Hydrogel modified microelectrode array device and preparation method thereof

Abstract

The invention discloses a hydrogel modified microelectrode array device and a preparation method thereof, the hydrogel modified microelectrode array device comprises a microelectrode array composite layer, a passivation layer, a hydrogel layer and a cell culture ring, the cell culture ring is arranged on the microelectrode array composite layer, and the passivation layer and the hydrogel layer are located in the cell culture ring; the microelectrode array composite layer comprises a microelectrode array with polydimethylsiloxane as a base material, at least part of the microelectrode array in the cell culture ring is exposed, and at least part of gaps of the exposed microelectrode array are filled with the passivation layer; the hydrogel layer grows on the surface of the polydimethylsilane, and a microgroove is formed in the exposed microelectrode array and passivation layer area to serve as a cell growth area. According to the invention, cell patterning can be realized on the microelectrode array, cell electrophysiological activity can be monitored for a long time, and the microelectrode array has the advantages of remarkable cell patterning promotion capability, high stability and robustness, flexible cell pattern design, excellent biological compatibility and simple process.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and specifically relates to a hydrogel-modified microelectrode array device and a preparation method thereof. Background Art

[0002] Cell electrophysiology research is an important field to explore the generation, conduction and regulation mechanisms of cell electrical signals. The methods currently used in cell electrophysiology research mainly include patch clamp technology, voltage clamp technology, and intracellular and extracellular recording methods. Microelectrode arrays, as one of the sensors for extracellular recording, can synchronously record electrophysiological signals of multiple cells or tissue sites with high throughput and high density. They have long-term stability and are non-invasive, providing a powerful tool for neuroscience research, retinal research, cardiomyocyte research, and drug development.

[0003] Cell patterning is a technology that controls the shape and size of cell adhesion. It provides a powerful tool for in-depth exploration of cell behavior, construction of in vitro disease models, accelerated drug screening and evaluation, and development of smart biomaterials. At present, the methods for realizing cell patterning on the surface of microelectrode arrays mainly include using soft lithography, laser etching, additive manufacturing and other technologies to construct three-dimensional microstructures to induce cell adhesion and growth at specific locations, or chemical modification or physical adsorption to construct molecular patterns to promote or repel cell adhesion. However, since the size of the electrodes exposed in the microelectrode array is usually in the micron order, the construction of three-dimensional microstructures for cell patterning on the microelectrode array requires a high-precision manufacturing process to match the electrode size, and the existing technology is difficult to achieve high-resolution three-dimensional micropatterns in the electrode area, and the construction of three-dimensional microstructures usually involves multiple process steps, such as photoresist coating, exposure, development, etching, etc., which limits the high-throughput manufacturing of microelectrode array devices with cell patterning functions.

[0004] Chemical modification and physical adsorption methods usually use the differences between electrode materials and substrate materials to introduce specific functional groups or molecules on the surface of microelectrode arrays to promote cell adhesion. However, the shape of the micropatterns that can be constructed by this method is limited. Usually, only independent cell adhesion areas can be constructed on a single electrode surface. Cells on different electrode surfaces cannot be directly connected, which limits its research on intercellular electrical signal transmission. In addition, this method is greatly affected by the surface morphology of the material, ambient temperature, etc., and its stability in long-term use is insufficient, which affects the cell patterning effect. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a hydrogel-modified microelectrode array device and a preparation method thereof.

[0006] In order to achieve the above purpose, the technical solution of the present invention is:

[0007] A hydrogel-modified microelectrode array device comprises a microelectrode array composite layer, a passivation layer, a hydrogel layer and a cell culture ring; the microelectrode array composite layer comprises a polydimethylsilane substrate, a patterned metal layer and a polydimethylsilane insulating layer in sequence, the patterned metal layer forms a microelectrode array, and the polydimethylsilane insulating layer partially covers the patterned metal layer; the cell culture ring is arranged on the microelectrode array composite layer, and the passivation layer and the hydrogel layer are located in the cell culture ring; wherein at least part of the microelectrode array in the cell culture ring is exposed, and the passivation layer fills at least part of the gap of the exposed microelectrode array; the hydrogel layer grows on the polydimethylsilane surface, and forms microgrooves in the exposed microelectrode array and passivation layer areas as cell growth areas.

