A micro-liquid dispensing micro-dish array device and its application

Abstract

This application discloses a microfluidic liquid dispensing micro-dish array device and its application. The microfluidic liquid dispensing micro-dish array device includes a microfluidic array chip and a sample introduction component. The chip includes a chip substrate, on which micro-dishes are arranged in an array and recessed into the chip substrate on a first surface. Each micro-dish includes a flow channel and a main channel. The flow channel has a first end and a second end. The first end of the flow channel is flush with the first surface of the chip substrate, and the height difference of the flow channel relative to the first surface increases sequentially from the first end to the second end. The second end of the flow channel communicates with the main channel. The angle between the extension direction of the flow channel from the first end to the second end and the first surface is θ, where 5°≤θ≤45°. The device provided in this application can generate droplets simply, quickly, and in high throughput. This device overcomes the problem that the growth of difficult-to-culture microorganisms is inhibited by dominant populations when cultured in large volumes. It allows difficult-to-culture microorganisms to obtain sufficient space and energy for growth in a small volume, and enables real-time monitoring of microorganisms in the chip to study their growth status and elucidate their physiological and biochemical characteristics.

Description

Technical Field

[0001] This application relates to the field of microfluidic chip analysis, specifically to a micro-liquid dispensing micro-dish array device and its application. Background Technology

[0002] Microfluidic chips are miniature laboratory devices that integrate microfluidic technology. Microfluidics is a technique for conducting experiments and analyses by controlling the flow of liquids in micrometer-scale channels. It scales down traditional laboratory operations to the micrometer scale, enabling efficient control and analysis of minute samples. This allows for the handling of minute amounts of liquid, reducing the volume of reaction systems and increasing sensitivity. Due to the small size of microfluidic chips, liquids within them can mix and react more rapidly, reducing reaction time. Currently, microfluidic chips have wide applications in the biomedical field, including cell analysis, DNA sequencing, and protein analysis. They can be used for single-cell analysis to study the behavior of biomolecules in the microenvironment, and also for chemical reactions and drug screening, enabling more efficient chemical synthesis and drug development.

[0003] Droplet generation technology is a method for generating and controlling droplets at the microscale, commonly used in microfluidic systems, drug delivery, and biological experiments. A droplet array refers to a matrix structure in which a series of tiny droplets are arranged in an ordered manner on a substrate or carrier. This structure has wide applications in many fields, including chemical analysis, biomedical research, and drug screening. There are various methods for generating droplet arrays, among which microfluidics is a common and effective approach. This technology relies on microchannels and microflow, using factors such as electric fields, pressure, and surface tension to precisely control the flow of liquid within the microchannels, thereby creating a uniformly sized and spaced droplet array. For example, inkjet technology sprays liquid onto a substrate using a printhead to form droplet arrays; chemically modifying surfaces to form droplet arrays is commonly used in applications such as biochip fabrication and DNA microarrays, where each droplet contains specific biomolecules; using oscillators or acoustic vibrations, liquids can be divided into uniform small droplets, which are then arranged into droplet arrays; photolithography and micromold techniques can create tiny grooves or microstructures on the substrate surface, allowing manipulation of liquids to form droplet arrays. Microfluidic chips, with their micrometer-scale channels and tiny reaction chambers, provide a microscale and highly controlled environment. This helps simulate the microscopic scale of microorganisms in their natural environment. Microfluidic chips can process a large number of tiny reaction units, enabling high-throughput experiments. This is highly beneficial for simultaneously testing multiple microbial samples under different conditions. Microfluidic chips typically require relatively small reaction volumes, which helps reduce the need for expensive reagents and microbial samples, thus lowering costs. Furthermore, microfluidic chips are easy to observe under a microscope, allowing real-time monitoring of microbial growth, interactions, and reactions.

[0004] Microfluidic chips may experience wear or contamination after prolonged use, affecting their stability and reliability. Durability is particularly critical for processing biological samples. Some samples can adversely affect the materials of microfluidic chips, leading to distorted experimental results. Currently, polydimethylsiloxane (PDMS) is a common material for microfluidic chips; however, PMS has poor strength and is easily deformed, limiting its use to laboratory research and failing to meet the demands of industrial applications. Furthermore, most microfluidic chips require external mechanical equipment and chemical surface modification to achieve droplet generation. Therefore, it is essential to provide a microfluidic chip with practical application value to address these problems. Summary of the Invention

[0005] Based on the above-mentioned defects and problems, this application provides a low-cost micro-liquid distribution micro-dish array device for the single-cell isolation and culture of difficult-to-culture microorganisms, overcoming the problem that the growth of difficult-to-culture microorganisms is inhibited by the dominant population when cultured in large volumes, so that difficult-to-culture microorganisms can obtain sufficient space and energy for growth in a small volume.

[0006] This application provides the following technical solution.

[0007] 1. A microfluidic liquid dispensing micro-dish array device, wherein the device comprises a microfluidic array chip and a sample introduction component, the chip comprising a chip substrate, and micro-dishes arranged in an array and recessed into the chip substrate on a first surface of the chip substrate, the micro-dishes comprising a flow channel and a main channel, the flow channel having a first end and a second end, the first end of the flow channel being flush with the first surface of the chip substrate, and the height difference of the flow channel relative to the first surface increasing sequentially from the first end to the second end, the second end of the flow channel communicating with the main channel; the angle between the extension direction of the flow channel from its first end to its second end and the first surface is θ, where 5°≤θ≤45°.

