A microneedle chip module, an adapter, and a culture monitoring device

Abstract

The present invention relates to the field of microneedle electrodes, and specifically relates to a microneedle chip module, an adapter, and a culture monitoring device. Among them, the microneedle chip module integrates a plurality of microneedle arrays on the surface of an electrode substrate, and the microneedle electrodes are inserted into tissue sections to record or stimulate target cells located in the inner cell layer. The adapter integrates a probe and a rear-stage device interface inside and outside a housing respectively, and uses the probe to connect the rear-stage device interface and the microneedle array, so as to provide protection for the microneedle chip module and the function of quickly connecting to the rear-stage device. The culture monitoring device integrates a culture dish on the surface of the electrode substrate, and provides nutrients and heat for 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 tissues, and high biocompatibility of rigid needle-shaped electrodes.

Description

Technical Field

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

[0002] The invention purpose of the in vitro microarray electrode MEA (microelectrode array) is to solve the problems of low throughput and complex operation of traditional electrophysiological techniques, and to achieve high-throughput, long-term monitoring and analysis of the cooperative electrical activities of population cells. The MEA technology is an extracellular recording technology. By integrating multiple electrodes in the form of an array on a chip, each electrode can perform extracellular recording or stimulation, so as to achieve synchronous recording of a group of cells.

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

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

[0005] However, planar electrodes highly depend on the attachment of brain organoids to the electrodes, can only detect surface signals, cannot maintain a good three-dimensional structure for a long time, and are prone to extensive cell migration on the electrode surface. Moreover, the outer cell layer in the same plane of tissue sections or cell clusters usually contains dead cells, non-discharging cells or non-discharging tissues, and the surface of tissue sections is often not flat. When planar microelectrode arrays are applied to extracellular recording or electrical stimulation of tissue sections or cell clusters, it is not only difficult to contact the target cells in the inner cell layer, but also difficult to fit most cells, resulting in most microelectrodes in the planar microelectrode array being difficult to collect discharge signals.

[0006] For needle electrodes, generally including rigid electrodes and flexible electrodes, rigid electrodes can be reused, but have poor biocompatibility, and flexible electrodes have better biocompatibility but higher costs. Although long-term stable detection of the electrophysiology of brain organoids 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 object of the present invention is to provide a microneedle chip module, an adapter, and a culture monitoring device, so as to solve the problems that in traditional extracellular recording or stimulation devices, the planar microelectrode array used is difficult to fit the surface of tissue sections and difficult to contact target cells in the inner cell layer, and the biocompatibility of the rigid needle-shaped electrodes used is poor, and the cost of the flexible needle-shaped electrodes used is high.

[0008] To solve the above technical problems, the present invention specifically provides the following technical solutions:

[0009] A microneedle chip module includes an electrode substrate, a substrate contact, and microneedle electrodes; a wire is arranged inside the electrode substrate, and the wire is used for electrically connecting the substrate contact and the microneedle electrodes; the substrate contact is arranged on one or both sides of the electrode substrate and is used for contacting a subsequent device, so as to obtain an electrical stimulation signal input by the subsequent device or transmit the collected electrical signal to the subsequent device; the microneedle electrodes are arranged on one side of the electrode substrate and are used for piercing target cells in the inner cell layer of a tissue section or cell cluster.

[0010] Furthermore, the diameter of the microneedle electrode is 25 μm, the length is from 20 μm to 5 μm, the material is a nitinol wire, the shape of the tip is planar or conical, an insulating layer is arranged on the surface of the microneedle electrode, and the tip of the microneedle electrode is not provided with an insulating layer.

[0011] Furthermore, the shape of the electrode substrate is a rectangular flat plate, and the material is a biocompatible insulating material.

