A through-hole sealing cover for high-throughput separation of single cell microparticles

Abstract

The present invention discloses a through-hole sealing cover for high-throughput separation of single cell microparticles, and its technical key points are: comprising a cover body, the cover body is horizontally divided into an upper half layer, a middle layer, and a lower half layer from top to bottom, the upper half layer is provided with multiple groups of sealing holes connected to the upper surface of the cover body, the middle layer is provided with multiple groups of air guide holes, and the lower half layer is provided with multiple groups of needle connection holes connected to the lower surface of the cover body; the air guide holes of the middle layer are connected and concentric with the sealing holes of the upper half layer, the air guide holes of the middle layer are connected and concentric with the needle connection holes of the lower half layer, and the sealing holes, air guide holes, and needle connection holes form a porous channel; the sealing holes are provided with a detachable sealing device for connecting with external flow path joint pipe fittings and achieving sealing. The present invention solves the technical problems of poor air tightness, high temperature resistance, and inability to repeatedly lock and stably control air tightness.

Description

Technical Field

[0001] The invention relates to the technical field of micro-droplets, microfluidic preparation and reagent mixing detection, and more specifically, to a through-hole sealing cover for high-throughput separation of single cell microparticles. Background Art

[0002] Microfluidics technology is one of the important branches of MEMS technology and one of the most rapidly developing multidisciplinary cross-scientific frontier technologies. It has important applications in life sciences, clinical medicine, chemistry and chemical engineering, pharmaceuticals, food hygiene, environmental testing and monitoring, information science and signal detection, and other disciplines.

[0003] Microfluidic technology usually uses microanalysis devices as the carrier for technology implementation, and microfluidic chips are the fastest growing of all types of microanalysis devices. Microfluidic chips use MEMS technology to process various microstructures on silicon, quartz, glass or polymer substrates, such as microchannels, microreaction pools, microelectrodes and other functional units, and then use microchannels to connect micropumps, microvalves, microreservoirs, microdetection elements and other components with fluid transportation, control and detection and monitoring functions, to maximize the integration of dilution, reagent addition, sampling, reaction, separation and dispersion, detection, monitoring and other processes on the chip into a micro-total analysis system. The area of ​​a microfluidic chip is usually several to tens of square centimeters, and its microchannel size is generally at the micron level or nearly millimeter level. When many chemical processes are carried out in microfluidic chips, the tiny volume brings many advantages: the microchannel size is reduced by one order of magnitude, and the reagent dosage will be reduced by three orders of magnitude; the diffusion rate of the fluid in the microchannel will be increased by two orders of magnitude, thereby greatly improving the reaction speed; at the same time, microfluidic chips also have the advantages of low cost, batch manufacturing, simple operation, good repeatability, and high reliability.

[0004] Most single-cell analyses require the separation of cells, separating group cells into individual dispersed cells for subsequent analysis. Common single-cell separation methods include: multiple solution dilutions, fluorescence-activated cell sorting systems, manual separation under microscope operation, laser tweezers separation, and microfluidic separation. Among them, microfluidic separation can achieve high-throughput single-cell separation. At present, there are two main separation methods based on microfluidics: single-cell droplet separation and single-cell microporous separation. Compared with the single-cell droplet separation method, single-cell microporous separation solves the problem of unstable water-oil droplet formation, and can quickly obtain high-throughput dispersed single cells on a large scale. However, the realization of single-cell microporous separation depends on the airtightness control of the multi-hole flow channel of the microfluidic system. The airtightness of the multi-hole flow channel restricts the single-cell microporous separation technology.

