A microfluidic biointerconnected neural network chip and its preparation method

Abstract

The present invention discloses a microfluidic bio-interconnected neural network chip and a preparation method thereof. The chip consists of a microelectrode array, a microfluidic structure, and a cell culture ring structure. The microelectrode array can detect and stimulate the activity of neurons; the microfluidic structure limits the growth direction of axons between cell culture partitions by setting cell culture partitions, microfluidic channel dimensions, microfluidic channel shapes, and fillet distribution, thereby achieving controllable low-power transmission of neuronal electrical information; the cell culture ring structure provides nutrients for cells in the cell culture chamber. The present invention provides a method for manufacturing a microfluidic bio-interconnected neural network chip, using fully exposed positive photoresist as a sacrificial layer for microfluidic structure peeling, thereby solving the problem of difficult demolding of thin-film microfluidic structures. The chip can be used to explore the response characteristics of neuronal networks to external stimuli, study the internal information transmission mode of neuronal networks, and other fields.

Description

Technical Field

[0001] The present invention relates to fields such as biosensors, neurobiology, micro-nano manufacturing technology, and bioelectronics. It relates to a method for fabricating a microfluidic biointerconnected neural network chip for achieving controllable transmission of neuronal electrical information. In particular, it relates to a microfluidic biointerconnected neural network chip and its fabrication method. Background Art

[0002] The brain is the most complex structure known to humans, capable of advanced functions such as learning, memory, and thinking. It is also the key to human perception of the world and control of the body. However, many questions remain about the brain's workings and internal structure, which calls for more in-depth research. At the same time, understanding the brain's workings will also help build more efficient artificial neural networks and provide new insights into the development of computer science.

[0003] One of the hot research directions in the brain is to start from the perspective of neuroelectrophysiology, using micro-nanofabrication technology to prepare microelectrode arrays for detecting and regulating neuronal activity. At the same time, combined with microfluidics technology, neural networks with specific structures and functions are constructed in vitro, and the characteristics of neurons and neural networks are studied in vitro, including neuronal discharge patterns, response patterns of neural networks to external stimuli, and information interaction characteristics between neurons.

[0004] Microfluidic bio-interconnected neural network chips fabricated using micro-nanofabrication technologies can customize the synaptic connections and growth directions of neurons, thereby creating diverse neural network structures and opening up new possibilities for building bio-computing chips. Different neural network structures exhibit distinct properties, providing richer material for studying the workings of neurons and the brain.

[0005] The neuronal network structure of existing microfluidic bio-interconnected neural network chips is not rich enough, making it difficult to control the direction of transmission of neuronal electrical information; the microfluidic structure produced using the SU-8 casting method is too thick, requiring separate punching operations to create cell culture chambers. Summary of the Invention

[0006] The microfluidic bio-interconnected neural network chip provided by the present invention can be used to explore neuronal networks fabricated using rectangular and four-channel microfluidic channel structures of varying sizes. By adjusting the length, shape, width, and corner distribution of the microfluidic channels, unidirectional growth of neuronal axons between different cell culture chambers can be achieved, thereby enabling controlled transmission of neuronal electrical information and enabling the construction of customized neuronal networks. The present invention provides a novel method for fabricating microfluidic structures, using negative photoresist SU-8 as a mold and a method for controlling the thickness of the PDMS layer by spin coating with a resin spinner. After mold casting, a thin-film microfluidic structure with a through-hole structure of tens to hundreds of microns in height can be obtained. Fully exposed positive resist is used as a sacrificial layer for peeling off the thin-film microfluidic structure, solving the problem of difficult peeling off the thin-film microfluidic structure after mold casting.

[0007] The present invention provides a microfluidic bio-interconnected neural network chip and its preparation method. The chip uses microfluidic technology to culture up to 64 independent neuronal networks on a microelectrode array. Each neural network consists of four cell culture chambers 7 and microfluidic channels 8 for connecting the cell culture chambers 7. Because the length of neuronal axons is generally much longer than that of neuronal dendrites, the length of the microfluidic channels is set so that longer neuronal axons in the cell culture chamber 7 can pass through the microfluidic channels 8 into the adjacent cell culture chamber 7, while shorter neuronal dendrites in the cell culture chamber 7 cannot pass through. At the same time, by setting the shape of the microfluidic channels 8 to a four-channel shape or a regular rectangle and designing a rounded corner distribution to guide axon growth, the growth direction of neuronal axons can be controlled to be unidirectional or bidirectional, thereby controlling the direction of neuronal electrical information transmission. The microelectrode array under the microfluidic structure can be used to detect and regulate neuronal activity. By designing different experimental paradigms, it is possible to further explore neuronal firing patterns, the response pattern of neuronal networks to external stimuli, and the characteristics of information exchange between neurons.