[0012] An adapter includes a microneedle chip module, and: a probe substrate, probes, a subsequent device interface, a fixing seat, and a pressing plate; a probe wire is arranged inside the probe substrate, and the probe wire is used for electrically connecting the probes and the subsequent device interface; the probes are arranged on the side of the probe substrate in contact with the electrode substrate, and the probes are used for contacting and electrically connecting the substrate contacts; the subsequent device interface is arranged on one side of the probe substrate; the fixing seat is provided with a limiting groove for embedding and restricting the electrode substrate; the pressing plate covers one side of the fixing seat and is used for connecting with the fixing seat to form a three-dimensional rectangular hollow shell, so as to protect the probe substrate, the probes, and the microneedle chip module inside.

[0013] Furthermore, a guide pin is arranged on the fixing seat, and a guide hole is arranged on the pressing plate. The guide pin and the guide hole are used for guiding the connection direction of the fixing seat and the pressing plate. A screw penetrates through the pressing plate and is screwed to the fixing seat, and the pressing plate and the fixing seat are connected by the force of the thread.

[0014] 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.

[0015] Furthermore, an exposure hole exposing the microneedle electrode is provided on the pressing plate, and an interface hole through which the interface of the subsequent device can pass is provided on one side of the fixing base.

[0016] A culture monitoring device includes a microneedle chip module or an adapter; wherein, a circular vertical wall surrounding the microneedle electrode for one week is provided on the side of the electrode substrate where the microneedle electrode is installed, so that the electrode substrate and the circular vertical wall form a culture dish.

[0017] Furthermore, it further includes a five-way joint, a semi-permeable membrane, a compression ring and a peristaltic pump. One end of the five-way joint is hermetically connected to the culture dish through a sealing ring. The semi-permeable membrane covers the remaining end of the five-way joint and fixes its position through the compression ring embedded in the port of the five-way joint, so that the electrode substrate, the culture dish and the semi-permeable membrane form a semi-sealed structure. The semi-permeable membrane allows CO2 and O2 to pass through. The peristaltic pump is connected to the remaining two ends of the five-way joint, and the peristaltic pump is used for changing the liquid in the culture dish.

[0018] Furthermore, it further includes a temperature sensor, a temperature control module and a heating table. The temperature sensor is inserted into the remaining end of the five-way joint for collecting the temperature of the culture medium inside the culture dish. The heating table is used for heating the culture medium. The temperature control module is connected to the temperature sensor and the heating table for controlling the opening and closing and power of the heating table to keep the temperature of the culture medium at the target temperature.

[0019] The present application has the following beneficial effects compared with the prior art:

[0020] There is provided a microneedle chip module, an adapter, and a culture monitoring device. Among them, the microneedle chip module integrates a plurality of microneedle arrays on the surface of an electrode substrate, and records or stimulates target cells located in the inner cell layer by piercing the tissue section with the microneedle electrode. The adapter integrates the probe and the interface of the subsequent device inside and outside a housing respectively, and uses the probe to connect the interface of the subsequent device and the microneedle array, so as to provide protection for the microneedle chip module and the function of quickly connecting to the subsequent device. The culture monitoring device integrates the culture dish on the surface of the electrode substrate, and provides nutrients and heat for the culture dish through the peristaltic pump and the 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-shaped electrodes. Description of the Drawings

[0021] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the accompanying drawings required for the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only exemplary. For those of ordinary skill in the art, without creative efforts, other implementation drawings can be obtained by extending the provided drawings.

[0022] Figure 1 A three-dimensional view of the microneedle chip module according to the first embodiment of the present invention and an enlarged view of its structure;

[0023] Figure 2 A three-dimensional view of the adapter according to the second embodiment of the present invention;

[0024] Figure 3 A top view of the culture dish according to the third embodiment of the present invention;

[0025] Figure 4 A three-dimensional view of the culture monitoring device according to the third embodiment of the present invention.