[0008] Optionally, the material of the passivation layer is at least one of silicon nitride, silicon carbide and aluminum oxide, and the thickness is 10 nm to 1 μm.

[0009] Optionally, the hydrogel layer has a thickness of 10 μm to 500 μm.

[0010] Optionally, the hydrogel layer is formed into one or a plurality of microgrooves that are not interconnected by patterning the exposed microelectrode array and the passivation layer area, wherein a single microgroove includes one or more exposed microelectrodes.

[0011] Optionally, the material of the patterned metal layer may be gold, platinum, or titanium, with a thickness of 50 nm to 500 nm.

[0012] Optionally, the monomer material of the hydrogel layer is at least one of acrylamide, polyethylene glycol diacrylate, and methacrylated gelatin.

[0013] Optionally, the polydimethylsilane in the cell culture ring has a doped surface layer of a hydrophobic photoinitiator.

[0014] The method for preparing the hydrogel-modified microelectrode array device comprises the following steps:

[0015] (1) forming a patterned metal layer on the polydimethylsilane-based bottom surface as a microelectrode array;

[0016] (2) patterning and depositing a passivation layer, wherein the passivation layer fills at least a portion of the gaps in the microelectrode array;

[0017] (3) depositing a polydimethylsilane insulating layer to cover a portion of the patterned metal layer;

[0018] (4) attaching a cell culture ring on the polydimethylsilane insulating layer, wherein the cell culture ring has an exposed microelectrode array and a passivation layer;

[0019] (5) doping the polydimethylsilane surface within the cell culture ring with a photoinitiator;

[0020] (6) Adding a hydrogel monomer solution into the cell culture ring, growing a hydrogel layer under the action of light, and forming microgrooves in the exposed microelectrode array and passivation layer areas of the hydrogel layer to serve as cell growth areas.

[0021] Optionally, in step (5), an organic solution containing a hydrophobic photoinitiator is added to the cell culture ring and allowed to stand, so that the hydrophobic photoinitiator is doped into the polydimethylsilane through swelling.

[0022] Optionally, the hydrophobic photoinitiator is benzophenone, and the organic solvent is one of alcohol and acetone.

[0023] Furthermore, the preparation method includes: placing a patterned hollowed-out mask plate on the surface of a microelectrode array with polydimethylsiloxane as the substrate, and using a magnetron sputtering process to spray a passivation layer; placing a patterned hollowed-out mask plate on the surface of a microelectrode array with polydimethylsiloxane as the substrate, and using an electrostatic spray process to spray a polydimethylsilane insulating layer; bonding a cell culture ring to the surface of a microelectrode array with polydimethylsiloxane as the substrate using glue; dripping an organic solution containing a hydrophobic photoinitiator into the cell culture ring; sucking out the organic solution containing the hydrophobic photoinitiator and washing it with pure water or alcohol; dripping an aqueous solution of a hydrogel monomer into the cell culture ring, and irradiating it with ultraviolet light to grow a hydrogel layer; sucking out the aqueous solution of the hydrogel monomer and washing it with pure water.

[0024] Among them, the pattern of the passivation layer can be changed by changing the shape of the hollowed-out area of ​​the mask; the material used for the electrostatic spray process to spray the polydimethylsilane insulating layer is a prepolymer of polydimethylsiloxane, which is polymerized and cured at room temperature to 120°C after spraying; the mass fraction of the hydrophobic photoinitiator in the organic solution containing the hydrophobic photoinitiator is 10% to 30%; the wavelength of the ultraviolet light is 285nm to 410nm, and the illumination time is 3min to 6h.

