Microneedle chip module, adapter and culture monitoring device

Abstract

The invention relates to the field of microneedle electrodes, in particular to a microneedle chip module, an adapter and a culture monitoring device.According to the microneedle chip module, a plurality of microneedle arrays are integrated on the surface of an electrode substrate, tissue slices are pierced through microneedle electrodes, and therefore target cells located in an inner cell layer are recorded or stimulated; according to the culture monitoring device, a probe and a back-stage equipment interface are respectively integrated inside and outside a shell through an adapter, the back-stage equipment interface and a microneedle array are connected through the probe, so that the functions of protecting and quickly connecting back-stage equipment are provided for a microneedle chip module, and a culture dish is integrated on the surface of an electrode substrate through a culture monitoring device; nutrient substances and heat are provided for the culture dish through the peristaltic pump and the heating table, so that the embodiment of the invention combines the advantages of low cost, easiness in penetrating into tissues and high biocompatibility of a rigid needle-shaped electrode.

Description

Technical Field

[0001] The invention relates to the field of microneedle electrodes, and in particular to a microneedle chip module, an adapter, and a culture monitoring device. Background Art

[0002] The purpose of inventing the in vitro microarray electrode MEA (microelectrode array) is to solve the problems of low throughput and complex operation of traditional electrophysiological technology, and to achieve high-throughput, long-term monitoring and analysis of the coordinated electrical activities of a group of cells. MEA technology is an extracellular recording technology that integrates multiple electrodes in the form of an array on a chip. Each electrode can perform extracellular recording or stimulation, thereby achieving synchronous recording of a group of cells.

[0003] Compared with traditional patch clamp technology, MEA technology has the following advantages: high throughput, able to record cell electrophysiological activities at multiple sites simultaneously, suitable for large-scale cell population research; long-term monitoring, supporting observations up to several days, suitable for chronic neurotoxicity research; non-destructive: using non-invasive recording methods to maintain the original physiological state of cells; high resolution, able to record extracellular microvoltage changes with high resolution, reflecting the real electrophysiological activities of cells in vivo.

[0004] Similarly, microelectrode arrays (MEA) provide technical means for functional detection of brain organoids. More and more electrodes are designed to detect their electrophysiology, such as planar electrodes, which can detect functional signals in the form of attachment or wrapping, and needle electrodes, which can acutely detect the electrophysiology of brain organoids at different developmental stages.

[0005] However, planar electrodes are highly dependent on the attachment of brain organoids to the electrodes and can only detect surface signals. They cannot maintain a good three-dimensional structure for a long time and are prone to extensive cell migration on the electrode surface. In addition, the outer cell layer on the same plane of tissue slices or cell clusters usually contains dead cells, non-discharging cells or non-discharging tissues, and the surface of tissue slices is often not planar. When planar microelectrode arrays are used for extracellular recording or electrical stimulation of tissue slices or cell clusters, it is not only difficult to contact the target cells in the inner cell layer, but also difficult to adhere to most cells, resulting in most of the microelectrodes in the planar microelectrode arrays having difficulty collecting discharge signals.

[0006] Needle electrodes generally include rigid electrodes and flexible electrodes. Rigid electrodes can be reused, but have poor biocompatibility, while flexible electrodes have better compatibility but are more expensive. Although long-term and stable detection of brain organoid electrophysiology based on needle electrodes is of great significance for revealing the functional development of brain organoids, there are still difficulties in practical applications. Summary of the invention

[0007] The purpose of the present invention is to provide a microneedle chip module, an adapter, and a culture monitoring device to solve the problems in traditional extracellular recording or stimulation equipment, that is, the planar microelectrode array used is difficult to fit the surface of the tissue slice and difficult to contact the target cells in the inner cell layer, and the rigid needle electrodes used have poor biocompatibility and the flexible needle electrodes used have high costs.