[0026] The reference numerals in the figures are respectively represented as follows:

[0027] 1 - Microneedle chip module; 11 - Electrode substrate; 12 - Substrate contact; 13 - Microneedle electrode;

[0028] 2 - Adapter; 21 - Probe substrate; 22 - Probe; 23 - Interface for subsequent equipment; 24 - Fixing seat; 25 - Pressing plate; 27 - Exposure hole; 26 - Screw;

[0029] 3 - Culture monitoring device; 31 - Circular vertical wall; 32 - Five-way joint; 33 - Semi-permeable membrane; 34 - Pressure ring; 35 - Peristaltic pump; 36 - Temperature sensor; 37 - Temperature control module; 38 - Heating table; 4 - Tissue press; 5 - Tissue section. Detailed implementation manners

[0030] The following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0031] In a traditional planar microelectrode array, the ends of all the microelectrodes are located in the same plane, and the microelectrodes are used for extracellular recording or stimulation.

[0032] 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.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] (First embodiment)

[0037] This embodiment provides a microneedle chip module 1.

[0038] refer to Figure 1 The microneedle chip module 1 includes: an electrode substrate 11, a substrate contact 12 and a microneedle electrode 13.

[0039] 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 .

[0040] 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.

[0041] 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.

[0042] In this embodiment, the substrate contact 12, the microneedle electrode 13, and the electrode wire are integrated onto a solid electrode substrate 11, which is easy to install, disassemble, replace, and mass-produce.

[0043] Furthermore, the manufacturing method and preparation materials of the microneedle chip module 1 are provided below.

[0044] The electrode substrate 11 is in the shape of a rectangular flat plate, and the electrode substrate 11 is made of an insulating material with good biocompatibility, such as silicon oxide, zirconium oxide, polyetheretherketone (PEEK), polyethylene (PE), polytetrafluoroethylene (PTFE), etc.

[0045] For anti-fooling design, one corner of the electrode substrate 11 is chamfered or marked (not shown in the figure), so as to facilitate the staff to install the electrode substrate 11 at the correct angle.

[0046] The electrode wire on the electrode substrate 11 is made of materials with low impedance and oxidation resistance, such as gold, platinum, iridium oxide, etc. The processing technology of the electrode wire can be completed by processes such as wire arrangement, etching, sputtering, printing, and printing.

[0047] The substrate contact 12 is arranged at the edge of the electrode substrate 11, and the microneedle electrode 13 is arranged at the center of the electrode substrate 11.

[0048] The diameter of the microneedle electrode 13 is 25μm, and the length is 20μm to 500μm. The shape of the tip of the nitinol wire is flat or conical, and the manufacturing process of the tip can adopt processes such as mechanical grinding, electrochemical corrosion, and photolithography.

[0049] The material of the microneedle electrode 13 is nitinol wire, and the nitinol wire has the advantages of shape memory function, superelasticity, good biocompatibility, and small damage to internal tissues. In addition, the material of the microneedle electrode 13 can also use conductive materials such as 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.

[0050] A layer of 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), silica gel, silicone rubber, etc.

[0051] The impedance of the tip of the microneedle electrode 13 is adjusted by processes such as exposure and gold plating. When the exposed tip of the microneedle electrode 13 contacts the surface of the nerve cell, the discharge activity of the nerve cell can be collected.

[0052] (Second Embodiment)

[0053] This embodiment provides an adapter 2 with a microneedle chip module 1.

[0054] The adapter 2 is used to fix and wrap the microneedle chip module 1 to protect the microneedle chip module 1 from accidental damage. At the same time, an interface capable of quickly connecting to and disconnecting from a subsequent device is provided on the adapter 2, thereby further improving the speed of connecting the microneedle chip module 1 and the subsequent device.

[0055] Specifically, referring to Figure 2 , the adapter 2 includes: a probe substrate 21, probes 22, a subsequent device interface 23, a fixing base 24, a pressing plate 25, and a screw 26.

[0056] Probe wires are arranged inside the probe substrate 21, and the probe wires are used to electrically connect the probes 22 and the subsequent device interface 23.

