Microfluidic devices and nerve cell action potential detection system

Abstract

The present invention relates to a microfluidic device, comprising a substrate, a microfluidic layer, a porous microelectrode array and a cell culture layer arranged in sequence from bottom to top, wherein the microfluidic layer is fixed on the substrate, at least part of the porous microelectrode array is fixed between the microfluidic layer and the cell culture layer, and the cell culture layer is fixed to the microfluidic layer; a first through hole and a second through hole are arranged on the cell culture layer, a first blind hole, a second blind hole and a microfluidic channel are arranged on a side of the microfluidic layer close to the cell culture layer, two ends of the microfluidic channel are respectively connected to the first blind hole and the second blind hole, the first through hole is aligned with the first blind hole, a portion of the porous microelectrode array located between the microfluidic layer and the cell culture layer is provided with a plurality of third through holes, the first through hole and the first blind hole are connected to each other through at least part of each of the third through holes, and the second through hole is aligned with the second blind hole and connected to each other.

Description

Technical Field

[0001] The present invention relates to the fields of neuroscience and biomedical engineering, and more specifically to a microfluidic device and a nerve cell action potential detection system. Background Art

[0002] In vitro detection of neuronal action potentials is of great significance in neuroscience research. Action potential is the basic unit of information transmission in nerve cells. By studying the generation, propagation and regulation mechanism of action potentials, we can deeply reveal the working principles of neural circuits and elucidate the pathological processes of nervous system diseases. In addition, in vitro detection technology has broad application potential in drug screening, development of new therapies, and early diagnosis of neurodegenerative diseases. Therefore, the development of efficient and accurate in vitro neuronal action potential detection technology is not only crucial to basic research in neuroscience, but also plays a key role in the diagnosis and treatment of neurological diseases.

[0003] Action potentials are triggered by the depolarization and repolarization process of neuronal membranes. When nerve cells receive external stimuli, the membrane potential rises rapidly from the resting potential (usually -60 to -70 mV). After exceeding the threshold potential, it triggers the opening of voltage-gated sodium ion channels, causing sodium ions to flow in rapidly, causing the membrane potential to quickly reach a positive value (+30 to +50 mV). This stage is called depolarization. Subsequently, sodium ion channels close, potassium ion channels open, potassium ions flow out, and the membrane potential returns to the resting level. This process is called repolarization. At present, the technologies for detecting neuronal action potentials in vitro are mainly divided into the following two categories:

[0004] 1. Patch-Clamp Technique

[0005] Patch clamp technology is the gold standard method for detecting neuronal action potentials. This technology can directly record the potential difference on both sides of the cell membrane and accurately measure the action potential of a single neuron. The main advantage of patch clamp technology is that the detection is highly accurate, and very subtle electrophysiological changes can be detected. It has extremely high time and voltage resolution (up to the millivolt level), and allows precise control of cell membrane voltage, which can study neuronal responses under different voltage conditions. However, this technology also has obvious limitations: it can only detect a single cell at a time, making it difficult to achieve group analysis of neural circuits and unable to meet the needs of large-scale experiments; it has high technical requirements, cumbersome operations, high experimental costs, and is difficult to record for a long time.

[0006] 2. Microelectrode Array (MEA)

[0007] Microelectrode array technology records the action potential discharges of neuronal groups through planar electrodes. Its main advantages include the ability to detect the electrical activity of a large number of neurons at the same time, which is helpful for the study of neural circuits. However, this technology also has some shortcomings. Because the contact surface between the electrode and the neuron is not tight enough, and the electrode is usually a planar structure, while the neural tissue is often a curved structure, the collected signal is weak and the signal-to-noise ratio is low; during the long detection process, the electrode and the neural tissue may shift, affecting the accuracy of the detection. Summary of the invention

[0008] The purpose of the present invention is to provide a microfluidic device and a nerve cell action potential detection system, which can not only meet the simultaneous detection of a large number of neurons, but also maintain a good acquisition signal-to-noise ratio, so as to solve the limitations of the existing technology and provide an advanced testing platform for the study of complex neural networks and neurological diseases.

