A low-cost ex vivo brain-machine interface chip batch preparation method and device

Abstract

The application discloses a low-cost ex vivo brain-machine interface chip batch preparation method and device, and belongs to the field of biosensors and micro-nano manufacturing technology.The method comprises sequentially performing cleaning, photolithography, sputtering of a metal layer, stripping, deposition of an insulating layer, overlaying, etching to expose electrode sites and pads, and preparing 3D electrodes through second overlaying and electrochemical deposition, and finally separating multiple independent microelectrode arrays through scribing or laser cutting.The aluminum wire pressure welding technology is adopted to connect the microelectrode pads and the adapter plate, the aluminum wire is embedded with high-temperature-resistant insulating glue, and cell culture rings are adhered.The application realizes batch preparation of multiple specifications of microelectrode arrays on a single substrate, has strong process compatibility, low cost and high efficiency, is convenient for microscopic observation and 3D electrode integration, and is suitable for ex vivo neuron network detection and organoid electrophysiology and electrochemical research.

Description

Technical Field

[0001] This invention belongs to the field of biosensor and micro / nano manufacturing technology, specifically relating to a low-cost method and apparatus for mass production of ex vivo brain-computer interface chips. Background Technology

[0002] Ex vivo microelectrode arrays are core components of ex vivo brain-computer interfaces, widely used in research on neural network information processing mechanisms, drug screening, and biological neural network computing. Compared to in vivo experiments, ex vivo research avoids complex ethical approvals and interference from individual differences, and has advantages such as controllable experimental conditions and high reproducibility, providing an important platform for basic neuroscience research and brain-like computing.

[0003] Currently, the fabrication of off-site microelectrode arrays mainly relies on two technical approaches. One is the traditional microelectromechanical system (MEMS) process, which fabricates planar electrode structures on an insulating substrate through steps such as photolithography, sputtering, and etching. However, this method typically only allows for the fabrication of a single device on a single substrate, resulting in low production efficiency and high costs. For example, while existing microelectrode array chip fabrication methods are mature, limitations in pad size and lead configuration make it difficult to achieve device miniaturization and high-density integration, thus restricting their potential for high-throughput applications.

[0004] Another category involves cutting-edge technologies based on complementary metal-oxide-semiconductor (CMOS) processes. These technologies enable high-density electrode integration and on-chip signal processing circuits, but face challenges such as high process barriers and high manufacturing costs. Furthermore, the multilayer metal wiring structures fabricated using CMOS processes result in opaque chips, severely hindering real-time morphological observation using conventional transmission optical microscopy during cell culture. Simultaneously, because electrodes are typically directly connected to built-in circuits, it is difficult to apply external potentials for effective electrochemical modification, thus limiting their application in the fabrication of three-dimensional electrode structures, which are crucial for improving cell-electrode coupling efficiency.

[0005] In summary, existing technologies suffer from significant shortcomings, both in terms of the low efficiency and high cost of traditional MEMS processes and the complexity, opacity, and poor compatibility with three-dimensional modification of advanced CMOS processes. Therefore, there is an urgent need in this field to develop a novel method for fabricating ex vivo brain-computer interface chips that can guarantee high performance, achieve mass production and low cost, and be compatible with three-dimensional structures and optical observation. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides a low-cost method and apparatus for mass production of ex vivo brain-computer interface chips. It employs traditional MEMS technology for mass processing on glass / silicon wafers, resulting in low technical barriers and costs. Furthermore, it is compatible with 3D electrode fabrication processes, effectively improving the fabrication efficiency and quality of ex vivo microelectrode arrays.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A low-cost method for mass production of ex vivo brain-computer interface chips, the method comprising:

[0009] Metal patterns of microelectrodes, microelectrode array pads, and leads connecting the microelectrodes and microelectrode array pads are formed on an insulating substrate through a first photolithography, sputtering, and lift-off process.