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

[0026] (1) The exposed patterned metal layer and the passivation layer are arranged to form microgrooves in the hydrogel layer, which can construct a variety of different three-dimensional microstructures. At the same time, the hydrogel layer is hydrophilic and inert to protein adsorption, thereby promoting the poor adhesion of cells on the hydrogel layer. The two together induce the cells to adhere and grow along the hydrogel microgrooves, which has a stronger cell patterning performance.

[0027] (2) The hydrogel layer grows from the polydimethylsiloxane surface of the microelectrode array composite layer using the principle of photoinduced grafting. Its molecular network deeply interpenetrates with polydimethylsiloxane and has good stability and robustness, which solves the problem of insufficient long-term stability of traditional chemical modification and physical adsorption.

[0028] (3) By combining the passivation layer with the patterned metal layer, the graphic structure of the hydrogel microgroove is constructed. The introduction of the passivation layer greatly broadens the boundaries of graphic design, making the microgroove pattern of the hydrogel layer flexible and changeable according to actual application requirements, achieving diversified design, and solving the graphic limitation caused by traditional chemical modification and physical adsorption that can only regulate electrode materials or substrate materials.

[0029] Other features and advantages of the present invention will be set forth in the following description, and in part will be apparent from the description, or may be learned by practicing the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Schematic diagram of the overall structure of the hydrogel-modified microelectrode array device of the embodiment;

[0031] Figure 2 A schematic diagram of the exploded structure of a hydrogel-modified microelectrode array device of an embodiment;

[0032] Figure 3 It is a top view of a cell culture ring of a hydrogel-modified microelectrode array device of an embodiment;

[0033] Figure 4 For example, the cell culture ring of the hydrogel-modified microelectrode array device is Figure 1 A partial enlarged view of the cross section along the A-A' direction;

[0034] Figure 5 is a process flow chart of a method for preparing a hydrogel-modified microelectrode array device according to an embodiment;

[0035] Figure 6 The figure is a relationship diagram between the thickness of the hydrogel layer and the ultraviolet irradiation time in the method for preparing the hydrogel-modified microelectrode array device of the embodiment;

[0036] Figure 7 1 is a physical picture of the hydrogel layer in the hydrogel-modified microelectrode array device of the embodiment, wherein a is a cross-sectional view of the hydrogel layer, and b is a surface morphology view of the hydrogel layer;

[0037] Figure 8 Schematic diagram of the colored structure of the hydrogel-modified microelectrode array device of the embodiment, wherein a is a colored isometric view, and b is a partial enlarged schematic diagram;

[0038] Fig. 9 1 is a physical picture of the hydrogel layer and the patterned metal layer in the hydrogel-modified microelectrode array device of the embodiment, wherein a is a top view of the hydrogel layer and the patterned metal layer; b is a top view near one of the microelectrodes; c is a three-dimensional reconstruction near one of the microelectrodes;

[0039] Fig.10 Schematic diagram of cell inoculation in a hydrogel-modified microelectrode array device of an embodiment, wherein a is a schematic diagram of the operation of adding a cell suspension, and b is a schematic diagram of cell growth. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely described below in conjunction with the accompanying drawings.

[0041] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] refer to Figures 1 to 4 The hydrogel-modified microelectrode array device of this embodiment includes a microelectrode array composite layer 1 with polydimethylsiloxane as the substrate, a passivation layer 2, a hydrogel layer 3 and a cell culture ring 4. The microelectrode array composite layer 1 includes a polydimethylsiloxane substrate 11, a patterned metal layer 12 and a polydimethylsilane insulating layer 13. The patterned metal layer 12 forms a microelectrode array. The polydimethylsilane insulating layer 13 partially covers the patterned metal layer 12 and leaves part of the patterned metal layer 12 exposed. In this embodiment, the patterned metal layer 12 includes two microelectrode arrays 12a arranged opposite to each other, the central area of ​​which is a working electrode, which is formed by the ends of a number of microelectrodes arranged in parallel and spaced apart, and the edge area is an electrode lead interface. The polydimethylsiloxane insulating layer 13 partially covers the upper surface of the patterned metal layer 12 to form the above two. The cell culture ring 4 is arranged on the microelectrode array composite layer 1, and the passivation layer 2 and the hydrogel layer 3 are located in the cell culture ring 4; wherein the working electrode area is located in the cell culture ring 4, and the passivation layer 2 fills the gaps in each exposed microelectrode array 12a. The hydrogel layer 3 grows on the polydimethylsilane surface (i.e., including the exposed polydimethylsiloxane substrate 11 and the surface of the polydimethylsilane insulating layer 13), and forms microgrooves 31 in the exposed microelectrode array 12a and the passivation layer 2 area as a cell growth area.