[0008] 2. The apparatus according to item 1, wherein the bottom of the flow channel is an arc-shaped surface recessed into the chip substrate, the maximum width of the arc-shaped surface is d, d is 0-8000μm and does not include 0, and the length of the bottom is 0-8000μm and does not include 0.

[0009] 3. The apparatus according to item 1, wherein the sidewall of the flow channel is a flat plate structure, and the angle between the sidewall of the flow channel and the first surface is greater than or equal to 90°.

[0010] 4. The apparatus according to item 3, wherein the sidewall of the flow guiding channel and the bottom of the flow guiding channel are connected by an arc-shaped transition structure; or

[0011] The sidewall of the flow guide channel is directly connected to the bottom of the flow guide channel.

[0012] 5. The apparatus according to item 1, wherein the bottom of the second end of the flow guide channel is connected to the bottom of the main channel, and the sidewall of the second end of the flow guide channel is connected to the sidewall of the main channel.

[0013] 6. The apparatus according to item 2, wherein the angle between the sidewall and the bottom of the main channel is α, where α is greater than or equal to 90°; or

[0014] The angle between the tangent of the sidewall of the main channel and the bottom is α, where α is greater than or equal to 90°.

[0015] 7. The apparatus according to item 6, wherein the bottom of the main channel is a plane or a curved surface, and the outer contour of the bottom of the main channel is a circle or an n-sided polygon, where n is greater than or equal to 3.

[0016] 8. The apparatus according to item 7, wherein when the outer contour of the bottom of the main channel is circular or regular n-gon, the length of its diameter or equivalent diameter is D, and d / D is 0-1, excluding 0.

[0017] 9. The apparatus according to item 8, wherein D is 10-8000 μm and its depth relative to the first surface is 10-3000 μm.

[0018] 10. The apparatus according to item 1, wherein the gap between adjacent microvessels is not less than 10 μm.

[0019] 11. The apparatus according to claim 1, wherein the chip substrate is made of one or more of polyethylene, polypropylene, polycarbonate, polyvinyl chloride, polystyrene, polydimethylsiloxane, cyclic olefin copolymer, polylactic acid, polyurethane, polymethyl methacrylate, glass, quartz, and resin.

[0020] 12. The apparatus according to any one of items 1-11, wherein the sample introduction assembly includes a holding portion, a connecting portion, and a liquid reservoir, the liquid reservoir being connected to the holding portion via the connecting portion.

[0021] 13. The device according to item 12, wherein the gripping part and the connecting part are integrally formed.

[0022] 14. The apparatus according to claim 12, wherein the connecting portion includes at least one set of connecting components, each set of connecting components including a first connecting portion and a second connecting portion, wherein the first connecting portion and the second connecting portion are both arc-shaped grooves or oblique grooves.

[0023] 15. The apparatus according to claim 14, wherein the liquid storage section includes a first liquid storage plate and a second liquid storage plate, a portion of the first liquid storage plate is connected to the first connecting portion, and a portion of the second liquid storage plate is connected to the second connecting portion.

[0024] The angle between the first liquid storage plate and the second liquid storage plate is α, where α is between 10° and 30°.

[0025] The minimum distance between the first liquid storage plate and the second liquid storage plate is L, where L is 0-1mm and does not include 0.

[0026] 16. The apparatus according to item 15, wherein an injection hole is provided on the first liquid storage plate, and the distance between the end of the first liquid storage plate away from the first connecting portion and the injection hole is 3-5 mm.

[0027] 17. The apparatus according to any one of items 1-11, wherein the sample introduction assembly includes an integrally formed grip and a liquid reservoir, the liquid reservoir including at least one set of liquid reservoir chambers.

[0028] 18. The apparatus according to item 17, wherein each group of liquid storage cavities includes a first liquid storage wall and a second liquid storage wall, wherein both the first liquid storage wall and the second liquid storage wall are arc-shaped plates or inclined plates.

[0029] 19. The apparatus according to claim 18, wherein the first liquid storage wall and the second liquid storage wall are both inclined plates, and the included angle formed between the first liquid storage wall and the second liquid storage wall is β, where β is 10°-30°, or

[0030] Both the first liquid storage wall and the second liquid storage wall are arc-shaped plates. The angle formed between the tangent of the arc surface of the first liquid storage wall and the tangent of the arc surface of the second liquid storage wall is β, where β is 10°-30°.

[0031] The minimum distance between the first liquid storage wall and the second liquid storage wall is L0, where L0 is 0-1mm and does not include 0.

[0032] 20. The apparatus according to item 18, wherein an injection hole is provided on the first liquid storage wall, and the distance between the end of the first liquid storage wall away from the grip portion and the injection hole is 3-5 mm.

[0033] 21. The micro-liquid dispensing micro-dish array device described in any one of items 1-20 is used in the preparation of droplet sensor arrays, optical lens arrays, large-scale single-cell culture, bacterial strain isolation, digital PCR quantitative analysis, as an arrayed storage system for droplet microfluidics, or as an arrayed screening system for droplet microfluidics.

[0034] 22. A method for single-cell droplet culture, wherein,

[0035] Provide a micro-liquid dispensing micro-dish array device according to any one of items 1-20;

[0036] The aqueous phase and oil phase are added sequentially to the reservoir of the sample introduction component;

[0037] Hold the sample introduction component perpendicular to the chip substrate so that the two come into contact, and the aqueous and oil phases seep out from the end of the reservoir.