[0010] A deposited insulating layer covers the metal pattern;

[0011] The insulating layer of the microelectrode and the pad region of the microelectrode array is removed by overlay and etching processes;

[0012] The insulating substrate is cut and separated according to a preset layout by dicing or laser cutting, thereby obtaining multiple independent chips including microelectrode arrays;

[0013] The microelectrode array pads corresponding to the independent chip are connected to the adapter board pads on the adapter board by aluminum wire bonding, and the adapter board is located below the independent chip;

[0014] The aluminum wire is coated with a high-temperature resistant insulating adhesive and a cell culture loop is adhered to the surface of the high-temperature resistant insulating adhesive.

[0015] On the other hand, the present invention provides a low-cost ex vivo brain-computer interface chip fabrication apparatus, comprising:

[0016] A metal patterning module is used to form metal patterns of microelectrodes, microelectrode array pads, and leads connecting the microelectrodes and microelectrode array pads on an insulating substrate through a first photolithography, sputtering, and lift-off process; and to deposit an insulating layer to cover the metal patterns;

[0017] A microelectrode array forming module is used to remove the insulating layer of the microelectrode and the pad area of ​​the microelectrode array through overlay and etching processes; and to cut and separate the insulating substrate according to a preset layout by dicing or laser cutting, thereby obtaining multiple independent chips including microelectrode arrays.

[0018] The packaging module is used to connect the microelectrode array pads corresponding to the independent chip to the adapter board pads on the adapter board by aluminum wire bonding, the adapter board being located below the independent chip; the aluminum wires are coated with high-temperature resistant insulating adhesive and cell culture loops are adhered to the surface of the high-temperature resistant insulating adhesive.

[0019] Thirdly, the present invention provides an electronic device, comprising: one or more processors; a memory for storing one or more programs; wherein, when the one or more programs are executed by the one or more processors, the one or more processors implement the aforementioned method for mass production of low-cost ex vivo brain-computer interface chips.

[0020] Fourthly, the present invention provides a computer-readable storage medium having executable instructions stored thereon, which, when executed by a processor, enable the processor to implement the aforementioned method for mass production of low-cost ex vivo brain-computer interface chips.

[0021] The beneficial effects of this invention are as follows:

[0022] It achieves high-efficiency and low-cost mass production: by replacing the traditional spring pin connection with aluminum wire pressure welding, the size of the solder pad is greatly reduced, allowing dozens to hundreds of independent microelectrode arrays to be arranged in high density on a single substrate. Combined with dicing / laser cutting for separation, the preparation efficiency is increased by tens to hundreds of times, and the cost per unit is significantly reduced.

[0023] It combines high performance with good observation compatibility: the MEMS process platform used has low requirements and controllable cost, and when transparent glass is used as the substrate, the entire device has excellent optical transmittance, which facilitates real-time and clear morphological observation of isolated cells or organoids using a variety of microscopic methods.

[0024] This approach effectively solves the challenges of 3D electrode integration: by introducing a second overlay process combined with subsequent electrochemical deposition, 3D electrode structures of specific heights can be conveniently constructed at designated microelectrode sites before dicing. This solution cleverly circumvents the limitations of CMOS processes in electrochemical modification, improves the coupling efficiency between electrodes and cells, and offers flexible and controllable process capabilities. Attached Figure Description

[0025] Figure 1 This is a flowchart of a low-cost, in vitro brain-computer interface chip mass production method according to the present invention;

[0026] Figure 2 A schematic diagram illustrating the fabrication process for creating 36 256-channel in vitro microelectrode arrays and 80 64-channel in vitro microelectrode arrays on a 4-inch glass slide.

