A suspended, flexible microelectrode array device, its manufacturing method, and its application.

Abstract

This invention relates to the field of bioengineering technology, specifically proposing a method for manufacturing a flexible, suspended microelectrode array device. The method involves fabricating a device frame with a culture tank, and then printing a suspended, ordered carrier fiber array above the culture tank using a suspended melt electrospinning process. Next, patterned metal layers for electrodes on the carrier fibers are sputtered, followed by electrostatic spraying to form a patterned insulating layer on the metal layer surface. Finally, the electrospun ordered fiber scaffold is integrated as a cell culture scaffold. The fabricated suspended, flexible microelectrode array device can maintain electrode-cell adhesion for extended periods during in vitro cell culture, enabling long-term detection of field potential signals. This invention's manufacturing method requires no material sacrifice, has a simple and efficient fabrication process, reduces costs, and has broad application prospects.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a suspended, flexible microelectrode array device, its manufacturing method, and its application. Background Technology

[0002] Drug development is a lengthy, costly, and high-failure-rate process. Because animal models cannot fully mimic the correct human response to drugs, approximately 92% of drugs proven effective in preclinical trials fail to pass clinical trials. Many cells in the human body possess electrophysiological properties, such as cardiomyocytes and nerve cells. Electrophysiological techniques based on in vitro cells can effectively test the mechanisms by which drugs affect cells. Microelectrode arrays, due to their high resolution and small size, offer significant advantages in detecting in vitro field potential signals in cells.

[0003] In traditional engineered tissue electrophysiological signal detection, microelectrode arrays are typically fabricated on rigid substrates using mature semiconductor technology. However, due to the significant difference in modulus between rigid substrates and the human tissue environment, cells cultured on rigid substrates struggle to adhere well to the electrodes, leading to mechanical mismatch. This results in cells and electrodes failing to maintain close contact for extended periods and easily detaching from the electrode surface. In recent years, the trend towards flexible electrodes in microelectrode arrays has effectively addressed this issue. By using materials with Young's modulus of only a few kPa and a few MPa, such as hydrogels and PDMS, as substrates, and performing field potential signal detection on these substrates, it is beneficial for reconstructing the electrophysiological signals of cells in vivo.

[0004] However, at the microstructural level, these flexible substrates cannot provide adequate topographic guidance and mechanical environment for some cells, such as muscle cells, leading to disordered cell growth orientation and making it difficult to truly simulate the real structure and mechanical environment in vivo. Therefore, it is necessary to construct low-stiffness flexible electrodes in vitro to ensure good contact between cells and electrodes while enabling three-dimensional culture and orderly growth of engineered tissues, promoting high maturity of in vitro cell culture. This requires a new type of microelectrode array device. Summary of the Invention

[0005] To address the aforementioned challenges, the present invention aims to construct a suspended, flexible microelectrode array device, its manufacturing method, and its application. This device not only provides a three-dimensional culture and orderly growth environment for engineered tissues but also solves the problem of long-term difficulty in detecting field potential signals in myocardial tissue by having flexible electrodes follow cell contraction.

[0006] This invention is achieved through the following technical solution:

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

[0008] Step 1: Manufacture a frame structure having a culture tank;

[0009] Step 2: Using thermoplastic polymer materials, a carrier fiber array is manufactured through melt electrospinning, and the carrier fiber array is suspended and arranged on the culture tank;

[0010] Step 3: A patterned metal layer is deposited by sputtering process. The metal layer is deposited on the surface of the carrier fiber and extends to the surface of the frame structure to form a conductive network.

[0011] Step 4: A patterned insulating layer is deposited on the metal layer using a spraying process, selectively exposing a portion of the metal layer on the surface of the carrier fiber as electrodes and a portion of the metal layer on the surface of the frame structure as pads to construct a microelectrode array;

[0012] Step 5: An ordered fiber scaffold is fabricated on the microelectrode array in the culture tank by electrospinning.

[0013] Optionally, in step 1, the frame structure is manufactured using a fused deposition modeling process, in which a thermoplastic polymer material is heated to a molten state, the nozzle inner diameter is 0.1–2 mm, the melting temperature is 100–300 °C, the nozzle moving speed is 30–300 mm / s, and the extrusion pressure is 20–250 kPa. The fused deposition thermoplastic polymer material 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, or thermoplastic polyurethane elastomer; the conditions of the melt electrospinning process are: nozzle inner diameter of 0.1–1 mm, melting temperature of 100–300 °C, nozzle moving speed of 150–300 mm / s, extrusion pressure of 20–250 kPa, melt electrospinning voltage of 1–8 kV, and distance between nozzle and collecting plate of 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 is 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, and the opposite ends are exposed under the insulating layer, so as to form two electrodes on each carrier fiber.