[0008] In order to solve the above technical problems, the present invention specifically provides the following technical solutions: A microneedle chip module comprises an electrode substrate, substrate contacts and microneedle electrodes; a wire is arranged inside the electrode substrate, and the wire is used to electrically connect the substrate contacts and the microneedle electrodes; the substrate contacts are arranged on one side or both sides of the electrode substrate, and are used to contact a subsequent device, so as to obtain an electrical stimulation signal input by the subsequent device, or to transmit the collected electrical signal to the subsequent device; the microneedle electrode is arranged on one side of the electrode substrate, and is used to penetrate into target cells in the inner cell layer of a tissue slice or a cell cluster.

[0009] Furthermore, the diameter of the microneedle electrode is 25 μm, the length is 20 μm to 5 μm, the material is nickel-titanium alloy wire, the shape of the tip is flat or conical, the surface of the microneedle electrode is provided with an insulating layer, and the tip of the microneedle electrode is not provided with an insulating layer.

[0010] Furthermore, the electrode substrate is in the shape of a rectangular flat plate and is made of an insulating material with biocompatibility.

[0011] An adapter comprises a microneedle chip module, as well as: a probe substrate, a probe, a post-stage device interface, a fixing seat and a pressing plate; a probe wire is arranged inside the probe substrate, and the probe wire is used to electrically connect the probe and the post-stage device interface; the probe is arranged on the side of the probe substrate that contacts the electrode substrate, and the probe is used to contact and electrically connect the substrate contact; the post-stage device interface is arranged on one side of the probe substrate; the fixing seat is provided with a limiting groove for embedding and constraining the electrode substrate; the pressing plate covers one side of the fixing seat, and is used to connect with the fixing seat to form a three-dimensional rectangular hollow shell, so as to protect the probe substrate, the probe, and the microneedle chip module inside.

[0012] Furthermore, a guide pin is provided on the fixing seat, and a guide hole is provided on the pressure plate. The guide pin and the guide hole are used to guide the connection direction of the fixing seat and the pressure plate. The screw passes through the pressure plate and is spirally connected to the fixing seat, and the pressure plate and the fixing seat are connected by the force of the thread.

[0013] Furthermore, the probe has elasticity, so that when the probe contacts the substrate contact, it can closely fit the substrate contact through its own resilience.

[0014] Furthermore, the pressing plate is provided with an exposure hole for exposing the microneedle electrode, and one side of the fixing seat is provided with an interface hole for the interface of the subsequent device to pass through.

[0015] A culture monitoring device includes a microneedle chip module or an adapter; wherein, a side of the electrode substrate on which the microneedle electrode is mounted is provided with a circular ring-shaped vertical wall surrounding the microneedle electrode, so that the electrode substrate and the circular ring-shaped vertical wall form a culture dish.

[0016] Furthermore, it also includes a five-way joint, a semipermeable membrane, a pressure ring and a peristaltic pump, one end of the five-way joint is tightly connected to the culture dish through a sealing ring, the semipermeable membrane covers the remaining end of the five-way joint, and fixes its own position by the pressure ring embedded in the port of the five-way joint, so that the electrode substrate, the culture dish and the semipermeable membrane form a semi-sealed structure, the semipermeable membrane allows CO2 and O2 to pass through, and the peristaltic pump is connected to the remaining two ends of the five-way joint, and the peristaltic pump is used to change the liquid in the culture dish.

[0017] Furthermore, it also includes a temperature sensor, a temperature control module and a heating platform. The temperature sensor is inserted into the remaining end of the five-way joint to collect the temperature of the culture solution inside the culture dish. The heating platform is used to heat the culture solution. The temperature control module connects the temperature sensor and the heating platform to control the opening and closing and power of the heating platform to keep the temperature of the culture solution at the target temperature.

[0018] Compared with the prior art, this application has the following beneficial effects: Provided are a microneedle chip module, an adapter, and a culture monitoring device, wherein the microneedle chip module integrates a plurality of microneedle arrays on the surface of an electrode substrate, and penetrates a tissue slice through the microneedle electrodes to record or stimulate target cells located in the inner cell layer; the adapter integrates a probe and a subsequent device interface inside and outside a shell, respectively, and uses the probe to connect the subsequent device interface and the microneedle array, thereby providing protection for the microneedle chip module and the function of quickly connecting to the subsequent device; the culture monitoring device integrates a culture dish on the surface of the electrode substrate, and provides nutrients and heat to the culture dish through a peristaltic pump and a heating table, so that the embodiments of the present invention combine the advantages of low cost, easy penetration into tissue, and high biocompatibility of rigid needle electrodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the implementation methods of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for the implementation methods or the description of the prior art. Obviously, the drawings in the following description are only exemplary, and for ordinary technicians in this field, other implementation drawings can be derived from the provided drawings without creative work.