[0038] The sample introduction component slides slowly in a specific direction, allowing the aqueous phase to enter the micro-vessel under the action of surface tension and be covered by the subsequent oil phase;

[0039] After the sample introduction component leaves the chip, the aqueous phase enters the micro-dish and forms droplets, while the oil phase covers the surface of the droplets to prevent them from evaporating.

[0040] Microscopic imaging is performed to obtain image information inside the micropores for subsequent analysis.

[0041] 23. The single-cell droplet culture method according to item 22, wherein the aqueous phase is a suspension containing bacteria or cells.

[0042] 24. The single-cell droplet culture method according to item 22, wherein the aqueous phase is a hydrogel solution that solidifies into a coagulated state in a micro dish at a specific temperature to support the fixation and growth of bacteria or cells into cell clusters or microcolonies in the gel.

[0043] 25. The single-cell droplet culture method according to item 22, wherein the micro dish is pre-filled with a solidified culture medium hydrogel that does not contain cells or bacteria, and a suspension containing bacteria or cells is introduced again by the coating method to achieve the growth of bacteria or cells on the surface of the hydrogel.

[0044] This application provides a micro-dish array device for micro-liquid distribution, which can generate droplets simply, quickly, and in high throughput. This device overcomes the problem that the growth of difficult-to-culture microorganisms is inhibited by dominant populations when cultured in large volumes, allowing difficult-to-culture microorganisms to obtain sufficient space and energy for growth in a small volume. It also enables real-time monitoring of microorganisms in the chip, study of their growth status, and elucidation of their physiological and biochemical characteristics. Attached Figure Description

[0045] Figure 1 A schematic diagram of the device provided in this application.

[0046] Figure 2 This is a schematic diagram of the structure of the microvessel provided in this application.

[0047] Figure 3 A partial structural schematic diagram of the microvessel provided in this application.

[0048] Figure 4A This is one of the structural schematic diagrams of the sample import component provided in this application.

[0049] Figure 4B The second schematic diagram of the sample import component provided in this application.

[0050] Figure 5 A flowchart illustrating the droplet preparation process using the apparatus provided in this application.

[0051] Figure 6 Results of droplet formation rate in droplet arrays of Examples 1 and 2 provided in this application.

[0052] Figure 7 Results of bacterial growth within droplets in Example 3 provided in this application.

[0053] Figure 8 Microscopic imaging results of high-throughput arrayed droplets provided in Example 4 of this application.

[0054] Figure 9 Frequency distribution results of the proportion of high-throughput arrayed droplets in Example 4 of this application.

[0055] Figure 10 The results of single-cell isolation and culture of the mixed bacterial solution provided in Example 5 of this application.

[0056] Figure 11 Array culture of colorectal cancer cells provided in Example 6 of this application.

[0057] Explanation of reference numerals in the attached figures

[0058] 1-Flow channel, 2-Main body channel, 3-Chip substrate, 4-Holding part, 5-Connecting part, 6-First connecting part, 7-Second connecting part, 8-First liquid storage plate, 9-Second liquid storage plate, 10-Injection hole, 11-First liquid storage wall, 12-Second liquid storage wall, 13-Liquid storage substrate. Detailed Implementation

[0059] The following description provides exemplary embodiments of this application, including various details to aid understanding, and should be considered merely exemplary. Therefore, those skilled in the art will recognize that various changes and modifications can be made to the embodiments described herein without departing from the scope and spirit of this application. Similarly, for clarity and brevity, descriptions of well-known functions and structures are omitted in the following description.

[0060] like Figures 1-3 As shown, this application provides a microfluidic liquid dispensing micro-dish array device. The device includes a microfluidic array chip and a sample introduction component. The microfluidic array chip includes a chip substrate. Micro-dishes are arranged in an array and recessed into the chip substrate on the first surface of the chip substrate. Each micro-dish includes a flow channel 1 and a main channel 2. The flow channel 1 has a first end and a second end. The first end of the flow channel 1 is flush with the first surface of the chip substrate, and the height difference of the flow channel 1 relative to the first surface increases sequentially from the first end to the second end. The second end of the flow channel 1 communicates with the main channel 2. The angle between the extension direction of the flow channel 1 from its first end to its second end and the first surface is θ, where 5°≤θ≤45°.

[0061] The guiding channel 1 is a ramp channel, providing a tilted plane for the droplet, allowing it to flow more smoothly down the ramp and accelerate towards the main channel with the help of gravity. Compared to the case without a guiding channel, the droplet can more easily overcome surface tension and other resistances, flowing directly into the main channel. The guiding channel can change the contact angle between the droplet and the surface, thereby reducing the overall surface energy of the droplet. The combination of the guiding channel and the main channel can form a structure similar to a capillary channel. When the droplet contacts the guiding channel, capillary force acts on the droplet, causing it to flow directionally into the main channel. When the tilt angle of the guiding channel is too small, the flow velocity of the droplet is relatively slow. This is because the component of gravity along the ramp direction is too small at a small tilt angle, and its acceleration effect on the droplet is not significant. In addition, due to the small component of gravity, the droplet may be more easily disturbed by other external forces (such as the lateral component of surface tension, airflow, etc.) during the flow process. However, when the tilt angle is too large, the droplets may flow too quickly due to gravity, resulting in too short a residence time on the slope, which may prevent some necessary physical processes from occurring. Furthermore, an excessively large angle reduces the flow stability of the droplets. The droplets may also be affected by factors such as air resistance, which can impact the efficiency of their flow into the main channel.