[0009] Based on the above purpose, the present invention provides a microfluidic device on one hand, comprising a substrate, a microfluidic layer, a porous microelectrode array and a cell culture layer arranged in sequence from bottom to top, the microfluidic layer is fixed on the substrate, at least a portion of the porous microelectrode array is fixed between the microfluidic layer and the cell culture layer, and the cell culture layer is fixed to the microfluidic layer; a first through hole and a second through hole are provided on the cell culture layer, a first blind hole, a second blind hole and a microfluidic channel are provided on a side of the microfluidic layer close to the cell culture layer, both ends of the microfluidic channel are respectively connected to the first blind hole and the second blind hole, the first through hole is aligned with the first blind hole, a portion of the porous microelectrode array located between the microfluidic layer and the cell culture layer is provided with a plurality of third through holes, the first through hole and the first blind hole are connected to each other through at least a portion of each third through hole, and the second through hole is aligned with the second blind hole and connected to each other.

[0010] Furthermore, a culture groove is provided on a side of the cell culture layer away from the microfluidic channel layer, and the first through hole is located in the culture groove.

[0011] Furthermore, the microfluidic layer and the cell culture layer are both made of polydimethylsiloxane material.

[0012] Furthermore, the porous microelectrode array includes a substrate, a first packaging layer, a wire layer, a pad layer and a second packaging layer arranged in sequence from bottom to top, the wire layer is used to form a plurality of electrode sites for neural signal collection, and the pad layer is used to transmit signals to a signal collection device.

[0013] Furthermore, the porous microelectrode array is divided into a first part and a second part, each third through hole is located in the first part, and the pad layer is located in the second part.

[0014] Furthermore, the first packaging layer and the second packaging layer are both made of SU-8 material.

[0015] Furthermore, sizes of the third through holes are all the same, partially the same, or different.

[0016] Furthermore, the third through holes are evenly arranged around the electrode sites.

[0017] Furthermore, the substrate, the microfluidic channel layer, the porous microelectrode array and the cell culture layer are fixed together in sequence by heating and curing a polydimethylsiloxane precursor solution.

[0018] On the other hand, the present invention provides a nerve cell action potential detection system, which includes a signal acquisition device, a pressure controller and the microfluidic device as described above, wherein the signal acquisition device is connected to the porous microelectrode array of the microfluidic device and is used to collect nerve cell action potential signals; the pressure controller is connected to the second through hole of the microfluidic device and is used to provide negative pressure to the second through hole so that the nerve tissue in the nerve cell culture fluid is tightly adsorbed on the porous microelectrode array.

[0019] The microfluidic device and the nerve cell action potential detection system of the present invention are provided with a plurality of third through holes on the porous microelectrode array, so that the nerve tissue can be tightly adsorbed on the porous microelectrode array under the action of negative pressure, so that the collected signal is stronger and the signal-to-noise ratio is higher, and a large number of nerve cells can be detected simultaneously, and the detection flux is high; the first packaging layer and the second packaging layer of the porous microelectrode array are made of SU-8 material, so a small amount of deformation can be produced to better fit the cells, thereby further improving the signal-to-noise ratio of the collected signal. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 A schematic diagram of the structure of a microfluidic device according to an embodiment of the present invention;

[0021] Figure 2 An exploded view of a microfluidic device according to an embodiment of the present invention;

[0022] Figure 3 4 is a cross-sectional view of a porous microelectrode array of a microfluidic device according to an embodiment of the present invention. DETAILED DESCRIPTION

[0023] The preferred embodiments of the present invention are given below in conjunction with the accompanying drawings and described in detail.