[0057] The probes 22 are arranged on the side of the probe substrate 21 that contacts the electrode substrate 11. The probes 22 have elasticity, so that when the probes 22 contact the substrate contacts 12, they can closely fit the substrate contacts 12 through their own resilience.

[0058] The subsequent device interface 23 is arranged on one side of the probe substrate 21 and is connected to the subsequent device through a plugging action.

[0059] The fixing base 24 is provided with a limiting groove for embedding and restricting the electrode substrate 11.

[0060] The pressing plate 25 covers one side of the fixing base 24 and is used to connect with the fixing base 24 to form a three-dimensional rectangular hollow shell, thereby protecting the probe substrate 21, the probes 22, and the microneedle chip module 1 inside it.

[0061] The screw 26 penetrates through the pressing plate 25 and is screwed to the fixing base 24. The pressing plate 25 and the fixing base 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.

[0062] In addition, it should also be noted that:

[0063] The probe substrate 21 is fixedly connected to the fixing base 24 or the pressing plate 25, or the probe substrate 21 and one of the fixing base 24 and the pressing plate 25 are integral parts. The specific position of the probe substrate 21 and the orientation of the probes 22 depend on the direction of the substrate contacts 12 on the electrode substrate 11.

[0064] The fixing base 24 is provided with guide pins, and the pressing plate 25 is provided with guide holes. The guide pins and the guide holes are used to guide the precise connection of the fixing base 24 and the pressing plate 25.

[0065] The pressing plate 25 is provided with an exposure hole 27 that exposes the microneedle electrode 13, and the tissue section 5 or cell cluster passes through the exposure hole 27 to contact the microneedle electrode 13.

[0066] One side of the fixing base 24 is provided with an interface hole through which the interface 23 of the subsequent device can pass.

[0067] (Third Embodiment)

[0068] This embodiment provides a culture monitoring device 3 equipped with a microneedle chip module 1.

[0069] Reference Figure 1 and Figure 3 , the culture monitoring device 3 includes a culture dish.

[0070] The culture dish and the electrode substrate 11 are an integral part, and its specific shape is an annular vertical wall 31 that surrounds the microneedle electrode 13 for one week. The exposure hole 27 provided on the pressing plate 25 is in clearance fit with the annular vertical wall 31.

[0071] The tissue press 4 is mainly composed of stainless steel wire and nylon wire. The tissue press 4 is used to fix the tissue section 5 during the experiment to avoid the displacement of the tissue press 4 caused by external factors. After the tissue section 5 and its tissue press 4 are placed inside the culture dish, the microneedle electrode 13 can easily penetrate into the inside of the tissue section 5 by relying on the self-gravity of the tissue press 4 and the tissue section 5.

[0072] Furthermore, referring to Figure 4 , the culture monitoring device 3 further includes: a five-way joint 32, a semi-permeable membrane 33, a compression ring 34, and a peristaltic pump 35.

[0073] One end of the five-way joint 32 is hermetically connected to the culture dish through a sealing ring.

[0074] The semi-permeable membrane 33 covers the remaining end of the five-way joint 32, and fixes its own position through the compression ring 34 embedded in the port of the five-way joint 32, so that the electrode substrate 11, the culture dish, and the semi-permeable membrane 33 form a semi-sealed structure. The semi-permeable membrane 33 allows CO2 and O2 to pass through, thus facilitating the long-term culture and monitoring of cell tissues.

[0075] The peristaltic pump 35 is connected to the remaining two ends of the five-way joint 32 and is used to change the liquid in the culture dish for the tissue, avoiding the damage caused by moving the tissue and the decline of the experimental effect.

[0076] In addition, the culture monitoring device 3 also has a constant temperature heating function. Specifically, the culture monitoring device 3 further includes: a temperature sensor 36, a temperature control module 37, and a heating table 38.

[0077] 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.

[0078] 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.

[0079] The heating stage 38 heats the culture solution via the fixing base 24 and the electrode substrate 11 .