[0062] In some implementations, 10° ≤ θ ≤ 45°.

[0063] In some implementations, 5° ≤ θ ≤ 35°.

[0064] In some implementations, 10°≤θ≤40°.

[0065] Specifically, θ can be 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, etc.

[0066] In this application, when it is necessary to prepare droplets, a liquid is dropped at the first end of the guide channel 1 or at a position close to the first end. The liquid falls into the main body channel 2 through the guide channel 1 to form droplets, filling the entire main body channel 2, thereby quickly forming high-throughput, uniform droplets.

[0067] In this application, the bottom of the flow channel 1 is an arc-shaped surface recessed into the chip substrate. The maximum width of the arc-shaped surface is d, which is 0-8000 μm and excludes 0. The length L1 of the bottom is 0-8000 μm and excludes 0. The appropriate maximum width and length L1 of the bottom of the flow channel 1 are determined based on the liquid flow rate and the size of the main channel volume. An excessively wide bottom of the flow channel 1 may cause droplet dispersion and reduce flow efficiency; an excessively narrow bottom may limit the droplet flow velocity. The selection of the length L1 should also consider the droplet volume and flow characteristics to ensure that the liquid maintains a stable flow state within the flow channel 1. If the length of the flow channel 1 is too short, the liquid may not have enough time and distance to accelerate and flow stably. In this case, the liquid may not be able to fully utilize the effects of gravity and the ramp, and the effect of flowing into the main channel may be poor. However, if the ramp channel length is too long, although it can provide a longer flow path for the droplets, it may also cause the droplets to experience more resistance during flow, such as air resistance and friction with the channel surface, thereby reducing the droplet flow velocity and efficiency. If the maximum width at the bottom of the ramp channel is too narrow, it may not be able to hold enough liquid, or it may cause the droplets to experience significant resistance during flow, affecting their flow speed and stability. If the maximum width at the bottom of the ramp channel is too wide, it will take up space in the micro-device.

[0068] In this paper, the maximum width of the bottom of the flow channel 1 refers to the fact that, since the bottom of the flow channel 1 is an inwardly concave arc surface, the bottom of the flow channel 1 has different widths depending on the depth of the concavity. The deeper the concavity, the smaller the corresponding width. The maximum width of the bottom of the flow channel 1 is the width at the connection point between it and the side wall of the flow channel 1.

[0069] In some embodiments, the bottom outer contour of the flow channel 1 can be a partial structure of a cylinder, specifically a structure obtained by obliquely cutting the chip substrate with a cylindrical cutting component at an angle θ to the first surface.

[0070] In some implementations, d is 50μm-8000μm.

[0071] In some implementations, d is 100μm-8000μm.

[0072] In some implementations, d is 500μm-8000μm.

[0073] Specifically, d can be 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm , 1000μm, 1500μm, 2000μm, 2500μm, 3000μm, 3500μm, 4000μm, 4500μm, 5000μm, 5500μm, 6000μm, 6500μm, 7000μm, 7500μm, 8000μm, etc.

[0074] In some implementations, L1 is 50 μm-8000 μm.

[0075] In some implementations, L1 is 100μm-8000μm.

[0076] In some implementations, L1 is 500μm-8000μm.

[0077] Specifically, L1 can be 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1000μm, 1500μm, 2000μm, 2500μm, 3000μm, 3500μm, 4000μm, 4500μm, 5000μm, 5500μm, 6000μm, 6500μm, 7000μm, 7500μm, 8000μm, etc.

[0078] In this paper, the direction from the first end to the second end of the flow channel 1 is the extension direction of the flow channel 1. The extension directions of the bottom and sidewalls of the flow channel 1 are consistent with the extension direction of the flow channel 1. The width of the bottom of the flow channel 1 is the length in the direction perpendicular to the extension direction of the bottom of the flow channel 1 (i.e., the width of the bottom of the flow channel 1 is the shorter side of the bottom). The height of the sidewall of the flow channel 1 is the length in the direction perpendicular to the extension direction of the sidewall of the flow channel 1 (i.e., the height of the sidewall of the flow channel 1 is the shorter side of the sidewall).

[0079] Furthermore, the sidewall of the flow channel 1 is a flat plate structure, and the angle between the sidewall of the flow channel 1 and the first surface is greater than or equal to 90°, preferably 90°-180°.

[0080] In some embodiments, the sidewall of the flow channel 1 is connected to its bottom via an arc-shaped transition structure. This arc-shaped transition structure allows for smoother flow of droplets from the flow channel into the main channel, reducing flow resistance. Compared to right-angle connections, the arc-shaped transition structure prevents abrupt changes in direction and velocity at corners, thus reducing energy loss. It also distributes stress evenly at the connection point, reducing stress concentration and improving structural stability and durability. Furthermore, the arc-shaped transition structure is relatively easier to implement in manufacturing and processing. Compared to right-angle connections, it is easier to achieve a smooth surface and accurate shape through molding, machining, or other manufacturing processes, reducing manufacturing difficulty and cost.

[0081] In some embodiments, the sidewall of the flow channel 1 is directly connected to the bottom of the flow channel 1.