[0008] To achieve this purpose, the present invention adopts the following technical solutions:

[0009] A microfluidic bio-interconnected neural network chip comprises a three-layer structure: a first layer of microelectrode array, a second layer of microfluidic structure, and a third layer of cell culture ring structure.

[0010] The first-layer microelectrode array includes: an insulating substrate 1, microelectrodes 2, counter electrodes 3, connecting wires 4, contact sites 5, and an insulating layer 6; the insulating substrate 1 is the supporting structure of the neural network chip; the microelectrodes 2 are divided into 64 groups, and the microelectrodes are located inside the cell culture chamber 7; the counter electrodes 3 and microelectrodes 2 are extended by connecting wires 4 and connected to the contact sites 5 on the periphery of the insulating substrate; the surfaces of all connecting wires 4 are covered with an insulating layer 6;

[0011] The second-layer microfluidic structure includes 256 groups of microfluidic channels 8 with specific shapes and 256 connected cell culture chambers 7, where every four adjacent cell culture chambers 7 are connected by microfluidic channels 8 to form an independent neuronal network unit 9. The first-layer microelectrode array and the second-layer microfluidic structure are bonded together using alcohol evaporation or polydimethylsiloxane as an adhesive material.

[0012] The third layer includes: a cell culture ring structure 10. The first layer of microelectrode array and the third layer of cell culture ring structure are attached by alcohol volatilization or using polydimethylsiloxane as an adhesive material.

[0013] Furthermore, the insulating substrate 1 is the substrate of the entire microfluidic bio-interconnected neural network chip; the microelectrodes 2 are 256 in total and divided into 64 groups; the microelectrodes 2 are located inside the cell culture chamber 8; the counter electrode 3 and the microelectrodes 2 are both extended by connecting wires 4 and connected to the contact sites 5 on the periphery of the insulating substrate 1; the surfaces of all connecting wires 4 are covered with an insulating layer 6;

[0014] Furthermore, the microfluidic structure has 256 groups of microfluidic channels 8 of specific shapes and 256 cell culture chambers 7 connected thereto, wherein every four adjacent cell culture chambers 7 are connected by microfluidic channels 8 to form an independent neuronal network unit 9, which structurally restricts the growth of neurons and functionally regulates the electrical information transmission of neurons.

[0015] Furthermore, the cell culture ring structure is used to accommodate nutrients required for neuron growth in the cell culture chamber 7 .

[0016] Furthermore, in the microelectrode array, the insulating substrate 1 is made of soda-lime glass; the microelectrode 2 is a conductive composite material with biocompatibility and good adhesion to the soda-lime glass substrate, preferably, the material of the microelectrode 2 is platinum and chromium; the insulating layer 6 is made of an insulating material with biocompatibility and good adhesion to the soda-lime glass substrate, preferably, the material of the insulating layer 6 is silicon dioxide and silicon nitride; the diameter of the microelectrode is distributed in the range of 4-35 μm, and the site spacing is distributed in the range of 500-700 μm.

[0017] Furthermore, the second layer of microfluidic chip and the third layer of cell culture ring are made of biocompatible polydimethylsiloxane.

[0018] Furthermore, the diameter of a single cell culture chamber 7, which integrates a microfluidic channel 8 in the second-layer microfluidic structure, is 200 μm. Four adjacent cell culture chambers 7 are connected in an orderly manner through the microfluidic channel 8 to form an independent neuronal network unit. The microfluidic channel 8 adopts a four-channel and ordinary rectangular shape, with a width of 400 μm-600 μm and a width of 2 μm-15 μm. At the same time, the connection between the microfluidic channel 8 and the cell culture chamber adopts a rounded connection. A large rounded structure of 10 μm-50 μm is set where it is necessary to control the entry of neuronal axons into the microfluidic channel 8, and a small rounded structure of 2 μm-6 μm is set where it is necessary to control the entry of neuronal axons into the microfluidic channel 8.

[0019] Furthermore, the inner dimension of the third layer of cell culture ring structure is slightly larger than that of the second layer of microfluidic structure, and is 2 mm to 10 mm in height, which is beneficial for in vitro culture of neurons.

[0020] Furthermore, the first layer of microelectrode array and the second layer of microfluidic structure are bonded together by alcohol volatilization adhesion; the first layer of microelectrode array and the third layer of cell culture ring structure are bonded together using polydimethylsiloxane as an adhesive material.

[0021] According to another aspect of the present disclosure, a method for preparing a microfluidic bio-interconnected neural network chip is provided, comprising preparing a first layer of microelectrode arrays, preparing a second layer of microfluidic structures, and preparing a third layer of cell culture ring structures.