[0024] like Figure 1 and Figure 2As shown, an embodiment of the present invention provides a microfluidic device, comprising a substrate 100, a microfluidic layer 200, a porous microelectrode array 300 and a cell culture layer 400 arranged in sequence from bottom to top, the microfluidic layer 200 is fixed on the substrate 100, at least part of the porous microelectrode array 300 is located between the microfluidic layer 200 and the cell culture layer 400 and is fixed to the microfluidic layer 200 and the cell culture layer 400 respectively, and the cell culture layer 400 is fixed to the microfluidic layer 200; a first through hole 410 and a second through hole 420 are provided on the cell culture layer 400, and the microfluidic layer 200 is close to the cell culture layer 400. A first blind hole 210, a second blind hole 220 and a microfluidic channel 230 are provided on one surface of 400, and both ends of the microfluidic channel 230 are respectively connected to the first blind hole 210 and the second blind hole 220, and the first through hole 410 is aligned with the first blind hole 210. The portion of the porous microelectrode array 300 located between the microfluidic channel layer 200 and the cell culture layer 400 covers the first through hole 410 and the first blind hole 210, and a plurality of third through holes are provided thereon, and the first through hole 410 and the first blind hole 210 are connected to each other through at least a portion of each third through hole, and the second through hole 420 is aligned with and connected to the second blind hole 220. When in use, the neural cell culture fluid can be injected into the first through hole 410, and a certain negative pressure can be applied at the second through hole 420. Under the negative pressure, the neural cell culture fluid will pass through the first through hole 410, the third through hole on the porous microelectrode array 300, the first blind hole 210, the microfluidic channel 230, the second blind hole 220 and the second through hole 420 in sequence, and flow out from the second through hole 420. Under the negative pressure, the neural tissue will be tightly adsorbed on the porous microelectrode array 300, and then the porous microelectrode array 300 can detect the electrical activities of a large number of neurons at the same time. Since the porous microelectrode array 300 is provided with a plurality of third through holes, a negative pressure can be applied at the second through hole 420, so that the neural tissue is tightly adsorbed on the porous microelectrode array 300 under the negative pressure. Compared with the existing solution of placing the neural tissue directly on the microelectrode array, the contact between the neural tissue and the porous microelectrode array 300 is closer, so the collected signal is stronger and the signal-to-noise ratio is higher.

[0025] In some embodiments, a culture groove 430 is provided on one side of the cell culture layer 400 away from the microfluidic layer 200, and the first through hole 410 is located in the culture groove 430. The culture groove 430 is used to contain a neural cell culture fluid. When in use, the neural cell culture fluid can be first injected into the culture groove 430, and then a negative pressure is applied at the second through hole 420. Under the action of the negative pressure, the neural cell culture fluid in the culture groove 430 will be sucked into the first through hole 410, and will pass through the third through hole, the first blind hole 210, the microfluidic channel 230, and the second blind hole 220 in sequence, and then flow out from the second through hole 420. At the same time, the neural tissue will also be tightly adsorbed on the porous microelectrode array 300 under the action of pressure.

[0026] In some embodiments, the culture tank 430 is a rectangular tank with a depth of 2 mm. The length and width can be set as needed. The diameters of the first through hole 410 and the first blind hole 210 are both 4 mm, the diameters of the second through hole 420 and the second blind hole 220 are both 3 mm, the depth and width of the microfluidic channel 230 are both 1 mm, and the length of the microfluidic channel 230 can be set as needed.

[0027] In some embodiments, the microfluidic layer 200 and the cell culture layer 400 are both made of polydimethylsiloxane (PDMS) material. Specifically, high-precision molds of the microfluidic layer 200 and the cell culture layer 400 can be prepared by 3D printing or computer numerical control (CNC) processing, and then the PDMS precursor solution (mixed by PDMS base monomer and curing agent in a ratio of 10:1) is poured on the prepared mold, and placed in a vacuum environment to remove bubbles, and then placed in an 80°C oven for 3 hours, PDMS is cured, and demolding and punching are performed to obtain the microfluidic layer 200 and the cell culture layer 400.

[0028] In some embodiments, the substrate 100 may be made of a glass material.