[0020] Figure 1 A three-dimensional diagram of a microneedle chip module according to a first embodiment of the present invention and an enlarged diagram of its structure; Figure 2 is a perspective view of an adapter according to a second embodiment of the present invention; Figure 3 is a top view of a culture dish according to a third embodiment of the present invention; Figure 4 It is a three-dimensional diagram of a culture monitoring device according to a third embodiment of the present invention.

[0021] The numbers in the figure represent the following: 1-microneedle chip module; 11-electrode substrate; 12-substrate contact; 13-microneedle electrode; 2-adapter; 21-probe substrate; 22-probe; 23-rear-stage equipment interface; 24-fixing seat; 25-pressing plate; 27-exposure hole; 26-screw; 3-culture monitoring device; 31-circular vertical wall; 32-five-way joint; 33-semipermeable membrane; 34-pressure ring; 35-peristaltic pump; 36-temperature sensor; 37-temperature control module; 38-heating table; 4-tissue pressing sheet; 5-tissue section. DETAILED DESCRIPTION

[0022] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] A conventional planar microelectrode array has all the microelectrodes with their ends located in the same plane, and the microelectrodes are used for extracellular recording or stimulation.

[0024] The outer cell layer on the same plane of a tissue slice or cell cluster usually contains dead cells, non-discharging cells or non-discharging tissue, and the surface of the tissue slice is often not flat. When a planar microelectrode array is used for extracellular recording or electrical stimulation of a tissue slice or cell cluster, it is not only difficult to contact the target cells in the inner cell layer, but also difficult to fit most of the cells, resulting in difficulty for most of the microelectrodes in the planar microelectrode array to collect discharge signals.

[0025] The basic idea to solve the above problem is to integrate a number of microneedle electrodes 13 in the form of an array on a chip. The common microneedle electrodes 13 have a length of 50μm to 100μm, which can penetrate into the tissue to collect electrical signals of target cells in the inner cell layer. The number of electrode channels, wire length, array spacing, and surface shape of the microneedle electrode 13 can be customized as needed, thereby improving the quality of signal acquisition and spatiotemporal resolution.

[0026] Furthermore, in order to achieve the recording or stimulation of tissue slices 5 or cell clusters of different thicknesses and shapes, the chip and several microneedle electrodes 13 are designed to be easy to install and disassemble, forming a microneedle chip module 1, so that the chip and several microneedle electrodes 13 can be simply and quickly connected or disconnected from the subsequent equipment.

[0027] Furthermore, in order to achieve long-term extracellular recording or stimulation, the chip is designed to be embedded in a culture container, so that the tissue slice 5 or cell cluster can be recorded or stimulated by the microneedle electrode 13 on the chip while being cultured in a nutrient solution.

[0028] (First embodiment) This embodiment provides a microneedle chip module 1.

[0029] refer to Figure 1 The microneedle chip module 1 includes: an electrode substrate 11, a substrate contact 12 and a microneedle electrode 13.

[0030] An electrode wire is disposed inside the electrode substrate 11 , and the electrode wire is used to electrically connect the substrate contact 12 and the microneedle electrode 13 .

[0031] The substrate contacts 12 are arranged on one or both sides of the electrode substrate 11 and are used to contact the probes 22 of the subsequent device, so as to obtain the electrical stimulation signal input by the subsequent device, or transmit the collected electrical signal to the subsequent device.

[0032] The microneedle electrode 13 is disposed on one side of the electrode substrate 11 and is used to penetrate into target cells in the inner cell layer of the tissue slice 5 or the cell cluster.

[0033] In this embodiment, the substrate contacts 12, the microneedle electrodes 13 and the electrode wires are integrated into a solid electrode substrate 11, which is easy to install, disassemble, replace and mass-produce.