[0022] Preparation of the first layer of microelectrode array includes the following steps:

[0023] (1) Using acetone, ethanol, and deionized water to clean the soda-lime glass insulating substrate 1 in sequence, and then washing it in concentrated sulfuric acid at 100° C. to obtain an insulating substrate 1 with a clean surface;

[0024] (2) Spin-coating a 1-3 μm thick positive photoresist layer on the cleaned insulating substrate 1. After photolithography development, the photoresist at the microelectrode 2, the counter electrode 3, the connecting wire 4, and the contact point 5 is dissolved in the developer, exposing the insulating substrate 1 underneath.

[0025] (3) Depositing a titanium metal layer on the photoresist-patterned insulating substrate by sputtering to increase the adhesion between the conductive layer and the insulating substrate 1. Then sputtering a platinum metal conductive layer;

[0026] (4) using a stripping process to sequentially clean and remove the conductive layer with the photoresist underneath in acetone, ethanol, and deionized water, leaving the required microelectrode 2, counter electrode 3, connecting wire 4, and contact site 5;

[0027] (5) depositing a silicon oxide or silicon nitride insulating layer 6 on the substrate by plasma enhanced chemical vapor deposition;

[0028] (6) Spin-coating a 2-3 μm thick photoresist positive layer on the silicon oxide or silicon nitride insulating layer 6. After photolithography and development, the photoresist at the microelectrode 2, the counter electrode 3, and the contact site 5 is dissolved in the developer, exposing the underlying silicon oxide or silicon nitride insulating layer 6.

[0029] (7) Plasma etching is used to remove the silicon oxide or silicon nitride insulating layer 6 at the microelectrode 2, the counter electrode 3 and the contact site 5 to expose the conductive metal layer underneath.

[0030] The second layer of microfluidic structure is prepared, comprising the following steps:

[0031] (1) Spin-coating a photoresist on a clean silicon wafer, patterning alignment marks for subsequent photolithography by photolithography development, and then forming platinum alignment marks on the silicon wafer using a sputtering process;

[0032] (2) Spin-coating a layer of negative photoresist with a thickness of 5 μm-10 μm on the silicon wafer with the alignment mark, and forming a negative photoresist in the shape of the microfluidic channel 8 on the silicon wafer through photolithography, development, and hardening processes;

[0033] (3) A layer of photoresist negative resist with a thickness of 100 μm to 200 μm is spin-coated on the silicon wafer with the negative resist in the shape of the microfluidic channel 8. A negative resist in the shape of the cell culture chamber 7 is formed on the silicon wafer through photolithography, development, and hardening processes. Thus, a mold for making a microfluidic structure is obtained.

[0034] (4) Spin-coat a layer of positive photoresist with a thickness of 1 μm-3 μm on the mold for making the microfluidic structure, and perform full exposure processing on a photolithography machine to serve as a sacrificial layer for making the microfluidic structure.

[0035] (5) After mixing the polydimethylsiloxane prepolymer and the catalyst, remove bubbles in a vacuum desiccator, and then spin-coat the polydimethylsiloxane on the photoresist sacrificial layer on a spinner. The thickness should match the height of the cell culture chamber 7, which is 100 μm-200 μm, and should not exceed the height of the cell culture chamber 7.

[0036] (6) The spin-coated polydimethylsiloxane is heated and cured.

[0037] (7) After the silicon wafer with the cured polydimethylsiloxane attached to the surface is immersed in a positive photoresist developer for a period of time, the photoresist under the polydimethylsiloxane is dissolved, and then the cured polydimethylsiloxane microfluidic structure is peeled off with tweezers in the positive photoresist developer.

[0038] (8) Oxygen plasma bombardment is used to remove possible defects on the polydimethylsiloxane microfluidic structure, and this method can be used to obtain a thinner polydimethylsiloxane microfluidic structure.

[0039] Preparing the third cell culture ring structure comprises the following steps:

[0040] (1) A high-precision 3D printer is used to produce a mold of the cell culture ring structure 10.

[0041] (2) After mixing the polydimethylsiloxane prepolymer and the catalyst, the bubbles are removed in a vacuum desiccator, and then the polydimethylsiloxane is poured into the mold of the cell culture ring structure 10.

[0042] (3) The cell culture ring structure 10 mold filled with polydimethylsiloxane is heated and cured, and then the cell culture ring structure is removed.

[0043] Furthermore, the method further includes the following steps: after completing the above process, spraying alcohol onto the adhesion surface of the first microelectrode array layer and the second microfluidic structure layer, then aligning them under a microscope, and completing the bonding of the first microelectrode array layer and the second microfluidic structure layer after the alcohol evaporates. Then, using polydimethylsiloxane as the adhesion material, liquid polydimethylsiloxane is spin-coated onto the surface of a clean silicon wafer using a spin coater to obtain a dimethylsiloxane layer of appropriate thickness, and then transferring the dimethylsiloxane layer to the adhesion surface of the third cell culture ring structure by embossing. The first microelectrode array layer and the third cell culture ring structure are then aligned, and finally, heat-curing is performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The above contents and other objects, features and advantages of the present disclosure will become more apparent through the following description of the embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0045] Figure 1 The schematic diagram shows the structure of the microfluidic bio-interconnected neural network chip according to an embodiment of the present disclosure.