[0029] like Figure 3 As shown, in some embodiments, the porous microelectrode array 300 includes a substrate 310, a first packaging layer 320, a wire layer 330, a pad layer 340 and a second packaging layer 350 arranged in sequence from bottom to top, the wire layer 330 may include chromium, gold, and chromium arranged in sequence from bottom to top, the pad layer 340 may include chromium, nickel, and gold arranged in sequence from bottom to top, the wire layer 330 is used to form a plurality of electrode sites for neural signal collection, and the pad layer 340 is connected to a customized signal collection device to realize electrical signal derivation; the porous microelectrode array 300 can be divided into two parts, namely Figure 3 The first part in the dotted box and the second part outside the dotted box, the first part is the part sandwiched between the cell culture layer 400 and the microfluidic layer 200, the first part is provided with a plurality of third through holes 360, the pad layer 340 is located on the second part, and the second part is located outside the cell culture layer 400 and the microfluidic layer 200, so as to facilitate the connection of the pad layer 340 with the signal acquisition device and export the electrical signal.

[0030] In some implementations, the sizes of the third through holes 360 may be all the same, for example, approximately equal to the size of the electrode site, or the sizes of the third through holes 360 may be partially the same, or the sizes of the third through holes 360 may be different. The third through holes 360 may be evenly arranged around the electrode sites so that the nerve tissue can be better attached to the electrode sites.

[0031] In some embodiments, the first packaging layer 320 and the second packaging layer 350 are both made of SU-8 material, which is a negative photoresist based on epoxy resin and is softer. The bottom of the first part of the porous microelectrode array 300 has no substrate, and only the first packaging layer 320 and the second packaging layer 350 cover the electrode sites of the wire layer 330. Therefore, the first part will be flexible and bendable, and the electrode can be formed into a curved structure, thereby increasing the contact area with the neural tissue and further improving the signal-to-noise ratio of the collected signal.

[0032] The assembly method of the microfluidic device of the embodiment of the present invention is as follows:

[0033] First, obtain the pre-prepared substrate 100, microfluidic layer 200, porous microelectrode array 300 and cell culture layer 400, and then apply PDMS precursor solution on the contact surfaces between these four components, for example, apply PDMS precursor solution on the upper surface of the substrate 100, the lower surface and upper surface of the microfluidic layer 200, the upper surface and lower surface of the porous microelectrode array 300, and the lower surface of the cell culture layer 400, and when applying, avoid the first through hole 410, the second through hole 420, the first blind hole 210, the second blind hole 220, the microfluidic channel 230 and the third through hole 360; then align the layers in sequence and stack them together, heat them after stacking to solidify the PDMS precursor solution, and realize the assembly between the components.

[0034] An embodiment of the present invention also provides a nerve cell action potential detection system, including a signal acquisition device, a pressure controller and a microfluidic device as described in the above embodiment, wherein the signal acquisition device is connected to the porous microelectrode array 300 of the microfluidic device for collecting nerve cell action potential signals; the pressure controller can be connected to the second through hole 420 through a trachea, for example, to provide a stable negative pressure to the second through hole 420, thereby tightly adsorbing the nerve tissue in the nerve cell culture fluid onto the porous microelectrode array 300, so that the two can fit tightly.

[0035] The working process of the nerve cell action potential detection system is as follows:

[0036] First, the nerve cell culture fluid is injected into the culture tank 430, and then a certain pressure (i.e., negative pressure) is applied to the second through hole 420 through the pressure controller to extract the culture fluid, so that the culture fluid flows from the culture tank 430 toward the second through hole 420. During the flow process, the nerve tissue will fit tightly with the porous microelectrode array under the action of negative pressure, and each electrode of the porous microelectrode array transmits the nerve cell potential signal to the signal acquisition device, and the nerve cell potential signal is acquired by the signal acquisition device.