[0027] Figure label:

[0028] 1. Insulating substrate; 2. Microelectrode array pads; 3. Microelectrode; 4. Adapter board; 5. Aluminum wire; 6. Adapter board pads; 7. Cell culture ring; 8. High-temperature resistant insulating adhesive. Detailed Implementation

[0029] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0030] like Figure 1 The diagram shows a flowchart of a low-cost, mass production method for ex vivo brain-computer interface chips according to the present invention. The core of this method lies in the mass production of multiple independent microelectrode arrays on an insulating substrate using standard MEMS processes. Specifically, this involves fabricating three types of 256-channel ex vivo microelectrode arrays with electrode spacings of 150 micrometers, 200 micrometers, and 300 micrometers on a 4-inch glass slide, including:

[0031] Step 1: Oxygen cleaning of glass or silicon wafers:

[0032] The selected 4-inch glass insulating substrate 1 was subjected to oxygen plasma cleaning. This step effectively removed organic contaminants from the substrate surface and activated the substrate surface, significantly enhancing the adhesion of photoresist in subsequent processes.

[0033] Step 2, First photolithography:

[0034] Using SPR220 photoresist, a layer of photoresist was uniformly coated onto a cleaned glass slide at a spin coater at a speed of 1700 r / min. Subsequently, exposure and development were performed on a photolithography machine to initially pattern the microelectrode array—including microelectrodes 3, microelectrode array pads 2, and the leads connecting the two.

[0035] Step 3, Sputtering:

[0036] A chromium adhesion layer and a platinum conductive layer are sequentially deposited on a patterned substrate using magnetron sputtering. Typical film thicknesses are 30 nm for Cr and 250 nm for Pt.

[0037] Step 4, peeling:

[0038] The sputtered substrate is immersed in an acetone solution for a stripping operation. This process dissolves the photoresist used in the first photolithography and removes the metal layer attached to it, ultimately leaving only the Cr / Pt metal layer in the patterned area to form microelectrodes 3, microelectrode array pads 2, and the leads connecting the two.

[0039] Step 5: ICP-CVD deposition of insulating layer:

[0040] A composite insulating dielectric layer is deposited on the entire device surface using inductively coupled plasma chemical vapor deposition (ICP-CVD). A typical layered structure is Si3N4 / SiO2 / Si3N4, with thicknesses of 300 nm / 400 nm / 300 nm, respectively. This insulating layer ensures the stability and reliability of electrical signal transmission.

[0041] Step 6, overlay engraving:

[0042] SPR220 photoresist was applied again at the same spin coating parameters (1700 r / min), followed by a second photolithography process. The purpose of this photolithography was to pattern the areas of microelectrode 3 and microelectrode array pad 2 that need to be exposed to the external environment.

[0043] Step 7, Etching:

[0044] The substrate with completed overlay is placed in a dielectric thin film etching system. By bombarding with plasma, the insulating layer above the microelectrode 3 and microelectrode array pad 2 areas is precisely removed, exposing the underlying metal layer. The insulating layer in other areas remains intact due to the protection of the photoresist SPR220.

[0045] Step 8, Second overlay (This step is performed only during the fabrication of the 3D electrode):

[0046] In fabricating the 3D electrode, AZ4903 photoresist was selected to form a cavity structure with a specific height in the microelectrode 3 region through photolithography. This cavity structure provides a mold for subsequent electrochemical deposition. During electrochemical deposition, the deposition material gradually fills the cavity structure to form a columnar 3D electrode with a certain height. The height of the 3D electrode can be controlled by parameters such as the time and voltage of the electrochemical deposition.

[0047] Step 9, scribing or laser cutting:

[0048] Using a dicing machine or laser cutting equipment, the entire insulating substrate 1 is cut and separated according to a pre-designed layout, thereby obtaining multiple independent, fully functional off-site microelectrode array chips in batches.

[0049] Step 10, Aluminum wire pressure welding:

[0050] The microelectrode array pads 2 on the cut individual detached microelectrode array chips are electrically connected to the corresponding adapter board pads 6 on the adapter board 4 using aluminum wire bonding technology. The adapter board 4 is located below the individual chip and is connected by aluminum wire 5. The purpose is to connect the detached microelectrode array sites to external electrophysiological signal acquisition systems, electrostimulation systems, and other devices. This method significantly reduces the pad size required by traditional spring-loaded pin connection methods, which is beneficial for device miniaturization.