[0046] Figure 2 The figure schematically shows the structure of the first-layer microelectrode array of the microfluidic bio-interconnected neural network chip according to an embodiment of the present disclosure.

[0047] Figure 3 The figure schematically shows the second-layer microfluidic structure of the microfluidic bio-interconnected neural network chip according to an embodiment of the present disclosure.

[0048] Figure 4 The flowchart of preparing a microelectrode array according to an embodiment of the present disclosure is schematically shown.

[0049] Figure 5 The flowchart of preparing the microfluidic structure according to the embodiment of the present disclosure is schematically shown.

[0050] In the figure: 1. Insulating substrate; 2. Microelectrode; 3. Counter electrode; 4. Connecting wire; 5. Contact site; 6. Insulating layer; 7. Cell culture chamber; 8. Microfluidic channel; 9. Neuronal network unit; 10. Cell culture ring structure. DETAILED DESCRIPTION

[0051] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. However, it should be understood that these descriptions are merely exemplary and are not intended to limit the scope of the present disclosure. In the detailed description below, for ease of explanation, many specific details are set forth to provide a comprehensive understanding of the embodiments of the present disclosure. However, it is apparent that one or more embodiments may also be implemented without these specific details. In addition, in the following description, descriptions of well-known structures and technologies are omitted to avoid unnecessary confusion of the concepts of the present disclosure.

[0052] The terms used herein are only for describing specific embodiments and are not intended to limit the present invention. The terms "comprise", "include", etc. used herein indicate the presence of the features, steps, operations and / or components, but do not exclude the presence or addition of one or more other features, steps, operations or components.

[0053] The present invention proposes a microfluidic bio-interconnected neural network chip for realizing controllable transmission of neuronal electrical information and a preparation method thereof.

[0054] like Figure 1 、 Figure 2 and Figure 3 As shown, the present invention has designed and fabricated a microfluidic bio-interconnected neural network chip for achieving controllable transmission of neuronal electrical information. The entire microfluidic bio-interconnected neural network chip consists of three layers: a microelectrode array on the first layer, a microfluidic structure on the second layer, and a cell culture ring structure on the third layer.

[0055] The first-layer microelectrode array includes: an insulating substrate 1 , microelectrodes 2 , counter electrodes 3 , connecting wires 4 , contact sites 5 and an insulating layer 6 .

[0056] The second layer of microfluidic structure includes: 256 groups of microfluidic channels 8 with specific shapes and 256 cell culture chambers 7 connected thereto, wherein every four adjacent cell culture chambers 7 are connected through the microfluidic channels 8 to form an independent neuronal network unit 9.

[0057] The third layer includes: a cell culture ring structure 10 .

[0058] The insulating substrate 1 is a soda-lime glass substrate. Microelectrodes 2, counter electrodes 3, connecting wires 4, contact sites 5, and an insulating layer 6 are disposed on the insulating substrate 1. The cell culture chamber 7, microfluidic channel 8, and cell culture ring structure 10 are attached to the insulating substrate 1 using alcohol or polydimethylsiloxane.

[0059] like Figure 1 As shown, the soda-lime glass insulating substrate 1 is the supporting structure of the entire microfluidic chip. It is 1 mm thick, approximately 6 cm long, and 6 cm wide. The microelectrodes 2 are divided into 64 groups, and the microelectrodes 2 are located inside the cell culture chamber 7. The counter electrode 3 and the microelectrodes 2 are both extended by connecting wires 4 and connected to the contact sites 5 on the periphery of the insulating substrate 1. The diameter of the microelectrodes 2 ranges from 4 μm to 35 μm, and the spacing between adjacent microelectrodes 2 is 600 μm. All connecting wires 4 are covered with an insulating layer 6.

[0060] The microfluidic channels 8 are used to connect different cell culture chambers 7 to achieve reliable connection of neuronal axons. There are a total of 256 groups of microfluidic channels 8 with specific shapes and sizes. The shapes include four-channel type and ordinary rectangular. The microfluidic channel length is 400μm-600μm, the microfluidic channel width is 2μm-15μm, and the microfluidic channel height is 2μm-7μm. The 256 groups of microfluidic channels are used to connect 256 cell culture chambers 7. The diameter of the cell culture chamber 7 is 200μm and the height is 50μm-200μm. Four adjacent cell culture chambers 7 are connected end to end through four groups of microfluidic channels to form an independent neuronal network unit 9. At the same time, the microfluidic channel 8 and the cell culture chamber are connected with rounded corners. A large rounded corner structure of 10μm-50μm is set where it is necessary to control the neuronal axons to enter the microfluidic channel 8, and a small rounded corner structure of 2μm-6μm is set where it is necessary to control the neuronal axons not to enter the microfluidic channel 8.