[0043] In this embodiment, the material of the patterned metal layer 12 is platinum, the material of the passivation layer 2 is silicon nitride, the material of the hydrogel layer 3 is polyacrylamide, and the material of the cell culture ring 4 is quartz glass.

[0044] In this embodiment, the working electrodes (i.e., the exposed microelectrode array 12a) are arranged in two rows, and the passivation layer 2 is a plurality of rectangular patterns, which are arranged between adjacent microelectrodes in the same row. The cell culture ring 4 is bonded to the upper surface of the microelectrode array composite layer 1 using Kraft silicone, and ensures that the working electrode and the passivation layer area are located inside the cell culture ring 4.

[0045] Due to the principle of photoinduced grafting, the hydrogel grows only at the interface where the aqueous solution of the hydrogel monomer contacts polydimethylsilane (PDMS). Therefore, in the cell culture ring 4, the surface of the polydimethylsilane insulating layer 13 and the polydimethylsiloxane substrate 11 is not covered by the patterned metal layer 12 and the passivation layer 2 to form a hydrogel layer 5 with a thickness of 10 μm to 500 μm, while at the interface of the area covered by the patterned metal layer 12 and the passivation layer 2, the hydrogel does not polymerize, and the hydrogel layer does not cover the working electrode and the passivation layer and form a patterned microgroove 31 with a depth consistent with the thickness of the hydrogel layer. In this embodiment, the patterned microgroove 31 is two long rectangles parallel to each other. Each long rectangle includes multiple working electrodes. It can be understood that by changing the graphic settings of the exposed microelectrode array 12a and the passivation layer 2 in the cell culture ring 4, other patterned microgrooves can be formed, such as single or multiple straight lines, broken lines, curves, rectangles, or irregular graphics.

[0046] The preparation method of the hydrogel-modified microelectrode array device is as follows: Figure 5 The steps shown include:

[0047] (1) taking a polydimethylsiloxane film with a thickness of 300 μm, and using a femtosecond laser to cut a square polydimethylsiloxane substrate 11 with a size of 40 mm and a width of 40 mm;

[0048] (2) Using laser cutting, a stainless steel plate with a thickness of 100 μm is cut, and its hollow pattern is consistent with the desired patterned metal layer 12 pattern, to obtain a metal layer mask. The mask is placed flat on the surface of the polydimethylsiloxane substrate 11, and metal platinum is sputtered onto the upper surface of the sample using a magnetron sputtering process. The mask is removed to obtain a patterned metal layer 12 with a thickness of 200 nm;

[0049] (3) Using laser cutting, a stainless steel plate with a thickness of 100 μm is cut, and its hollow pattern is consistent with the desired passivation layer 2 pattern, to obtain a passivation layer mask. The mask is placed flat on the sample surface obtained in step (2), and a silicon nitride material is sputtered onto the sample surface using a magnetron sputtering process. The mask is removed to obtain a patterned silicon nitride passivation layer 2 with a thickness of 200 nm;

[0050] (4) Using laser cutting, a stainless steel plate with a thickness of 100 μm is cut, and its hollow pattern is consistent with the desired polydimethylsiloxane insulating layer 13 pattern, to obtain a polydimethylsiloxane insulating layer mask. The mask is placed flat on the sample surface obtained in step (3), and a polydimethylsiloxane prepolymer is sprayed onto the sample surface using an electrostatic spray process. The mask is removed to obtain a polydimethylsiloxane insulating layer 13 that partially covers the patterned metal layer 12, thereby obtaining a microelectrode array composite layer having a passivation layer 3 on the surface;