[0017] Optionally, the metal layer is a stack of an adhesion-enhancing layer and a conductive layer, wherein the adhesion-enhancing layer is made of Ti, and the conductive layer is made of either Pt or Au. The thickness of the adhesion-enhancing 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 selected from biocompatible insulating materials, 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 spraying process is an electrostatic spraying process, the needle inner diameter is 0.11 mm, the receiving distance is 5-20 cm, the solution propulsion speed is 100-2000 μL / h, and the voltage at the nozzle is 5-30 kV.

[0019] The organic solvents in the spray solution include, but are not limited to, one or more of anhydrous ethanol, dimethylformamide, and acetone; the content of insulating material in the spray solution is 1-5 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: needle inner diameter of 0.25 mm, voltage at the nozzle of 5-30 kV, solution propulsion speed of 100-1000 μL / h, and receiving distance of 10-30 cm.

[0021] The organic solvents in the electrospinning solution include, but are not limited to, one or more of the following solvents: glacial acetic acid, chloroform, dimethylformamide, and acetone; the content of ordered fiber scaffold material in the electrospinning solution is 15–25 wt%.

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

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

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

[0025] The beneficial effects of this invention are as follows:

[0026] This invention provides a method for manufacturing a suspended, flexible microelectrode array device. It utilizes suspended melt electrospinning to create flexible, micron-scale suspended fibers as electrode carriers. Metal and insulating layers are patterned on the carrier fiber array using sputtering and electrostatic spraying processes, respectively, ultimately achieving selective exposure of electrodes and pads. The fabricated suspended, flexible microelectrode array device can maintain long-term adhesion between electrodes and cells during in vitro cell culture, enabling long-term detection of field potential signals. It also ensures three-dimensional, orderly cell growth during culture, allowing for the construction of corresponding in vivo physiological structures and mechanical environments based on the growth requirements of different cells. This method solves the problems of difficult three-dimensional directional arrangement of cells in vitro and the difficulty of long-term detection of field potential signals, showing broad application prospects. The manufacturing method of this invention requires no material sacrifice, has a simple and efficient preparation process, and can use a single mask to manufacture electrodes of different sizes, reducing the cost of mask manufacturing and further enhancing its broad application potential. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating the manufacturing process of the suspended, flexible microelectrode array device as an example.

[0028] Figure 2 The diagram shows a schematic and a partially enlarged view of the suspended, flexible microelectrode array device as an example.

[0029] Figure 3 The images shown are physical photos and SEM images of the suspended, flexible microelectrode array device used in this embodiment.

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

[0031] Figure 5 This is a schematic diagram illustrating the detection of multi-day field potential signals in cardiomyocytes on a suspended, flexible microelectrode array device, as an example. Detailed Implementation

[0032] The present invention will be further explained below with reference to the accompanying drawings and specific embodiments.

[0033] A schematic diagram of the fabrication method of the suspended, flexible microelectrode array device in the embodiment is shown below. Figure 1 As shown, the structural schematic diagram and partially enlarged schematic diagram of the fabricated suspended, flexible microelectrode array device are as follows. Figure 2 As shown, combined with Figure 1 and Figure 2 A detailed description of the embodiments follows.

[0034] See Figure 1Step I: Thermoplastic polylactic acid (PLA) material is added to the heating barrel of the 3D printer and heated to a molten state at 190°C. The frame structure 1 with the culture tank 11 is printed using the fused deposition modeling (FDM) process. FDM process conditions: nozzle inner diameter 400μm, nozzle movement speed 50mm / s, extrusion pressure 100kPa, and melt temperature 190°C.

[0035] See Figure 1 Step II: Thermoplastic polyurethane elastomer (TPU) is added to the 3D printer barrel and heated to a molten state at 250°C. A suspended melt electrospinning process is used to create a suspended, flexible, ordered micro / nanoscale fiber carrier fiber array. The carrier fiber array is arranged in an orderly manner in the culture tank 11. The conditions for the suspended melt electrospinning process are: nozzle inner diameter 200 μm, nozzle moving speed 250 mm / s, melt temperature 250°C, nozzle voltage 2.4 kV, receiving distance 3 mm, and extrusion pressure 50 kPa. The carrier fibers 2 in the array are arranged in parallel at intervals, with both ends overlapping the surface of the frame structure 1 at the edge of the culture tank 11; the diameter of the carrier fibers 2 is 10 μm, and the spacing is 900 μm.

[0036] See Figure 1 In steps III and IV, a stainless steel mask is used to selectively expose the areas of the metal layer to be sputtered. A 5nm layer of Ti and a 250nm layer of Pt are deposited sequentially using a magnetron sputtering machine 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 frame structure 1. Specifically, along the extension direction of the carrier fiber 2, the metal layer 3 forms two spaced sections 3a and 3b on each carrier fiber 2. The spacer region (i.e., the area where no metal is deposited) is preferably located in the middle of the carrier fiber 2, with a length of, for example, 500μm. The opposite ends of the two sections 3a and 3b extend to the surface of the frame structure 1, and the distance between the opposite ends is the length of the spacer region.