[0005] The seals required for single-cell microporous separation are the communication medium between the single-cell microporous separation microfluidic body and the external flow channel. They have complex structures, often high manufacturing costs, low assembly yields, and cannot meet the 100-degree high temperature and airtightness requirements of practical applications. At the same time, the existing single-cell microporous separation technology mainly relies on manual operation, which has many disadvantages, such as:

[0006] 1. The manual operation process is prone to errors;

[0007] 2. Manual operation takes a long time and has low efficiency;

[0008] 3. Manual preparation relies on operating experience and has high requirements for operators;

[0009] 4. Manual operation cannot achieve a large amount of sample preparation work;

[0010] 5. The consistency of cell concentration in manually operated samples is poor;

[0011] 6. The channel is tiny.

[0012] In view of the fact that the existing single-cell microporous separation technology is still at the manual operation stage, there are many uncontrollable internal and external factors. Therefore, designing a new automated structure for the existing single-cell microporous microfluidic separation system to solve the above problems plays an important role in research and technological progress. Summary of the invention

[0013] In view of the shortcomings of the prior art, the purpose of the present invention is to provide a through-hole sealing cover for high-throughput separation of single cell microparticles, aiming to solve the technical problems of poor air tightness, high temperature resistance and inability to repeatedly lock the air tightness for consistent and stable control of the existing microfluidic system.

[0014] The above-mentioned object of the present invention is achieved by the following technical scheme: a through-hole sealing cover for high-throughput separation of single cell microparticles, comprising a cover body, wherein the cover body is horizontally divided into an upper half layer, a middle layer, and a lower half layer from top to bottom, wherein the upper half layer is provided with a plurality of sealing holes connected to the upper surface of the cover body, the middle layer is provided with a plurality of air guide holes, and the lower half layer is provided with a plurality of needle connection holes connected to the lower surface of the cover body;

[0015] The air guide holes of the middle layer are connected and concentric with the sealing holes of the upper layer, the air guide holes of the middle layer are connected and concentric with the needle connection holes of the lower layer, and the sealing holes, air guide holes and needle connection holes form a porous channel;

[0016] The sealing hole is provided with a detachable sealing device for connecting with external flow path joint pipe fittings and achieving sealing.

[0017] By adopting the above technical solution, the porous channel is suitable for positive and negative pressure airtight environments, and is provided with multiple groups of positive and negative pressure changes that can be controlled by the outside world, which are finally output to the inner hole of the microneedle through the closed porous channel. Regardless of whether the outside temperature changes within 100°C or the outside air pressure changes within 300Kpa, the array porous channel seal does not leak, and can also meet the requirements of the single-cell microporous microfluidic separation system. First, when the upper half-layer sealing hole is docked and locked with the external flow path connector through the detachable sealing device, the inner hole of the upper half-layer is connected to the outside world to seal the fluid channel, and the needle connection hole of the lower half-layer fixes the microneedle to ensure that the microneedle will not leak when it is slightly deformed by axial force and radial force; the needle connection hole and the middle layer through hole are directly connected to form a sealed fluid channel. Finally, the sealed porous channel formed by the upper inner hole, the middle through hole and the lower inner hole can be used for positive pressure fluid channel as well as negative pressure fluid channel. This scheme completely changes the manual operation idea of ​​the single-cell microporous microfluidic separation system. The single-cell microporous microfluidic separation system is changed from the original cell sample collection port and the reagent injection port sharing one port to the reagent injection port and the cell collection port being two different ports, one for input and one for output. This makes it possible for the single-cell microporous microfluidic separation system to be operated automatically instead of manually, thus promoting the commercialization of high-throughput single-cell microporous microfluidic separation systems.

[0018] Preferably, the detachable sealing device comprises a threaded sleeve embedded in the sealing hole, which is used to cooperate with the threaded sleeve on the external flow path joint pipe fitting.

[0019] By adopting the above technical solution, the threaded connection between the screw sleeve embedded in the inner hole of the upper half layer and the plastic part of the external flow path joint is reliably sealed, the number of locking and loosening times is high, and the manufacturing cost is low.

[0020] Preferably, a rubber sealing ring is provided at the bottom of the sealing hole, and one end of the air guide hole close to the sealing hole passes through the rubber sealing ring.