[0051] (5) Using Kraft silica gel, the cell culture ring is bonded to the upper surface of the sample obtained in step (4), and the working electrode and the passivation layer area are ensured to be located inside the cell culture ring 4;

[0052] (6) dropping 800 μL of an acetone solution of a hydrophobic photoinitiator benzophenone into the cell culture ring on the surface of the microelectrode array composite layer, the concentration of benzophenone being 20 wt %, and standing at room temperature for 10 min;

[0053] (7) The acetone solution of benzophenone is sucked out and washed with pure water or alcohol. At this time, due to the swelling effect, some benzophenone molecules in the solution move to the inside of the polydimethylsiloxane;

[0054] (8) 800 μL of acrylamide hydrogel monomer aqueous solution was dropped into the cell culture ring on the surface of the microelectrode array composite layer, the concentration of the hydrogel monomer was 30 wt%, and a UV lamp with a power of 60 W and a wavelength of 365 nm was used to vertically irradiate for 15 min to 60 min at a distance of 20 mm from the sample surface to grow a hydrogel layer 3; the thickness of the hydrogel layer 3 and the illumination time were as shown in Figure 6 As shown, its thickness ranges from about 100 μm to 300 μm; the obtained hydrogel layer 3 is as shown Figure 7 As shown, it can be seen that the hydrogel grows and polymerizes uniformly in the polydimethylsiloxane, and there is no obvious interface between the two, indicating that an interpenetrating molecular network is formed between the hydrogel and the polydimethylsiloxane;

[0055] (9) The aqueous solution of the hydrogel monomer is aspirated and the sample is washed with pure water to obtain a hydrogel-modified microelectrode array device.

[0056] Figure 8 The colored structure diagram of the prepared hydrogel-modified microelectrode array device shows that the bottom of the microgrooves 31 of the hydrogel layer 3 exposes the alternating patterned metal layer 12 and the passivation layer 2. In this embodiment, the thickness of the patterned metal layer 12 and the silicon nitride passivation layer 2 constructed in step (2) and step (3) is much smaller than the thickness of the hydrogel layer 3, and also much smaller than the characteristic scale of the cell. Therefore, the effect of the thickness of the patterned metal layer 12 and the silicon nitride passivation layer 2 on the cell patterning can be ignored. Fig. 9As shown in the physical picture, it can be seen that the hydrogel layer grows only in the area not covered by the patterned metal layer 12 and the silicon nitride passivation layer 2, and forms concave microgrooves in the patterned metal layer 12 and the silicon nitride passivation layer 2 area.

[0057] In the present embodiment, in step (6) and step (7), since the acetone-based organic solvent can only swell high molecular polymers such as polydimethylsiloxane, the hydrophobic photoinitiator benzophenone molecules will only move to the interior of the polydimethylsiloxane under the swelling effect, and will not move to the patterned metal layer 12 and the passivation layer 2.

[0058] In this embodiment, in step (8), the acrylic hydrogel monomer undergoes a polymerization reaction under the combined action of ultraviolet light and a hydrophobic photoinitiator. Due to the insolubility of the hydrophobic photoinitiator benzophenone in water, the polymerization reaction only occurs near the interface between the polydimethylsiloxane containing benzophenone and the acrylamide hydrogel monomer aqueous solution, and the long chains of some hydrogel molecules are staggered and constructed inside the polydimethylsiloxane, resulting in a firm and stable grafting of the hydrogel layer and the polydimethylsiloxane, thereby improving the stability and robustness of the hydrogel layer. Since the sputtered patterned metal layer 12 and the passivation layer 2 are closely connected to the polydimethylsiloxane, the direct contact between the acrylamide hydrogel monomer aqueous solution and the polydimethylsiloxane is hindered, and the patterned metal layer 12 and the passivation layer 2 do not contain the hydrophobic photoinitiator benzophenone, so in the area covered by the patterned metal layer 12 and the passivation layer 2, the hydrogel will not undergo a polymerization reaction, forming a vacancy, thereby constructing the hydrogel microgroove 31.