[0061] For example, four adjacent cell culture chambers 7 are connected end-to-end using a four-channel microfluidic channel 8. The cell culture chamber 7 has a diameter of 200 μm and a height of 150 μm, and the distance between adjacent cell culture chambers 7 is 600 μm. The four-channel microfluidic channel 8 is 400 μm long, 6 μm wide, and 5 μm high. The connection between the microfluidic channel 8 and the cell culture chamber is connected using rounded corners. A large rounded corner structure of 50 μm is set where it is necessary to control the entry of neuronal axons into the microfluidic channel 8, and a small rounded corner structure of 3 μm is set where it is necessary to control the entry of neuronal axons into the microfluidic channel 8. After the microelectrode array a and the microfluidic structure b are aligned and attached, each cell culture chamber 7 contains a microelectrode 2 with a diameter of 30 μm.

[0062] In the microelectrode array, the insulating substrate 1 is made of soda-lime glass; the microelectrode 2 is a conductive composite material with biocompatibility and good adhesion to the soda-lime glass substrate, preferably, the material of the microelectrode 2 is a composite material of platinum and chromium; the insulating layer 6 is made of an insulating material with biocompatibility and good adhesion to the soda-lime glass substrate, preferably, the material used for the insulating layer 6 is silicon dioxide and silicon nitride.

[0063] The second layer of microfluidic chip and the third layer of cell culture ring structure are made of biocompatible polydimethylsiloxane.

[0064] The preparation of microfluidic bio-interconnected neural network chips can be divided into three parts: the preparation of the first layer microelectrode array, the preparation of the second layer microfluidic structure and the preparation of the third layer cell culture ring structure.

[0065] The specific preparation process of the first layer microelectrode array of the present invention is as follows: Figure 4 , described in detail as follows:

[0066] Step a) using acetone, ethanol, deionized water sequentially clean the soda-lime glass insulating substrate 1, and then into 100 ℃ concentrated sulfuric acid to obtain a clean surface of the insulating substrate 1, the cleaned insulating substrate 1 is dried and placed on a spinning machine, a 2μm layer of positive photoresist SPR220 is spin-coated on the insulating substrate 1;

[0067] Step b) After the insulating substrate 1 after the rejection of the glue is exposed in contact with the photolithography machine into a concentration of 0.6% NaOH developer, the microelectrode 2, the counter electrode 3, the connecting wire 4 and the contact site 5 at the photoresist is dissolved in the developer, exposing the insulating substrate 1 below;

[0068] Step c) depositing a 30 nm thick titanium layer on the photoresist-patterned insulating substrate 1 by sputtering to improve the adhesion between the conductive layer and the substrate. Then, depositing a 250 nm thick platinum conductive layer by sputtering;

[0069] Step d) using a stripping process in acetone, ethanol and deionized water to remove the conductive layer of the photoresist below, leaving the desired microelectrode 2, the counter electrode 3, the connecting wire 4 and the contact site 5;

[0070] Step e) by plasma enhanced chemical vapor deposition method on the substrate sequentially deposited a layer of 300nm thick silicon oxide and a layer of 500nm thick silicon nitride as an insulating layer 6;

[0071] Step f): Spin-coat a 2 μm thick positive photoresist SPR220 on the silicon oxide or silicon nitride insulating layer 6 .

[0072] Step g) after the insulating substrate 1 after the rejection of the glue contact exposure on the photolithography machine into a concentration of 0.6% NaOH developer, photolithography development after the microelectrode 2, the electrode 3 and the contact site 5 at the photoresist is dissolved in the developer, exposing the underlying silicon oxide and silicon nitride insulating layer 6;

[0073] Step h). Plasma etching is used to remove the silicon oxide or silicon nitride insulating layer 6 at the microelectrode 2, the counter electrode 3 and the contact site 5, exposing and retaining the conductive metal layer underneath.

[0074] The specific preparation process of the second layer microfluidic structure of the present invention is as follows: Figure 5 , described in detail as follows:

[0075] Step a) Spin-coat a 2 μm thick layer of positive photoresist SPR220 on a clean silicon wafer;

[0076] Step b) after the silicon wafer after the rejection of the contact exposure on the photolithography machine into a concentration of 0.6% NaOH developer to remove excess photoresist;

[0077] Step c) depositing a 30 nm thick titanium layer on the photoresist-patterned insulating substrate 1 by sputtering to improve the adhesion between the conductive layer and the substrate. Then, depositing a 250 nm thick platinum conductive layer by sputtering;

[0078] Step d) using a stripping process in acetone, ethanol and deionized water to remove the conductive layer below the photoresist, leaving the desired alignment mark pattern;

[0079] Step e) Spin-coating a layer of 7.5 μm thick negative photoresist SU-82005 on the silicon wafer;