[0051] Step 11, Packaging:

[0052] High-temperature resistant insulating adhesive 8 is used to cover and protect the aluminum wire 5 bonding points, while the cell culture ring 7 is firmly adhered to the designated area of ​​the microelectrode array (such as the upper surface). The adapter plate below the microelectrode array is perforated. When a transparent glass substrate is used, various microscopic methods can be used to observe the isolated cells, thus completing the final packaging of the device.

[0053] Using the above process, three types of 256-channel ex vivo microelectrode arrays with electrode spacings of 150 μm, 200 μm, and 300 μm were fabricated on a 4-inch glass slide (e.g., Figure 2 (As shown). The prepared in vitro microelectrode array was then electrochemically modified using an electrochemical workstation. A solution of gold nanoparticles was placed in a cell culture loop, and a 3D electrode with a height of approximately 30 micrometers was modified on the electrode surface using a chronoamperometry method (-1.1V, 30min). The AZ4903 photoresist used for the 3D electrode fabrication was then removed in acetone solution, yielding a 3D in vitro microelectrode array with a certain height.

[0054] After autoclaving the ex vivo microelectrode array designed and fabricated in this invention, PDL (poly-L-lysine), a neuronal adhesion and growth promoter, was coated onto the electrode surface. Primary hippocampal neurons were isolated from ICR rat embryos at 15-18 days of gestation and seeded in cell culture loops. After culturing the neurons in a CO2 incubator for 2-3 weeks, the neuronal network began to mature. The neuronal network was detected and analyzed using electrophysiological monitoring instruments to explore the firing activity characteristics of the neuronal network at different spatial scales.

[0055] After autoclaving the 3D ex vivo microelectrode array designed and fabricated according to this invention, mature brain organoids were transferred to the surface of the 3D ex vivo microelectrode array. The neuronal network was then detected and analyzed using an electrophysiological monitoring instrument. Different concentrations of the epilepsy modeling drug 4-ap were added to the culture medium of the brain organoids, and the modeling effect of 4-ap was studied based on the neuronal network signals detected by the electrophysiological monitoring instrument. The brain organoids were electrically stimulated using a Multi Channel Systems STG4002 electrostimulator through the ex vivo microelectrode sites, and the neuronal network was then detected and analyzed using a 128-channel electrophysiological monitoring instrument to observe the effect of electrical stimulation on the brain organoids.

[0056] On the other hand, the present invention provides a low-cost, in vitro brain-computer interface chip mass production apparatus, the various modules of which can implement the various steps of the aforementioned method, specifically including:

[0057] A metal patterning module is used to form metal patterns of microelectrodes, microelectrode array pads, and leads connecting the microelectrodes and microelectrode array pads on an insulating substrate through a first photolithography, sputtering, and lift-off process; and to deposit an insulating layer to cover the metal patterns;

[0058] A microelectrode array forming module is used to remove the insulating layer of the microelectrode and the pad area of ​​the microelectrode array through overlay and etching processes; and to cut and separate the insulating substrate according to a preset layout by dicing or laser cutting, thereby obtaining multiple independent chips including microelectrode arrays.

[0059] The packaging module is used to connect the microelectrode array pads corresponding to the independent chip to the adapter board pads on the adapter board by aluminum wire bonding, the adapter board being located below the independent chip; the aluminum wires are coated with high-temperature resistant insulating adhesive and cell culture loops are adhered to the surface of the high-temperature resistant insulating adhesive.

[0060] Thirdly, the present invention provides an electronic device, comprising: one or more processors; a memory for storing one or more programs; wherein, when the one or more programs are executed by the one or more processors, the one or more processors implement the aforementioned method for mass production of low-cost ex vivo brain-computer interface chips.

[0061] Fourthly, the present invention provides a computer-readable storage medium having executable instructions stored thereon, which, when executed by a processor, enable the processor to implement the aforementioned method for mass production of low-cost ex vivo brain-computer interface chips.