[0021] By adopting the above technical solution, the rubber sealing ring further increases the air tightness of the sealing hole, and the middle layer through hole penetrates to the center of the rubber sealing ring, ensuring that the middle layer through hole and the upper layer through hole are sealed without leakage, further optimizing the structure.

[0022] Preferably, the diameter of the air guide hole is smaller than the diameter of the sealing hole and the needle connection hole.

[0023] By adopting the above technical solution, the diameter of the air guide hole is small, which facilitates the installation of the sealing holes and the needle connection holes at both ends in a tight fit, thereby further improving the air tightness.

[0024] Preferably, the number of the sealing holes, air guide holes and needle connection holes is 8-96.

[0025] By adopting the above technical solution, the number of porous channels can be modified and adjusted according to specific experiments and needs.

[0026] Preferably, the needle connection hole is used for sliding connection with an external microneedle, and a sealing adhesive is injected between the needle connection hole and the outer wall of the microneedle.

[0027] By adopting the above technical solution, since the needle connection hole is larger than the diameter of the air guide hole in the middle layer, the microneedle wall is close to the middle layer, and sealing adhesive is injected into the lower half layer of needle connection holes and the outer wall of the microneedle to further improve the air tightness.

[0028] Preferably, the cover body is made of non-metallic sheet material.

[0029] By adopting the above technical solution, the non-metallic sheet material can be modified polystyrene, reinforced linear polyester, polytetrafluoroethylene, polyimide, modified polyphenylene ether PPO and other high temperature resistant engineering plastics.

[0030] Preferably, the cover body may be in various shapes such as a cuboid, a cube, a cylinder, etc.

[0031] By adopting the above technical solution, sealing covers of different shapes can be designed according to the supporting platform or processing platform of the test instrument.

[0032] Preferably, the lower surface of the cover body can be connected to a sealing gasket for preventing leakage, and the lower surface of the cover body is provided with a fixing hole for fixing the sealing gasket.

[0033] By adopting the above technical solution, in order to prevent leakage of the test solution, the sealing gasket at the bottom of the cover body is preferably matched with the cover body and can be fixed through the additional fixing holes, thereby further optimizing the structure.

[0034] Preferably, the sealing cover is provided with limiting holes around it, and the limiting holes are used to fix the sealing cover to the test instrument.

[0035] By adopting the above technical solution, in order to prevent the sealing cover from deflecting, the additional limiting holes further improve the stability of the overall structure.

[0036] In summary, the beneficial effects of the present invention are as follows: This scheme completely changes the manual operation idea of ​​the single-cell microporous microfluidic separation system. The single-cell microporous microfluidic separation system is changed from the original cell sample collection port and the reagent injection port sharing one port to the reagent injection port and the cell collection port being two different ports, one for input and one for output. This makes it possible for the single-cell microporous microfluidic separation system to be operated automatically instead of manually, thus promoting the commercialization process of high-throughput single-cell microporous microfluidic separation systems. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1A schematic diagram for showing the overall structure of this embodiment.

[0038] Figure numerals: 1. upper surface of cover body; 2. lower surface of cover body; 3. sealing hole; 4. air guide hole; 5. needle connection hole; 6. fixing hole; 7. limiting hole; 11. cover body. DETAILED DESCRIPTION

[0039] The specific implementation of the present invention will be described in detail below with reference to the accompanying drawings.

[0040] Example: Figure 1 As shown, a through-hole sealing cover for high-throughput separation of single cell microparticles includes a cover body 11, which is made of a non-metallic plate with a thickness of 10-30CM. The non-metallic plate material can be modified polystyrene, reinforced linear polyester, polytetrafluoroethylene, polyimide, modified polyphenylene ether PPO and other high-temperature resistant engineering plastics. The shape of the non-metallic plate after processing can be various shapes such as a cuboid, a cube, a cylinder, etc.