[0080] Step f) after the silicon wafer after the rejection of the glue is exposed on the photolithography machine before baking on a hot plate, and then placed in a PGMEA developer to remove excess photoresist, leaving a photoresist having a microfluidic channel structure;

[0081] Step g) spin coating a layer of 160 μm thick negative photoresist SU-82075 on the silicon wafer;

[0082] Step h) after the silicon wafer after the rejection of the glue contact exposure on the photolithography machine before baking on a hot plate, and then placed in PGMEA developer to remove excess photoresist, leaving a photoresist having a microfluidic chamber structure;

[0083] Step i) Spin-coating a 2 μm thick layer of positive photoresist SPR220 on the silicon wafer;

[0084] Step j). Placing the silicon wafer on a photolithography machine for full exposure without using a mask;

[0085] Step k) Place the silicon wafer on a spin coater and spin coat a layer of PDMS;

[0086] Step 1). After the PDMS on the silicon wafer surface is cured, it is peeled off using tweezers in a 1% NaOH developer to obtain a separated PDMS microfluidic structure.

[0087] The specific preparation process of the third-layer cell culture ring structure of the present invention is described in detail as follows:

[0088] Step 1. Create a 3D model of the cell culture ring structure mold in SolideWorks, slice the 3D model using Cura slicing software, and then use a high-precision 3D printer to produce the cell culture ring structure mold.

[0089] Step 2. After mixing the polydimethylsiloxane prepolymer and the catalyst in a mass ratio of 10:1, the air bubbles were removed in a vacuum desiccator, and then the polydimethylsiloxane was poured into the mold of the cell culture ring structure.

[0090] Step 3. After removing bubbles from the cell culture ring structure mold filled with polydimethylsiloxane in a vacuum desiccator, place it in an 80-degree Celsius oven for curing for 30 minutes, and demold it after solidification.

[0091] After completing the above process, alcohol is sprayed onto the adhesion surface of the first microelectrode array and the second microfluidic structure. They are then aligned under a microscope. After the alcohol evaporates, the first and second microelectrode arrays are bonded together. Polydimethylsiloxane is then used as the adhesion material. Liquid polydimethylsiloxane is spin-coated onto a clean silicon wafer using a spinner to obtain a dimethylsiloxane layer of appropriate thickness. This layer is then transferred to the adhesion surface of the third cell culture ring structure by embossing. The first and second microelectrode arrays are then aligned and cured by heat.

[0092] The steps for using a microfluidic biointerconnected neural network chip for achieving controllable transmission of neuronal electrical information are as follows:

[0093] Step 1. Before using the chip, add deionized water to the cell culture ring structure 10 and vibrate it ultrasonically for 15 minutes to allow the deionized water to fill the microfluidic channel 8.

[0094] Step 2. Remove the deionized water in the cell culture ring structure 10 and add the polylysine solution into the cell culture chamber 7. After the solution fills the microfluidic channel 8, wait for the polylysine to modify the surface for 30 minutes, and then remove the solution.

[0095] Step 3. Add the primary neuronal cell solution to the top of the cell culture chamber 7 and let it sink freely. After culturing for 3-7 days, the axons of the neurons in the adjacent cell culture chambers 7 connected by the microfluidic channel 8 will form functional connections in the microfluidic channel 8.

[0096] Example 1:

[0097] Primary cortical excitatory neurons isolated from embryos of ICR rats at 15-18 days of gestation were cultured in the cell culture rings of the chip. Under gravity, the cultured primary cortical excitatory neurons automatically descended into the cell culture chamber below, where they continued to grow. After 2-3 weeks of culture in a CO2 incubator, the neuronal network on the microfluidic biointerconnected neural network chip began to mature.

[0098] The neural network was tested using a 128-channel electrophysiological testing instrument from Blackrock, USA. The testing steps can be divided into the following steps:

[0099] Step 1. Record the neuronal activity of the neural network without external interference as a reference for analyzing neuronal activity.

[0100] Step 2. Use a Multichannel electrophysiological stimulation device to stimulate a node in the neural network and continuously record the neural activity of the neural network. Focus on the transmission of neuronal spike signals within the neural network, including the transmission order and firing frequency of spike signals.

[0101] Step 3. Add glutamate, a chemical that affects neuronal activity, to a node in the neural network and continuously record the neural activity of the network. Again, focus on the transmission of spike signals within the network, including the order and frequency of spike signal transmission.

[0102] Step 4. Use the offline data analysis software Offline Sorter and NeuroExplore to classify the waveforms of the neural network, count the spikes emitted by the neural network before and after stimulation, and use Python programming to further explore the activity characteristics of the neural network after stimulation.

[0103] Example 2:

[0104] The density is 10 5 / mL undifferentiated PC12 cells were seeded and cultured in fresh F-12K medium, and a suitable environment and growth factors for cell growth were provided. Nerve growth factor was used to promote cell differentiation and cultured for six days to obtain a neuronal network with a certain degree of maturity.