[0082] Furthermore, the bottom of the second end of the flow channel 1 is connected to the bottom of the main channel 2, and the sidewall of the second end of the flow channel 1 is connected to the sidewall of the main channel 2. This connection provides a continuous and unobstructed flow path for the droplet. When the droplet flows from the flow channel into the main channel, the connection between the sidewall and the bottom allows the droplet to transition smoothly, avoiding sudden changes in direction or interruptions. The continuous flow path helps reduce energy loss during the flow process, improving the efficiency and stability of the flow. In addition, the connection between the sidewall and the bottom can be achieved through one-time molding or a simple assembly process, simplifying the manufacturing process.

[0083] In this application, the sidewall of the main channel 2 is an arc-shaped surface or a plane.

[0084] When the sidewall of the main channel 2 is arc-shaped, the angle between the tangent of the sidewall and its bottom is α, where α is greater than or equal to 90°. When α is greater than or equal to 90°, the guiding effect of the arc-shaped sidewall on the droplet is more pronounced. Under the influence of gravity, the droplet slides down the arc-shaped sidewall. Due to the larger angle, the supporting force from the sidewall on the droplet is relatively small, making it easier for the droplet to flow downwards into the main channel. When α is too small, the guiding effect of the arc-shaped sidewall on the droplet weakens, and the droplet may remain in the channel for a longer time, or even become blocked. This reduces the flow efficiency of the droplet and affects the normal operation of the system.

[0085] In some embodiments, the angle between the tangent of the sidewall of the main channel 2 and its bottom is 90°≤α≤150°.

[0086] In some embodiments, the tangent of the sidewall of the main channel 2 forms a 90° angle with its bottom.

[0087] ≤α≤135°.

[0088] In some embodiments, the tangent of the sidewall of the main channel 2 forms a 90° angle with its bottom.

[0089] ≤α≤120°.

[0090] Specifically, α can be 90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, etc.

[0091] When the sidewall of the main channel 2 is planar, the angle between the sidewall and its bottom is α, where α is greater than or equal to 90°. A larger angle makes the manufacturing and processing of the main channel easier. For example, in micromachining technology, right-angled or obtuse-angled structures can be more easily formed through processes such as photolithography and etching. In contrast, manufacturing acute-angled structures requires higher precision and more complex processes, increasing manufacturing difficulty and cost. However, when the angle is too large, special processing techniques or molds may be required. This increases manufacturing difficulty and cost, and may reduce production efficiency.

[0092] In some embodiments, the angle between the sidewall of the main channel 2 and its bottom is 90°≤α≤150°.

[0093] In some embodiments, the angle between the sidewall of the main channel 2 and its bottom is 90°≤α≤135°.

[0094] In some embodiments, the angle between the sidewall of the main channel 2 and its bottom is 90°≤α≤120°.

[0095] Specifically, α can be 90°, 95°, 100°, 105°, 110°, 115°, 120°, 125°, 130°, 135°, 140°, 145°, 150°, etc.

[0096] Furthermore, the bottom of the main channel is a plane or a curved surface. For example, the curved surface can be a curved surface that protrudes in the direction of the first surface, or a curved surface that is recessed in the direction away from the first surface.

[0097] Furthermore, such as Figure 3 As shown, the outer contour of the bottom of the main channel 2 is a circle or an n-sided polygon, where n is greater than or equal to 3, preferably a circle or a regular n-sided polygon, such as an equilateral triangle, a square, a regular hexagon, a regular octagon, a regular dodecagon, etc.

[0098] Furthermore, when the outer contour of the bottom of the main channel 2 is circular or regular n-gon, its diameter or equivalent diameter length is D, and d / D is 0-1, excluding 0.

[0099] When the bottom of the main channel has different shapes and the diameter can vary within a certain range, it can adapt to a variety of different application scenarios and requirements. A smaller d / D value may increase the difficulty and cost of manufacturing. For high-precision machining processes, smaller dimensional tolerances and higher machining accuracy are required, which may extend the production cycle and increase production costs. When the d / D value is too large, it may lead to a reduction in the fluid flow area within the main channel, thereby increasing fluid resistance.

[0100] In some embodiments, when the outer contour of the bottom of the main channel 2 is circular, its diameter is D.

[0101] In some embodiments, when the bottom of the main channel 2 is a regular n-gon, its equivalent diameter (the equivalent diameter here refers to the diagonal) is D.

[0102] In some implementations, d / D is 0.1-1.

[0103] In some implementations, d / D is 0.1-0.8.

[0104] In some implementations, d / D is 0.1-0.5.

[0105] Specifically, d / D can be 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, etc.

[0106] In some embodiments, the bottom of the main channel 2 is a plane.

[0107] In some embodiments, the bottom of the main channel 2 is convex.

[0108] Furthermore, D is 10-8000 μm, and its depth h relative to the first surface is 10-3000 μm.

[0109] Specifically, D can be 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm , 1000μm, 1500μm, 2000μm, 2500μm, 3000μm, 3500μm, 4000μm, 4500μm, 5000μm, 5500μm, 6000μm, 6500μm, 7000μm, 7500μm, 8000μm, etc.

[0110] Specifically, h can be 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 200 μm, 300 μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1000μm, 1500μm, 2000μm, 2500μm, 3000μm, etc.