[0041] like Figure 1 As shown, the cover body 11 is horizontally divided into an upper half layer, a middle layer and a lower half layer from top to bottom, the upper half layer is provided with a plurality of sealing holes 3 connected to the upper surface 1 of the cover body, the middle layer is provided with a plurality of air guide holes 4, the lower half layer is provided with a plurality of needle connection holes 5 connected to the lower surface 2 of the cover body, the air guide holes 4 in the middle layer are connected and concentric with the sealing holes 3 in the upper half layer, the air guide holes 4 in the middle layer are connected and concentric with the needle connection holes 5 in the lower half layer, and the sealing holes 3, air guide holes 4 and needle connection holes 5 form a porous channel.

[0042] like Figure 1 As shown, a detachable sealing device is provided on the sealing hole 3, which is used to connect with the external flow path joint pipe fitting and achieve sealing. The detachable sealing device includes a screw sleeve embedded in the sealing hole 3, preferably a screw sleeve made of stainless steel, which is used to cooperate with the thread on the external flow path joint pipe fitting. The screw sleeve embedded in the inner hole of the upper layer and the thread connection of the external flow path joint plastic part are reliable and sealed, with a high number of locking and loosening times and low manufacturing cost. A rubber sealing ring is provided at the bottom of the sealing hole 3, and the end of the air guide hole 4 close to the sealing hole 3 passes through the rubber sealing ring. The rubber sealing ring further increases the air tightness of the sealing hole 3, and the middle layer through hole passes through the center of the rubber sealing ring to ensure that the middle layer through hole and the upper layer through hole are sealed without leakage.

[0043] like Figure 1As shown, the diameter of the air guide hole 4 is smaller than the diameter of the sealing hole 3 and the needle connection hole 5. The diameter of the air guide hole 4 is small, which is convenient for the sealing holes 3 and the needle connection holes 5 at both ends to be installed in a close fit, thereby further improving the air tightness. The number of sealing holes 3, air guide holes 4, and needle connection holes 5 can be 8-96. The number of porous channels can be modified and adjusted according to specific experiments and needs. The needle connection hole 5 is used to be connected with the external microneedle in a sliding fit, and a sealing adhesive is injected between the needle connection hole 5 and the outer wall of the microneedle. Since the needle connection hole 5 is larger in diameter than the air guide hole 4 of the middle layer, the microneedle needle wall is close to the middle layer, and the sealing adhesive is injected into the lower half layer of the needle connection hole 5 and the outer wall of the microneedle, and the cross section of the needle is close to the bottom of the needle connection hole 5 at the bottom of the sealing cover. After drying naturally for 6 hours, the sealing hole 3 on the upper part of the sealing cover, the air guide hole 4 in the middle of the sealing cover, and the needle connection hole 5 on the lower part of the sealing cover form a sealed porous channel. The sealed porous channel can be used for a positive pressure fluid channel or a negative pressure fluid channel.

[0044] like Figure 1 As shown, the lower surface 2 of the cover body can be connected to a sealing gasket for preventing leakage, and the lower surface 2 of the cover body is provided with a fixing hole 6 for fixing the sealing gasket. In order to prevent leakage of the test solution, the sealing gasket at the bottom of the cover body 11 is preferably matched with the cover body 11, and can be fixed through the additional fixing hole 6. The sealing cover is provided with limiting holes 7 around it, and the limiting holes 7 are used to fix the sealing cover to the single-cell microporous microfluidic separation automation instrument. In order to prevent the deviation of the sealing cover, the additional limiting holes 7 further improve the stability of the overall structure. The limiting hole 7 and the needle connection hole 5 can be connected by different routes by opening side holes.