[0062] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above descriptions are merely specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A low-cost method for mass production of ex vivo brain-computer interface chips, characterized in that, The method includes: Multiple microelectrode array patterns with different electrode spacings are simultaneously formed on the same insulating substrate (1) through a first photolithography, sputtering and stripping process. Each microelectrode array pattern includes a microelectrode (3), a microelectrode array pad (2), and a metal pattern of leads connecting the microelectrode (3) and the microelectrode array pad (2). A deposited insulating layer covers the metal pattern; The insulating layer of the microelectrode (3) and the microelectrode array pad (2) area is removed by overlay and etching processes; The insulating substrate (1) is cut and separated according to a preset layout by dicing or laser cutting, thereby obtaining multiple independent chips including microelectrode arrays at the same time; For each of the aforementioned individual chips, execute the following respectively: The microelectrode array pad (2) corresponding to the independent chip is connected to the adapter plate pad (6) on the adapter plate (4) by aluminum wire (5) pressure welding. The adapter plate (4) is located below the independent chip. The aluminum wire (5) is coated with high-temperature resistant insulating adhesive (8) and a cell culture loop (7) is adhered to the surface of the high-temperature resistant insulating adhesive (8). The adapter plate (4) has through holes in the area corresponding to the area below the cell culture ring (7).

2. The method for mass production of low-cost ex vivo brain-computer interface chips according to claim 1, characterized in that, Before the insulating substrate (1) is cut and separated according to a preset layout by dicing or laser cutting, the step of fabricating 3D electrodes is also included: A second overlay is performed to form a photoresist cavity in the region of the microelectrode (3); A 3D structure is formed on the microelectrode (3) by electrochemically depositing conductive material into the cavity; The photoresist is then removed.

3. The method for mass production of low-cost ex vivo brain-computer interface chips according to claim 1, characterized in that, Each of the segmented individual chips can fully perform the functions of in vitro cell detection and regulation.

4. The method for mass production of low-cost ex vivo brain-computer interface chips according to claim 1, characterized in that, The insulating substrate (1) is a glass sheet or a silicon sheet.

5. The method for mass production of low-cost ex vivo brain-computer interface chips according to claim 2, characterized in that, The electrochemical deposition is performed by depositing gold nanoparticles using a chronoamperometry method.

6. The method for mass production of low-cost ex vivo brain-computer interface chips according to claim 1, characterized in that, The metal layer formed by the sputtering process is a composite layer of a chromium adhesion layer and a platinum conductive layer.

7. A low-cost, in vitro brain-computer interface chip mass production apparatus, used to perform the method according to any one of claims 1-6, characterized in that, include: A metal patterning module is used to form a metal pattern of microelectrodes (3), microelectrode array pads (2), and leads connecting the microelectrodes (3) and microelectrode array pads (2) on an insulating substrate (1) through a first photolithography, sputtering and stripping process; and to deposit an insulating layer to cover the metal pattern; The microelectrode array forming module is used to remove the insulating layer of the microelectrode (3) and the microelectrode array pad (2) area by overlay and etching processes; and to cut and separate the insulating substrate (1) according to a preset layout by dicing or laser cutting, thereby obtaining multiple independent chips including microelectrode arrays. The packaging module is used to connect the microelectrode array pad (2) corresponding to the independent chip to the adapter plate pad (6) on the adapter plate (4) by pressure welding with aluminum wire (5), the adapter plate (4) being located below the independent chip; the aluminum wire (5) is covered with high temperature resistant insulating adhesive (8) and a cell culture loop (7) is adhered to the surface of the high temperature resistant insulating adhesive (8).

8. An electronic device, characterized in that, include: One or more processors; Memory, used to store one or more programs; When one or more programs are executed by the one or more processors, the one or more processors implement the low-cost in vitro brain-computer interface chip mass production method according to any one of claims 1-6.

9. A computer-readable storage medium, characterized in that, It stores executable instructions, which, when executed by a processor, enable the processor to implement the low-cost, mass production method for ex vivo brain-computer interface chips as described in any one of claims 1-6.

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