[0105] The neural network was tested using a 128-channel electrophysiological testing instrument from Blackrock, USA. The testing steps can be divided into the following steps:

[0106] Step 1. Record the neuronal activity of the neural network without external interference as a reference for analyzing neuronal activity.

[0107] Step 2. Enter the experimental phase and select a cell culture chamber node with relatively strong neuronal activity. When the neuronal activity in this area exceeds a certain threshold, use Multichannel's electrophysiological stimulation instrument to perform a 0.5V tetanic stimulation on this node in the neuronal network. The experiment lasts for five minutes and the neural activity of the neuronal network is continuously recorded.

[0108] Step 3. Use the offline data analysis software Offline Sorter and NeuroExplore to classify and preliminarily analyze the waveforms of the neural network. Use Python programming to further explore the neuronal activity of the neural network before and after stimulation to determine whether the neural network has learning function.

[0109] The above embodiments are used to illustrate the present invention rather than to limit the present invention. Any modifications and changes made to the present invention within the spirit of the present invention and the protection scope of the claims shall fall within the protection scope of the present invention.

Claims

1. A microfluidic biointerconnected neural network chip, characterized in that: The microfluidic biointerconnected neural network chip comprises a three-layer structure: a first-layer microelectrode array, a second-layer microfluidic structure, and a third-layer cell culture ring structure (10); wherein the first-layer microelectrode array comprises: an insulating substrate (1), microelectrodes (2), counter electrodes (3), connecting wires (4), contact sites (5), and an insulating layer (6); the insulating substrate (1) is a supporting structure of the microfluidic biointerconnected neural network chip; the microelectrodes (2) are divided into 64 groups, and the microelectrodes (2) are located inside the cell culture chamber (7); the counter electrodes (3) and the microelectrodes (2) are extended and connected to the contact sites (5) on the periphery of the insulating substrate (1) through connecting wires (4); the surfaces of all connecting wires (4) are covered with an insulating layer (6); The second layer of microfluidic structure includes: 256 groups of microfluidic channels (8) and 256 cell culture chambers (7) connected thereto, wherein every four adjacent cell culture chambers (7) are connected through the microfluidic channels (8) to form an independent neuronal network unit (9); The third layer includes: a cell culture ring structure (10); The first layer of microelectrode array and the second layer of microfluidic structure were bonded by alcohol evaporation, and the first layer of microelectrode array and the third layer of cell culture ring structure were bonded by polydimethylsiloxane as the adhesive material. The diameter of a single cell culture chamber (7) integrated with a microfluidic channel (8) is 200 μm. Four adjacent cell culture chambers (7) are connected in an orderly manner through the microfluidic channel (8) to form an independent neuronal network unit (9). The shape of the microfluidic channel (8) is a four-channel type and a common rectangle. The length of the microfluidic channel (8) is 400 μm-600 μm and the width is 2 μm-15 μm. At the same time, the connection between the microfluidic channel (8) and the cell culture chamber (7) is connected by a rounded corner. A large rounded corner structure of 10 μm-50 μm is set at the place where the neuronal axons need to be controlled to enter the microfluidic channel (8), and a small rounded corner structure of 2 μm-6 μm is set at the place where the neuronal axons need to be controlled not to enter the microfluidic channel (8).

2. The microfluidic bio-interconnected neural network chip according to claim 1, characterized in that: The insulating substrate (1) is made of soda-lime glass; The microelectrode (2) is made of a conductive composite material having biocompatibility and good adhesion to the insulating substrate (1); The material used for the insulating layer (6) is an insulating material that is biocompatible and has good adhesion to the insulating substrate (1).

3. The microfluidic bio-interconnected neural network chip according to claim 1, characterized in that: The diameter of the microelectrodes (2) ranges from 4 to 35 μm, and the inter-site spacing ranges from 500 to 700 μm.

4. The microfluidic bio-interconnected neural network chip according to claim 1, characterized in that: The materials of the second-layer microfluidic structure and the third-layer cell culture ring structure are biocompatible polydimethylsiloxane.

5. The microfluidic bio-interconnected neural network chip according to claim 1, characterized in that: The inner size of the third-layer cell culture ring structure is slightly larger than that of the second-layer microfluidic structure, and is 2 mm to 10 mm in height, which is conducive to in vitro culture of neurons.