[0045] Working principle and process: The composed porous channels are suitable for positive pressure and negative pressure airtight environments, and are equipped with multiple groups. The positive and negative pressure changes that can be controlled by the outside world are finally output to the inner holes of the microneedles through the closed porous channels. Regardless of whether the outside temperature changes within 100°C or the outside air pressure changes within 300Kpa, the array porous channel seal does not leak, and can also meet the requirements of the single-cell microporous microfluidic separation system. First, when the upper half-layer sealing hole 3 is docked and locked with the external flow path connector through the detachable sealing device, the inner hole of the upper half layer is connected to the outside world to seal the fluid channel, and the needle connection hole 5 of the lower half layer fixes the microneedle to ensure that the microneedle will not leak when it is slightly deformed by axial force and radial force; the needle connection hole 5 is directly connected to the middle layer through the hole to form a sealed fluid channel. Finally, the sealed porous channel formed by the upper inner hole, the middle through hole and the lower inner hole can be used for positive pressure fluid channel as well as negative pressure fluid channel. This scheme completely changes the manual operation idea of ​​the single-cell microporous microfluidic separation system. The single-cell microporous microfluidic separation system is changed from the original cell sample collection port and the reagent injection port sharing one port to the reagent injection port and the cell collection port being two different ports, one for inlet and one for outlet, so that the single-cell microporous microfluidic separation system can be operated automatically instead of manual operation.

[0046] This specific embodiment is merely an explanation of the present invention and is not a limitation of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the present embodiment as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by the patent law.

Claims

1. A through-hole sealing cover for high-throughput separation of single cell microparticles, characterized in that: The invention comprises a cover body (11), wherein the cover body (11) is horizontally divided into an upper layer, a middle layer and a lower layer from top to bottom, wherein the upper layer is provided with a plurality of sealing holes (3) connected to the upper surface of the cover body (11), the middle layer is provided with a plurality of air guide holes (4), and the lower layer is provided with a plurality of needle connection holes (5) connected to the lower surface of the cover body (11); The air guide holes (4) of the middle layer are connected and concentric with the sealing holes (3) of the upper layer, and the air guide holes (4) of the middle layer are connected and concentric with the needle connection holes (5) of the lower layer, and the sealing holes (3), the air guide holes (4) and the needle connection holes (5) form a porous channel; The sealing hole (3) is provided with a detachable sealing device for connecting with an external flow path joint pipe and achieving sealing; The detachable sealing device comprises a threaded sleeve embedded in the sealing hole (3) and used for matching and connecting with threads on an external flow path joint pipe; A rubber sealing ring is provided at the bottom of the sealing hole (3), and one end of the air guide hole (4) close to the sealing hole (3) passes through the rubber sealing ring; The diameter of the air guide hole (4) is smaller than the diameters of the sealing hole (3) and the needle connection hole (5); The needle connection hole (5) is used for sliding connection with an external microneedle, and a sealing adhesive is injected between the needle connection hole (5) and the outer wall of the microneedle.

2. A through-hole sealing cover for high-throughput separation of single cell microparticles according to claim 1, characterized in that: The number of the sealing holes (3), the air guide holes (4), and the needle connection holes (5) is 8-96.

3. A through-hole sealing cover for high-throughput separation of single cell microparticles according to claim 1, characterized in that: The cover body (11) is made of non-metallic plate material.

4. A through-hole sealing cover for high-throughput separation of single cell microparticles according to claim 1, characterized in that: The cover body (11) is in the shape of a cuboid, a cube or a cylinder.

5. The through-hole sealing cover for high-throughput separation of single cell microparticles according to claim 1, characterized in that: The lower surface of the cover body (11) is connected to a sealing gasket for preventing leakage, and the lower surface of the cover body (11) is provided with a fixing hole (6) for fixing the sealing gasket.

6. A through-hole sealing cover for high-throughput separation of single cell microparticles according to claim 1, characterized in that: The sealing cover is provided with limiting holes (7) around its periphery, and the limiting holes (7) are used to fix the sealing cover to the test instrument.

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