[0111] In this application, the gap between adjacent microvessels is not less than 10 μm. In some embodiments, the gap between adjacent microvessels is 10-8000 μm; in others, it is 10-1000 μm; and in still others, it is 10-50 μm. Larger gaps occupy more chip area, resulting in a reduction in the number of microvessels that can be accommodated on a chip of the same size. This not only wastes valuable chip space but may also increase chip manufacturing costs. In microfluidic experiments, precise control of liquid flow is crucial for achieving experimental objectives. If the gap is too large, the liquid may form irregular flow paths between the microvessels, potentially leading to uneven liquid flow. When the gap between adjacent microvessels is too small, the reduced distance between them may increase the risk of cross-contamination. Smaller gaps place higher demands on chip manufacturing processes. Precise control of the gap between microvessels requires higher processing precision and technical expertise, which may increase chip manufacturing costs.

[0112] The spacing and volume of the droplets prepared by the device in this application are determined by the gap between the micro-dins and the volume of the main channel. When the gap between the micro-dins increases, the spacing between the generated droplets also increases accordingly. Conversely, when the gap between the micro-dins decreases, the spacing between the droplets also decreases. The volume of the main channel is directly related to the volume of the generated droplets. Generally, a larger main channel can hold more liquid, thus generating larger droplets. Conversely, a smaller main channel limits the amount of liquid it can hold, thus generating smaller droplets. During the chip design stage, different volumes of main channels can be selected according to experimental requirements. By adjusting the size, shape, and depth of the main channel, its volume can be changed, thereby controlling the volume of the generated droplets.

[0113] Specifically, the gap between adjacent microplates can be 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 200μm, 300μm, 400μm, 500μm, 600μm, 700μm, 800μm, 900μm, 1000μm, 1500μm, 2000μm, 2500μm, 3000μm, 3500μm, 4000μm, 4500μm, 5000μm, 5500μm, 6000μm, 6500μm, 7000μm, 7500μm, 8000μm, etc.

[0114] In this application, the chip substrate is made of one or more of the following materials: polyethylene, polypropylene, polycarbonate, polyvinyl chloride, polystyrene, polydimethylsiloxane, cyclic olefin copolymer, polylactic acid, polyurethane, polymethyl methacrylate, glass, quartz, and resin.

[0115] In this application, as Figure 4A As shown, the sample introduction component includes a holding part 4, a connecting part 5, and a liquid storage part. The holding part 4 and the connecting part 5 are integrally formed, and the liquid storage part is connected to the holding part 4 through the connecting part 5.

[0116] The connecting part 5 includes at least one set of connecting components. The number of connecting components in the connecting part 5 can be 1, 2, 3, 4, 5, 6, etc., which can be determined according to actual needs.

[0117] In some embodiments, the connecting portion 5 has two sets of connecting components, one set of which is used to engage with the reservoir for storing the oil phase, and the other set of connecting components is used to engage with the reservoir for storing the aqueous phase. The distance between two adjacent sets of connecting components is 5-15 mm, for example, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, etc.

[0118] Two sets of connecting components are arranged side by side, and the gap between two adjacent sets of connecting components is greater than the gap between adjacent microvessels.

[0119] Furthermore, each set of connecting components includes a first connecting portion 6 and a second connecting portion 7. Both the first connecting portion 6 and the second connecting portion 7 have two extending directions: a first extending direction and a second extending direction. The first extending direction of both the first connecting portion 6 and the second connecting portion 7 extends from the end of the connecting portion 5 near the gripping portion 4 to the end away from the gripping portion 4, and their second extending direction extends from the first side surface of the connecting portion 5 to the second side surface. The first extending direction can be its width direction, and the second extending direction can be its length direction.

[0120] The first connecting portion 6 and the second connecting portion 7 are continuously distributed in their first extending direction.

[0121] In some embodiments, the first connecting portion 6 and the second connecting portion 7 are continuously distributed in their second extending direction.

[0122] In some embodiments, the first connecting portion 6 and the second connecting portion 7 can be discretely distributed along their second extension direction, that is, the first connecting portion 6 has multiple first connecting portion 6 units along its second extension direction, and the second connecting portion 7 has multiple second connecting portion 7 units along its second extension direction. The distance between two adjacent connecting components is consistent with the distance between two adjacent microvessels. This design facilitates the precise addition of liquid samples into the microvessels.

[0123] Furthermore, both the first connecting portion 6 and the second connecting portion 7 are arc-shaped grooves or oblique grooves.

[0124] In some embodiments, both the first connecting portion 6 and the second connecting portion 7 are arc-shaped grooves. These arc-shaped grooves can more naturally conform to the edges of the liquid storage plates, especially for materials with a certain degree of elasticity, achieving a tighter fit and improving the stability of the fixation. Furthermore, the arc shape can guide the insertion of the first liquid storage plate 8 and the second liquid storage plate 9, making the insertion process smoother; simultaneously, when it is necessary to remove the plates, it is easier to apply force to push the plates out of the first connecting portion 6 and the second connecting portion 7.

[0125] The longitudinal cross-sections of the first connecting part 6 and the second connecting part 7 form the shape of "()" or "[]" or "\ / ".

[0126] In some embodiments, both the first connecting part 6 and the second connecting part 7 are inclined grooves. The design of the inclined grooves allows the liquid storage plates to be placed at a certain angle, so that the two adjacent liquid storage plates form a certain angle at the bottom, making the bottom a slit opening, which constitutes a sample introduction component.

[0127] The longitudinal cross-section of the first connecting part 6 and the second connecting part 7 forms a V-shaped shape, in which the bottom of the V is not closed.

