV, the solution propulsion speed is 100 to 1000 μL / h, and the receiving distance is 10 to 30 cm.

9. A suspended, flexible microelectrode array device manufactured by the method for manufacturing a suspended, flexible microelectrode array device according to any one of claims 1 to 8, characterized in that: The invention comprises a frame structure, wherein the frame structure has a culture tank, a suspended and flexible microelectrode array is arranged in the culture tank, and an ordered fiber support is arranged on the microelectrode array.

10. Use of the suspended, flexible microelectrode array device according to claim 9 in the culture of myocardial cells, characterized in that: The field potential signal of the cardiomyocytes is detected by the microelectrode array.

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