[0037] Weigh 3g of ethyl cellulose powder into 97g of anhydrous ethanol solution to prepare a 3% (w / w) ethyl cellulose solution. Stir the solution on a magnetic stirrer until it becomes clear. (See also...) Figure 1 In steps V and VI, areas requiring an insulating layer are selectively exposed using a stainless steel mask, and the insulating layer 4 is deposited by electrostatic spraying using an electrostatic spraying system. The electrostatic spraying process conditions are: needle inner diameter 0.18 mm, receiving distance 10 cm, solution propulsion speed 1200 μL / h, and nozzle voltage 8 kV. The insulating layer 4 covers the metal layer 3, exposing the opposite ends of sections 3a and 3b (i.e., not covered by insulating material) to form electrodes. The ends of the metal layer on the frame structure surface are exposed as pads, constructing a microelectrode array, thereby forming two electrodes on each carrier fiber 2.

[0038] Polycaprolactone (PCL) and anhydrous ethanol were mixed thoroughly at a mass ratio of 2:8 to form an electrospinning solution. (See also...) Figure 1 Step VII involves constructing an ordered fiber scaffold 5 on the microelectrode array using electrospinning. The deposition conditions are: needle inner diameter of 0.25 mm, nozzle voltage of 10 kV, solution propulsion speed of 500 μL / h, and receiving distance of 10 cm. The ordered fiber scaffold 5 consists of parallel, spaced fibers with a diameter of 1.19 ± 0.20 μm and a fiber density of 250 pcs / mm. The fiber extension direction is perpendicular to the extension direction of the carrier fiber 2. The engineered tissue of the ordered fiber scaffold 5 provides a structural and mechanical environment for three-dimensional culture and ordered growth. Furthermore, since the ordered fiber scaffold 5 is formed directly on the suspended, flexible surface of the microelectrode array, it allows for good connectivity and synchronous movement.

[0039] A suspended, flexible microelectrode array device manufactured through the above steps is as follows: Figure 3 As shown. See also Figure 1 Step VIII: To facilitate in vitro culture of myocardial tissue, a culture loop was attached to the device surface using Kafte glue to restrict the culture of cardiomyocytes within the loop. Cardiomyocytes derived from pluripotent stem cells (induced pluripotent stem cells) obtained through in vitro culture and induced differentiation were seeded onto the device surface. After seeding, culture medium was added for further culture. The growth of cardiomyocytes over 10 days and immunofluorescence staining are shown below. Figure 4 As shown, the cells exhibit good activity and orderly growth.

[0040] Sections 3a and 3b of metal layer 3 extend to the pads on the surface of frame structure 1, connecting to a field potential signal detection system. The system uses two electrodes to detect the field potential signal of the myocardial cells on the device for up to ten days. The field potential signal results are as follows: Figure 5 As shown, from left to right, the electrophysiological signals of myocardial tissue monitored by the microelectrode on days 2, 6, and 10 are displayed. The field potential signal of myocardial tissue increases with the increase of culture days, and the maturity of cells in terms of electrophysiology increases accordingly. At the same time, the device shows good long-term culture capability.

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

Claims

1. A method for manufacturing a suspended, flexible microelectrode array device, characterized in that, The steps include: Step 1: Manufacture a frame structure having a culture tank; Step 2: Using thermoplastic polymer materials, a carrier fiber array is manufactured through melt electrospinning, and the carrier fiber array is suspended and arranged on the culture tank; Step 3: A patterned metal layer is deposited by sputtering process. The metal layer is deposited on the surface of the carrier fiber and extends to the surface of the frame structure to form a conductive network. Step 4: A patterned insulating layer is deposited on the metal layer using a spraying process, selectively exposing a portion of the metal layer on the surface of the carrier fiber as electrodes and a portion of the metal layer on the surface of the frame structure as pads to construct a microelectrode array; Step 5: An ordered fiber scaffold is fabricated on the microelectrode array in the culture tank by electrospinning.

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 using a fused deposition process, in which thermoplastic polymer material is heated to a molten state, the nozzle inner diameter is 0.1–2 mm, the melting temperature is 100–300 °C, the nozzle moving speed is 30–300 mm / s, and the extrusion pressure is 20–250 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: nozzle inner diameter of 0.1-1 mm, melting temperature of 100-300 °C, nozzle moving speed of 150-300 mm / s, extrusion pressure of 20-250 kPa, melt electrospinning voltage of 1-8 kV, and distance between nozzle and collecting plate of 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 is 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 segments on each carrier fiber; the opposite ends of the two segments extend to the surface of the frame structure, 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 adhesive layer and a conductive layer. The adhesive layer is made of Ti, and the conductive layer is made of either Pt or Au.

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

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

9. A suspended, flexible microelectrode array device manufactured by the manufacturing method of any one of claims 1 to 8, characterized in that: The system includes a frame structure with a culture tank containing a suspended, flexible microelectrode array, and an ordered fiber scaffold on the microelectrode array.

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

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