[0128] In this document, the longitudinal sections of the first connecting part 6 and the second connecting part 7 are the first side or the second side of the connecting part 5.

[0129] Furthermore, the liquid storage section includes a first liquid storage plate 8 and a second liquid storage plate 9, wherein a portion of the first liquid storage plate 8 is connected to the first connecting portion 6, and a portion of the second liquid storage plate 9 is connected to the second connecting portion 7.

[0130] The portion of the first liquid storage plate 8 extending into the first connecting portion 6 is fixed within the first connecting portion 6, and the portion of the second liquid storage plate 9 extending into the second connecting portion 7 is fixed within the second connecting portion 7. The snap-fit ​​method is not further limited, as long as it achieves the function required in this application.

[0131] The included angle between the first liquid storage plate 8 and the second liquid storage plate 9 is α, which is between 10° and 30°, for example, 10°, 15°, 20°, 25°, 30°, etc. If the included angle α is too small, the liquid may have difficulty falling smoothly into the micro-pits of the chip substrate. Due to the narrow gaps, the liquid may experience significant surface tension upon contact with the chip substrate. Secondly, if the included angle is too small, the minimum distance between the first liquid storage plate 8 and the second liquid storage plate 9 (i.e., the minimum distance between the first liquid storage plate 8 and the second liquid storage plate 9 is the liquid outlet of the storage section) will be too small, resulting in a limited volume of stored liquid, making it difficult to support the generation of a droplet for an entire chip. Conversely, if the angle is too large, the distance between the first connecting part 6 and the second connecting part 7 will increase, allowing for the storage of excessive liquid and increasing the length of the sample introduction component, thus affecting usability.

[0132] In this document, the included angle between the first liquid storage plate 8 and the second liquid storage plate 9 is the angle formed by the intersection of the extending direction of the first liquid storage plate 8 and the extending direction of the second liquid storage plate 9.

[0133] The minimum distance between the first liquid storage plate 8 and the second liquid storage plate 9 is L, which is 0-1mm and does not include 0. For example, it can be 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, etc.

[0134] The width of the first liquid storage plate 8 outside the first connecting portion 6 is 1 / 3 to 1 / 2 of the width of the first liquid storage plate 8. The width of the second liquid storage plate 9 outside the second connecting portion 7 is 1 / 3 to 1 / 2 of the width of the second liquid storage plate 9. This design is to ensure that the minimum distance L between the first liquid storage plate 8 and the second liquid storage plate 9 is sufficient to hold a certain volume of liquid. If the distance is too small, the volume held will be too small; if the distance is too large, more of the liquid storage plate will be exposed above the connecting portion, making it prone to deformation and affecting the experiment.

[0135] The lengths of the first liquid storage plate 8 and the second liquid storage plate 9 are slightly greater than the lengths of their corresponding chip substrates.

[0136] The first liquid storage plate 8 and the second liquid storage plate 9 can be rectangular plates, parallelogram-shaped plates, etc. The length of the first liquid storage plate 8 and the second liquid storage plate 9 refers to the length of the side edge closest to the chip substrate. The width of the first liquid storage plate 8 and the second liquid storage plate 9 is the length perpendicular to the side edge.

[0137] The liquid storage capacity of the liquid storage section formed by the first liquid storage plate 8 and the second liquid storage plate 9 is 0-500μL, excluding 0. For example, it can be 40μL, 60μL, 80μL, 100μL, 120μL, 140μL, 160μL, 180μL, 200μL, 220μL, 240μL, 260μL, 280μL, 300μL, 320μL, 340μL, 360μL, 380μL, 400μL, 420μL, 440μL, 460μL, 480μL, 500μL, etc.

[0138] Furthermore, an injection hole 10 is provided on the first liquid storage plate 8. The distance between the end of the first liquid storage plate 8 away from the first connecting part 6 and the injection hole 10 is 3-5 mm, for example, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, etc. This ensures that the liquid storage part can accommodate a certain amount of experimental volume. Through the injection hole 10, the aqueous phase or oil phase can be added into the liquid storage part. The bottom of the liquid storage part of the sample introduction component presents a very small long slit opening, which is very small in size. The surface tension of the liquid acts as a barrier at the slit, preventing the liquid from easily flowing out through the slit. Secondly, regarding the sides of the liquid storage part, there is surface tension between the liquid and the first liquid storage plate 8 and the second liquid storage plate 9. This surface tension causes the liquid to form a curved liquid surface on the sides of the first liquid storage plate 8 and the second liquid storage plate 9, which, like a "film," binds the liquid in the gap between the first liquid storage plate 8 and the second liquid storage plate 9. When the handheld sample introduction component is always perpendicular to the chip substrate, the liquid will not easily flow out from the side of the reservoir.

[0139] The diameter or equivalent diameter of the injection hole 10 is 1-5mm, for example, it can be 1mm, 2mm, 3mm, 4mm, 5mm, etc. If it is too large, it will occupy too much space of the first liquid storage plate 8 and the second liquid storage plate 9. If it is too small, it will make it difficult for the pipette tip to be inserted into the inlet hole, affecting the injection.

[0140] In this application, as Figure 4B As shown, the sample introduction component includes an integrally formed gripping part 4 and a liquid storage part, wherein the liquid storage part includes at least one set of liquid storage chambers.

[0141] The number of liquid storage chambers can be 1, 2, 3, 4, 5, 6, etc., which can be determined according to actual needs.