[0059] The method of using the hydrogel-modified microelectrode array device is as follows: use a pipette to draw 600 μL of a cell suspension containing 300,000 cardiomyocytes and drop it into a cell culture ring 4, and place it in a cell culture incubator for culture. After 4 to 24 hours, use a microscope to observe the cell sedimentation, migration and adsorption. When it is observed that the cells 5 are mainly distributed in the microgrooves 31 of the hydrogel layer 3, as shown in FIG. Fig.10 As shown, it shows that the cell patterning is initially formed and forms a good interface with the patterned metal layer 12 and the passivation layer 2. An oscilloscope or data acquisition device is connected to the electrode lead interface at the edge of the patterned metal layer 12 to measure the field potential signal caused by the cell action potential.

[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some or all of the technical features thereof may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A hydrogel-modified microelectrode array device, characterized in that: The invention comprises a microelectrode array composite layer, a passivation layer, a hydrogel layer and a cell culture ring; the microelectrode array composite layer comprises a polydimethylsilane substrate, a patterned metal layer and a polydimethylsilane insulating layer in sequence, the patterned metal layer forms a microelectrode array, and the polydimethylsilane insulating layer partially covers the patterned metal layer; the cell culture ring is arranged on the microelectrode array composite layer, and the passivation layer and the hydrogel layer are located in the cell culture ring; wherein at least part of the microelectrode array in the cell culture ring is exposed, and the passivation layer fills at least part of the gap of the exposed microelectrode array; the hydrogel layer grows on the polydimethylsilane surface, and forms microgrooves in the exposed microelectrode array and passivation layer areas as cell growth areas.

2. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The material of the passivation layer is at least one of silicon nitride, silicon carbide and aluminum oxide, and the thickness is 10nm-1μm.

3. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The thickness of the hydrogel layer is 10 μm to 500 μm.

4. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The hydrogel layer is formed into one or a plurality of microgrooves which are not interconnected by patterning the exposed microelectrode array and the passivation layer area, wherein a single microgroove includes one or more exposed microelectrodes.

5. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The thickness of the patterned metal layer is 50nm-500nm.

6. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The monomer material of the hydrogel layer is at least one of acrylamide, polyethylene glycol diacrylate, and methacrylated gelatin.

7. The hydrogel-modified microelectrode array device according to claim 1, characterized in that: The polydimethylsilane in the cell culture ring has a doped surface layer of a hydrophobic photoinitiator.

8. A method for preparing a hydrogel-modified microelectrode array device according to any one of claims 1 to 7, characterized in that: The steps include: (1) forming a patterned metal layer on the polydimethylsilane-based bottom surface as a microelectrode array; (2) patterning and depositing a passivation layer, wherein the passivation layer fills at least a portion of the gaps in the microelectrode array; (3) depositing a polydimethylsilane insulating layer to cover a portion of the patterned metal layer; (4) attaching a cell culture ring on the polydimethylsilane insulating layer, wherein the cell culture ring has an exposed microelectrode array and a passivation layer; (5) doping the polydimethylsilane surface within the cell culture ring with a photoinitiator; (6) Adding a hydrogel monomer solution into the cell culture ring, growing a hydrogel layer under the action of light, and forming microgrooves in the exposed microelectrode array and passivation layer areas of the hydrogel layer to serve as cell growth areas.

9. The method for preparing a hydrogel-modified microelectrode array device according to claim 8, characterized in that: In step (5), an organic solution containing a hydrophobic photoinitiator is added into the cell culture ring and allowed to stand, so that the hydrophobic photoinitiator is doped into the polydimethylsilane through swelling.

10. The method for preparing a hydrogel-modified microelectrode array device according to claim 9, characterized in that: The hydrophobic photoinitiator is benzophenone, and the organic solvent is one of alcohol and acetone.

Images