[0034] Furthermore, the following provides a method for making the microneedle chip module 1 and preparation materials.

[0035] The electrode substrate 11 is in the shape of a rectangular plate and is made of insulating materials with good biocompatibility, such as silicon oxide, zirconium oxide, polyetheretherketone (PEEK), polyethylene (PE), polytetrafluoroethylene (PTFE), and the like.

[0036] For foolproof design, one corner of the electrode substrate 11 is cut or marked (not shown in the figure), so that the staff can install the electrode substrate 11 at the correct angle.

[0037] The electrode wires on the electrode substrate 11 are made of low-impedance, oxidation-resistant materials such as gold, platinum, and iridium oxide. The processing technology of the electrode wires can be completed by wiring, etching, sputtering, printing, and other processes.

[0038] The substrate contact 12 is disposed at the edge of the electrode substrate 11 , and the microneedle electrode 13 is disposed at the center of the electrode substrate 11 .

[0039] The diameter of the microneedle electrode 13 is 25 μm, and the length is 20 μm to 500 μm. The tip of the nickel-titanium alloy wire is flat or conical, and the tip can be made by mechanical grinding, electrochemical corrosion, photolithography, and other processes.

[0040] The material of the microneedle electrode 13 is nickel-titanium alloy wire, which has the advantages of shape memory function, superelasticity, good biocompatibility and little damage to body tissues. In addition, the material of the microneedle electrode 13 can also use titanium / titanium alloy, copper (or gold-plated copper), platinum, iridium / iridium oxide, platinum-iridium alloy, Pedot, gold, carbon / graphene, silver, silver / silver chloride, and some other alloys and other conductive materials.

[0041] An insulating layer is deposited on the surface of the microneedle electrode 13 , and the insulating layer is made of a flexible thin film material with biocompatibility, such as polyimide (PI), parylene, polydimethylsiloxane (PDMS), polyurethane (PU), silicone, silicone rubber, etc.

[0042] The tip of the microneedle electrode 13 is exposed, gold-plated, and other processes to adjust its impedance. When the tip of the exposed microneedle electrode 13 contacts the surface of a nerve cell, the discharge activity of the nerve cell can be collected.

[0043] (Second embodiment) This embodiment provides an adapter 2 having a microneedle chip module 1 .

[0044] The adapter 2 is used to fix and wrap the microneedle chip module 1, thereby protecting the microneedle chip module 1 from accidental damage. At the same time, the adapter 2 is provided with an interface that can be quickly connected and disconnected with the subsequent equipment, thereby further improving the speed of connecting the microneedle chip module 1 and the subsequent equipment.

[0045] Specifically, refer to Figure 2 The adapter 2 includes: a probe substrate 21 , a probe 22 , a rear-stage device interface 23 , a fixing seat 24 , a pressing plate 25 and a screw 26 .

[0046] A probe wire is disposed inside the probe substrate 21 , and the probe wire is used to electrically connect the probe 22 and the subsequent device interface 23 .

[0047] The probe 22 is disposed on a surface of the probe substrate 21 that contacts the electrode substrate 11 . The probe 22 is elastic, so that when the probe 22 contacts the substrate contact 12 , it can closely adhere to the substrate contact 12 through its own resilience.

[0048] The subsequent device interface 23 is disposed on one side of the probe substrate 21 and is connected to the subsequent device by plugging and unplugging.

[0049] The fixing seat 24 is provided with a limiting groove for embedding and constraining the electrode substrate 11 .

[0050] The pressing plate 25 covers one side of the fixing seat 24 and is used to be connected with the fixing seat 24 to form a three-dimensional rectangular hollow shell, so as to protect the probe substrate 21, the probe 22 and the microneedle chip module 1 inside.

[0051] The screw 26 passes through the pressing plate 25 and is spirally connected to the fixing seat 24. The pressing plate 25 and the fixing seat 24 are connected by the force of the thread. Rotating the screw 26 can quickly connect or separate the pressing plate 25, thereby quickly installing, removing or replacing the microneedle chip module 1.