6. A method for preparing the microfluidic biointerconnected neural network chip according to any one of claims 1 to 5, characterized in that: The method comprises preparing a first layer of microelectrode array, preparing a second layer of microfluidic structure and preparing a third layer of cell culture ring structure; Preparation of the first layer of microelectrode array includes the following steps: Step a). Using acetone, ethanol, and deionized water to clean the soda-lime glass insulating substrate (1) in sequence, and then washing it in concentrated sulfuric acid at 100°C to obtain an insulating substrate (1) with a clean surface. After drying the cleaned insulating substrate (1), the substrate is placed on a spin coater and a 2 μm layer of positive photoresist SPR220 is spin-coated on the insulating substrate (1); Step b). The insulating substrate (1) after the spin coating is subjected to contact exposure on a photolithography machine and then placed in a 0.6% NaOH developer. The photoresist at the microelectrode (2), the counter electrode (3), the connecting wire (4) and the contact point (5) is dissolved in the developer, exposing the insulating substrate (1) below. Step c). A 30 nm thick titanium metal layer is deposited on the photoresist patterned insulating substrate (1) by sputtering to increase the adhesion between the conductive layer and the substrate, and then a 250 nm thick platinum metal conductive layer is sputtered; Step d). Using a stripping process, the conductive layer with the photoresist underneath is removed by washing in acetone, ethanol and deionized water in sequence, leaving the required microelectrode (2), counter electrode (3), connecting wire (4) and contact site (5); Step e) depositing a 300 nm thick silicon oxide layer and a 500 nm thick silicon nitride layer as an insulating layer (6) on the substrate by plasma enhanced chemical vapor deposition. Step f) spin coating a 2 μm thick photoresist positive resist SPR220 on the silicon oxide or silicon nitride insulating layer (6); Step g). The insulating substrate (1) after the spin coating is subjected to contact exposure on a photolithography machine and then placed in a 0.6% NaOH developer. After photolithography development, the photoresist at the microelectrode (2), the counter electrode (3) and the contact point (5) is dissolved in the developer, exposing the underlying silicon oxide and silicon nitride insulating layers (6); Step h) using plasma etching to remove the silicon oxide or silicon nitride insulating layer (6) at the microelectrode (2), the counter electrode (3) and the contact site (5), exposing and retaining the conductive metal layer underneath; The second layer of microfluidic structure is prepared, comprising the following steps: Step a) spin-coating a layer of 2 μm thick positive photoresist SPR220 on a clean silicon wafer; Step b) after the silicon wafer is thrown into the photolithography machine for contact exposure and then placed in a 0.6% concentration of NaOH developer to remove excess photoresist; Step c). A 30 nm thick titanium metal layer is deposited on the photoresist patterned insulating substrate (1) by sputtering to increase the adhesion between the conductive layer and the substrate, and then a 250 nm thick platinum metal conductive layer is sputtered; Step d) using a stripping process in acetone, ethanol and deionized water sequentially clean and remove the conductive layer of the photoresist underneath, leaving the desired alignment mark pattern; Step e) spin-coating a layer of 7.5 μm thick negative photoresist SU-8 2005 on the silicon wafer; Step f) after the silicon wafer is thrown on the photolithography machine for contact exposure before baking on a hot plate, and then placed in a PGMEA developer to remove excess photoresist, leaving a photoresist having a microfluidic channel structure; Step g) spin-coating a layer of 160μm thick negative photoresist SU-8 2075 on the silicon wafer; Step h) after the silicon wafer is thrown off the contact exposure on the photolithography machine before baking on a hot plate, and then placed in a PGMEA developer to remove excess photoresist, leaving a photoresist having a microfluidic chamber structure; Step i) spin-coating a 2 μm thick layer of positive photoresist SPR220 on the silicon wafer; Step j) placing the silicon wafer on a photolithography machine for full exposure without using a mask; Step k) Place the silicon wafer on a spin coater and spin-coat a layer of PDMS; Step l) After the PDMS on the silicon wafer surface is cured, it is peeled off using tweezers in a 1% NaOH developer to obtain a separated PDMS microfluidic structure; Preparing the third cell culture ring structure comprises the following steps: Step (1) using a high-precision 3D printer to produce a mold of the cell culture ring structure (10); Step (2) mixing the polydimethylsiloxane prepolymer and the catalyst and removing bubbles in a vacuum desiccator, and then pouring the polydimethylsiloxane into a mold of the cell culture ring structure (10); Step (3) heats and solidifies the mold of the cell culture ring structure (10) filled with polydimethylsiloxane, and then removes the cell culture ring structure (10).

7. The method according to claim 6, characterized in that The method further comprises the following steps: after completing the above process, spraying alcohol in the form of a spray on the adhesion surface of the first layer microelectrode array and the second layer microfluidic structure, then aligning them under a microscope, and completing the bonding of the first layer microelectrode array and the second layer microfluidic structure after the alcohol evaporates; then using polydimethylsiloxane as an adhesion material, spin-coating liquid polydimethylsiloxane on the surface of a clean silicon wafer using a spinner to obtain a dimethylsiloxane layer of appropriate thickness, then transferring the dimethylsiloxane layer to the adhesion surface of the third layer cell culture ring structure by embossing, then aligning the first layer microelectrode array and the third layer cell culture ring structure, and finally heating and curing.

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