[0037] In the microfluidic device and the nerve cell action potential detection system of the embodiments of the present invention, a plurality of third through holes are provided on the porous microelectrode array 300, so that the nerve tissue can be tightly adsorbed on the porous microelectrode array 300 under the action of negative pressure, so that the collected signal is stronger and the signal-to-noise ratio is higher, and a large number of nerve cells can be detected simultaneously, with a high detection flux; the first packaging layer 320 and the second packaging layer 350 of the porous microelectrode array 300 are made of SU-8 material, so a small amount of deformation can be produced to better fit the cells, thereby further improving the signal-to-noise ratio of the collected signal.

[0038] The above is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. The above embodiment of the present invention can also be modified in various ways. That is, all simple, equivalent changes and modifications made according to the claims and the description of the present invention fall within the scope of protection of the claims of the present invention. The contents not described in detail in the present invention are all conventional technical contents.

Claims

1. A microfluidic device, characterized in that: The invention comprises a substrate, a microfluidic layer, a porous microelectrode array and a cell culture layer arranged in sequence from bottom to top, wherein the microfluidic layer is fixed on the substrate, a part of the porous microelectrode array is fixed between the microfluidic layer and the cell culture layer, and the cell culture layer is fixed to the microfluidic layer; a first through hole and a second through hole are arranged on the cell culture layer, a first blind hole, a second blind hole and a microfluidic channel are arranged on a side of the microfluidic layer close to the cell culture layer, two ends of the microfluidic channel are respectively connected to the first blind hole and the second blind hole, the first through hole is aligned with the first blind hole, and the porous microelectrode array is located in the A plurality of third through holes are provided on the portion between the microfluidic channel layer and the cell culture layer, the first through hole and the first blind hole are interconnected through portions of the third through holes, and the second through hole is aligned with the second blind hole and is interconnected; the first through hole is used to inject a neural cell culture solution, and the second through hole is used to apply a negative pressure, so that the neural cell culture solution passes through the first through hole, the third through hole, the first blind hole, the microfluidic channel, the second blind hole and the second through hole in sequence through the negative pressure, and under the action of the negative pressure, the neural tissue in the neural cell culture solution is tightly adsorbed on the porous microelectrode array; The porous microelectrode array comprises a substrate, a first packaging layer, a wire layer, a pad layer and a second packaging layer arranged in sequence from bottom to top, the wire layer is used to form a plurality of electrode sites for neural signal collection, and the pad layer is used to transmit signals to a signal collection device; The porous microelectrode array is divided into a first part and a second part, each third through hole is located in the first part, and the pad layer is located in the second part; the bottom of the first part has no substrate to have flexibility.

2. The microfluidic device according to claim 1, characterized in that: A culture groove is provided on a side of the cell culture layer away from the microfluidic channel layer, and the first through hole is located in the culture groove.

3. The microfluidic device according to claim 1, characterized in that: The microfluidic layer and the cell culture layer are both made of polydimethylsiloxane material.

4. The microfluidic device according to claim 1, characterized in that: The first packaging layer and the second packaging layer are both made of SU-8 material.

5. The microfluidic device according to claim 1, characterized in that: The sizes of the third through holes are all the same, partially the same, or different.

6. The microfluidic device according to claim 1, characterized in that: The third through holes are evenly arranged around the electrode sites.

7. The microfluidic device according to claim 1, characterized in that: The substrate, the microfluidic channel layer, the porous microelectrode array and the cell culture layer are fixed together in sequence by heating and curing a polydimethylsiloxane precursor solution.

8. A nerve cell action potential detection system, characterized in that: It comprises a signal acquisition device, a pressure controller and a microfluidic device as described in any one of claims 1 to 7, wherein the signal acquisition device is connected to the porous microelectrode array of the microfluidic device and is used to collect nerve cell action potential signals; the pressure controller is connected to the second through hole of the microfluidic device and is used to provide negative pressure to the second through hole so that the neural tissue in the neural cell culture fluid is tightly adsorbed on the porous microelectrode array.

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