[0052] In addition, it should be noted that: The probe substrate 21 is fixedly connected to the fixing seat 24 or the pressing plate 25 , or the probe substrate 21 is an integral part with one of the fixing seat 24 and the pressing plate 25 . The specific position of the probe substrate 21 and the orientation of the probe 22 depend on the direction of the substrate contact 12 on the electrode substrate 11 .

[0053] The fixing seat 24 is provided with a guide pin, and the pressing plate 25 is provided with a guide hole. The guide pin and the guide hole are used to guide the fixing seat 24 and the pressing plate 25 to be accurately connected.

[0054] The pressing plate 25 is provided with an exposing hole 27 exposing the microneedle electrode 13 , and the tissue slice 5 or the cell cluster passes through the exposing hole 27 to contact the microneedle electrode 13 .

[0055] One side of the fixing base 24 is provided with an interface hole for the subsequent device interface 23 to pass through.

[0056] (Third embodiment) This embodiment provides a culture monitoring device 3 having a microneedle chip module 1 .

[0057] refer to Figure 1 and Figure 3 , the culture monitoring device 3 includes a culture dish.

[0058] The culture dish and the electrode substrate 11 are an integral piece, and its specific shape is a circular ring-shaped vertical wall 31 surrounding the microneedle electrode 13 . The exposed hole 27 provided on the pressing plate 25 is gap-matched with the circular ring-shaped vertical wall 31 .

[0059] The tissue pressing sheet 4 is mainly composed of stainless steel wire and nylon wire. The tissue pressing sheet 4 is used to fix the tissue slice 5 during the experiment to prevent the displacement of the tissue pressing sheet 4 caused by external factors. After the tissue slice 5 and its tissue pressing sheet 4 are placed inside the culture dish, the microneedle electrode 13 can easily penetrate into the inside of the tissue slice 5 by relying on the self-gravity of the tissue pressing sheet 4 and the tissue slice 5.

[0060] For further reference, Figure 4 The culture monitoring device 3 also includes: a five-way connector 32, a semipermeable membrane 33, a pressure ring 34, and a peristaltic pump 35.

[0061] One end of the five-way joint 32 is tightly connected to the culture dish via a sealing ring.

[0062] The semipermeable membrane 33 covers the remaining end of the five-way joint 32 and is fixed in position by a pressure ring 34 embedded in the port of the five-way joint 32, so that the electrode substrate 11, the culture dish and the semipermeable membrane 33 form a semi-sealed structure. The semipermeable membrane 33 allows CO2 and O2 to pass through, thereby facilitating long-term culture and monitoring of cell tissues.

[0063] The peristaltic pump 35 is connected to the remaining two ends of the five-way connector 32 to replace the liquid in the tissue in the culture dish to avoid damage caused by moving the tissue and a decrease in the experimental effect.

[0064] In addition, the culture monitoring device 3 also has a constant temperature heating function. Specifically, the culture monitoring device 3 also includes: a temperature sensor 36 , a temperature control module 37 and a heating platform 38 .

[0065] The temperature sensor 36 is inserted into the remaining end of the five-way connector 32 to collect the temperature of the culture solution inside the culture dish.

[0066] The temperature control module 37 is connected to the temperature sensor 36 and the heating platform 38, and is used to control the opening and closing and power of the heating platform 38 to keep the temperature of the culture solution constant at around 30°, so as to prevent the culture solution temperature from being too high or too low and affecting signal collection.

[0067] The heating stage 38 heats the culture solution via the fixing base 24 and the electrode substrate 11 .

[0068] The above embodiments are only exemplary embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is defined by the claims. Those skilled in the art may make various modifications or equivalent substitutions to the present invention within the essence and protection scope of the present invention, and such modifications or equivalent substitutions shall also be deemed to fall within the protection scope of the present invention.

Claims

1. A microneedle chip module, characterized in that: It comprises an electrode substrate (11), a substrate contact (12) and a microneedle electrode (13); A conductive wire is arranged inside the electrode substrate (11), and the conductive wire is used to electrically connect the substrate contact (12) and the microneedle electrode (13); The substrate contact (12) is arranged on one side or both sides of the electrode substrate (11) and is used to contact a subsequent device, thereby obtaining an electrical stimulation signal input by the subsequent device, or transmitting the collected electrical signal to the subsequent device; The microneedle electrode (13) is arranged on one side of the electrode substrate (11) and is used to penetrate into target cells in the inner cell layer of a tissue slice (5) or a cell cluster.