[0080] 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. An adapter, characterized in that, Comprising: A microneedle chip module, and: a probe substrate (21), a probe (22), a post-stage device interface (23), a fixing base (24), and a pressing plate (25); The microneedle chip module includes an electrode substrate (11), a substrate contact (12), and microneedle electrodes (13); Wires are arranged inside the electrode substrate (11) for electrically connecting the substrate contact (12) and the microneedle electrodes (13); The substrate contact (12) is arranged on one or both sides of the electrode substrate (11) for contacting a post-stage device, thereby obtaining an electrical stimulation signal input by the post-stage device, or transmitting the collected electrical signal to the post-stage device; The microneedle electrodes (13) are arranged on one side of the electrode substrate (11) for piercing target cells in the inner cell layer of a tissue slice (5) or cell cluster; Probe wires are arranged inside the probe substrate (21) for electrically connecting the probe (22) and the post-stage device interface (23); The probe (22) is arranged on the side of the probe substrate (21) in contact with the electrode substrate (11), and the probe (22) is used for contacting and electrically connecting the substrate contact (12); The post-stage device interface (23) is arranged on one side of the probe substrate (21); The fixing base (24) is provided with a limiting groove for embedding and restricting the electrode substrate (11); The pressing plate (25) covers one side of the fixing base (24) and is used for connecting with the fixing base (24) to form a three-dimensional rectangular hollow housing, thereby protecting the probe substrate (21), the probe (22), and the microneedle chip module inside.

2. A microneedle chip module according to claim 1, characterized in that The diameter of the microneedle electrode (13) is 25 μm, the length is 20 μm to 500 μm, the material is nitinol wire, the shape of the tip is flat or conical, an insulating layer is provided on the surface of the microneedle electrode (13), and no insulating layer is provided at the tip of the microneedle electrode (13).

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

4. An adapter according to claim 1, characterized in that Guide pins are provided on the fixing base (24), guide holes are provided on the pressing plate (25), the guide pins and the guide holes are used to guide the connection direction of the fixing base (24) and the pressing plate (25), and screws (26) penetrate through the pressing plate (25) and are screwed to the fixing base (24), and the pressing plate (25) and the fixing base (24) are connected by the force of the thread.

5. An adapter according to claim 1, 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.

6. An adapter according to claim 1, characterized in that An exposure hole (27) exposing the microneedle electrode (13) is provided on the pressing plate (25), and an interface hole through which the subsequent device interface (23) can pass is provided on one side of the fixed seat (24).

7. A culture monitoring device, characterized in that it includes the adapter according to any one of claims 1-6; wherein, a circular vertical wall (31) surrounding the microneedle electrode (13) for one week is provided on the side of the electrode substrate (11) where the microneedle electrode (13) is installed, so that the electrode substrate (11) and the circular vertical wall (31) form a culture dish.

8. The culture monitoring device according to claim 7, characterized in that it further includes a five-way joint (32), a semi-permeable membrane (33), a compression ring (34) and a peristaltic pump (35). One end of the five-way joint (32) is hermetically connected to the culture dish through a sealing ring. The semi-permeable membrane (33) covers the remaining end of the five-way joint (32), and fixes its position through the compression ring (34) embedded in the port of the five-way joint (32), so that the electrode substrate (11), the culture dish and the semi-permeable membrane (33) form a semi-sealed structure. The semi-permeable 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), and the peristaltic pump (35) is used for changing the liquid in the culture dish.

9. The culture monitoring device according to claim 8, characterized in that it further includes a temperature sensor (36), a temperature control module (37) and a heating table (38). The temperature sensor (36) is inserted into the remaining end of the five-way joint (32) for collecting the temperature of the culture solution inside the culture dish. The heating table (38) is used for heating the culture solution. The temperature control module (37) is connected to the temperature sensor (36) and the heating table (38) for controlling the opening and closing and power of the heating table (38) to keep the temperature of the culture solution at the target temperature.

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