2. A microneedle chip module according to claim 1, characterized in that: The microneedle electrode (13) has a diameter of 25 μm and a length of 20 μm to 5 μm. The material is nickel-titanium alloy wire. The shape of the tip is flat or conical. The surface of the microneedle electrode (13) is provided with an insulating layer, and the tip of the microneedle electrode (13) is not provided with an insulating layer.

3. A microneedle chip module according to claim 2, characterized in that: The electrode substrate (11) is in the shape of a rectangular flat plate and is made of an insulating material with biocompatibility.

4. An adapter, characterized in that: A microneedle chip module comprising any one of claims 1 to 3, as well as: a probe substrate (21), a probe (22), a post-stage equipment interface (23), a fixing seat (24) and a pressing plate (25); A probe wire is arranged inside the probe substrate (21), and the probe wire is used to electrically connect the probe (22) and the subsequent device interface (23); The probe (22) is arranged on a side of the probe substrate (21) that contacts the electrode substrate (11), and the probe (22) is used to contact and electrically connect the substrate contact (12); The rear-stage device interface (23) is arranged on one side of the probe substrate (21); The fixing seat (24) is provided with a limiting groove for embedding and constraining the electrode substrate (11); The pressing plate (25) covers one side of the fixing seat (24) and is used to be connected to the fixing seat (24) to form a three-dimensional rectangular hollow shell, thereby protecting the probe substrate (21), the probe (22), and the microneedle chip module inside.

5. An adapter according to claim 4, characterized in that: The fixing seat (24) is provided with a guide pin, and the pressing plate (25) is provided with a guide hole, the guide pin and the guide hole are used to guide the connection direction of the fixing seat (24) and the pressing plate (25), the screw (26) passes through the pressing plate (25) and is screwed to the fixing seat (24), and the pressing plate (25) and the fixing seat (24) are connected by the force of the thread.

6. An adapter according to claim 4, characterized in that: The probe (22) has elasticity, so that when the probe (22) contacts the substrate contact (12), it can closely fit the substrate contact (12) through its own resilience.

7. An adapter according to claim 4, characterized in that: The pressing plate (25) is provided with an exposure hole (27) for exposing the microneedle electrode (13), and one side of the fixing seat (24) is provided with an interface hole for the subsequent device interface (23) to pass through.

8. A culture monitoring device, characterized in that: The invention comprises a microneedle chip module according to any one of claims 1 to 3, or an adapter according to any one of claims 4 to 7; wherein a side of the electrode substrate (11) on which the microneedle electrode (13) is mounted is provided with a circular vertical wall (31) surrounding the microneedle electrode (13), so that the electrode substrate (11) and the circular vertical wall (31) form a culture dish.

9. A culture monitoring device according to claim 8, characterized in that: It also comprises a five-way joint (32), a semipermeable membrane (33), a pressure ring (34) and a peristaltic pump (35); one end of the five-way joint (32) is tightly connected to the culture dish via a sealing ring; the semipermeable membrane (33) covers the remaining end of the five-way joint (32) and fixes its position via the pressure ring (34) embedded in the port of the five-way joint (32), so that the electrode substrate (11), the culture dish and the semipermeable membrane (33) form a semi-sealed structure; the semipermeable membrane (33) allows CO2 and O2 to pass through; the peristaltic pump (35) is connected to the remaining two ends of the five-way joint (32); the peristaltic pump (35) is used to replace the liquid in the culture dish.

10. A culture monitoring device according to claim 9, characterized in that: It also includes a temperature sensor (36), a temperature control module (37) and a heating platform (38), wherein the temperature sensor (36) is inserted into the remaining end of the five-way joint (32) and is used to collect the temperature of the culture solution inside the culture dish, and the heating platform (38) is used to heat the culture solution. The temperature control module (37) is connected to the temperature sensor (36) and the heating platform (38) and is used to control the opening and closing and power of the heating platform (38) so that the temperature of the culture solution is maintained at a target temperature.

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