[0142] In some embodiments, the liquid storage section has two sets of liquid storage chambers, one set for storing the oil phase and the other set for storing the aqueous phase. The distance between two adjacent sets of liquid storage chambers is 5-15 mm, for example, 5 mm, 6 mm, 7 mm, 8 mm, 9 mm, 10 mm, 11 mm, 12 mm, 13 mm, 14 mm, 15 mm, etc.

[0143] Two sets of liquid storage chambers are arranged side by side, and the gap between two adjacent sets of liquid storage chambers is greater than the gap between adjacent microvessels.

[0144] Furthermore, the liquid storage cavity includes a liquid storage base 13, which is connected to the gripping part 4, and the first liquid storage wall 11 and the second liquid storage wall 12 of each group of liquid storage cavities are connected side by side to the end of the liquid storage base 13 away from the gripping part 4.

[0145] Both the first liquid storage wall 11 and the second liquid storage wall 12 are arc-shaped plates or inclined plates.

[0146] In some embodiments, the first liquid storage wall 11 and the second liquid storage wall 12 are both inclined plates, and the included angle formed between the first liquid storage wall 11 and the second liquid storage wall 12 is β, which is 10°-30°, for example, 10°, 15°, 20°, 25°, 30°, etc.

[0147] In this paper, the included angle β formed between the first liquid storage wall 11 and the second liquid storage wall 12 is the included angle formed by the intersection of the extension direction of the first liquid storage wall 11 and the extension direction of the second liquid storage wall 12.

[0148] Both the first liquid storage wall 11 and the second liquid storage wall 12 are arc-shaped plates. The angle formed between the tangent of the arc surface of the first liquid storage wall 11 and the tangent of the arc surface of the second liquid storage wall 12 is β, where β is 10°-30°, for example, it can be 10°, 15°, 20°, 25°, 30°, etc.

[0149] An angle β that is too small may make it difficult for the liquid to fall smoothly into the micro-pits of the chip substrate. Due to the narrow gaps, the liquid may be subject to significant surface tension upon contact with the chip substrate. Secondly, an angle that is too small will result in a small minimum distance between the first liquid storage wall 11 and the second liquid storage wall 12 (i.e., the minimum distance between the first liquid storage wall 11 and the second liquid storage wall 12 is the liquid outlet of the liquid storage section), limiting the volume of liquid that can be stored and making it difficult to support the generation of a droplet for an entire chip. On the other hand, an angle that is too large means that the distance between the first liquid storage wall 11 and the second liquid storage wall 12 increases, allowing for the storage of too much liquid and increasing the length of the sample introduction component, thus affecting usability.

[0150] The liquid storage cavity has a larger upper part and a smaller lower part, that is, the size of the liquid storage cavity on the side closer to the liquid storage substrate 13 is larger than the size of the liquid storage cavity on the side farther away from the liquid storage substrate 13.

[0151] Furthermore, the minimum distance between the first liquid storage wall 11 and the second liquid storage wall 12 is L0, where L0 is 0-1mm and does not include 0. For example, it can be 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1mm, etc.

[0152] The storage capacity of each of the aforementioned liquid storage sections is 0-500 μL, excluding 0, for example, 40 μL, 60 μL, 80 μL, 100 μL, 120 μL, 140 μL, 160 μL, 180 μL, 200 μL, 220 μL, 240 μL, 260 μL, 280 μL, 300 μL, 320 μL, 340 μL, 360 μL, 380 μL, 400 μL, 420 μL, 440 μL, 460 μL, 480 μL, 500 μL, etc.

[0153] Furthermore, an injection hole 10 is provided on the first liquid storage wall 11. The distance between the end of the first liquid storage wall 11 away from the holding part 4 and the injection hole 10 is 3-5 mm, for example, 3 mm, 3.5 mm, 4 mm, 4.5 mm, 5 mm, etc. This ensures that the liquid storage part can accommodate a certain amount of experimental volume. Through the injection hole 10, the aqueous phase or oil phase can be added into the liquid storage part. The bottom of the liquid storage part of the sample introduction component presents a very small long slit opening, which is very small in size. The surface tension of the liquid acts as a barrier at the slit, preventing the liquid from easily flowing out through the slit. Secondly, regarding the sides of the liquid storage part, there is surface tension between the liquid and the first liquid storage wall 11 and the second liquid storage wall 12. This surface tension causes the liquid to form a curved liquid surface on the sides of the first liquid storage wall 11 and the second liquid storage wall 12, which, like a "film," binds the liquid in the gap between the first liquid storage wall 11 and the second liquid storage wall 12. When the handheld sample introduction component is always perpendicular to the chip substrate, the liquid will not easily flow out from the side of the reservoir.

[0154] Furthermore, the diameter or equivalent diameter of the injection hole 10 is 1-5mm, for example, it can be 1mm, 2mm, 3mm, 4mm, 5mm, etc. If it is too large, it will occupy too much space of the first liquid storage wall 11 and the second liquid storage wall 12, and if it is too small, it will make it difficult for the pipette tip to be inserted into the inlet hole, affecting the injection.

[0155] In this application, as Figure 5As shown, the ends of the first liquid storage plate 8 and the second liquid storage plate 9 on the sample introduction component are sample inlets. Liquid is added through the sample inlets to the first end or near the first end of the guide channel 1 on the chip substrate. The liquid falls into the main body channel 2 through the guide channel 1 to form droplets, filling the entire main body channel 2, thereby quickly forming high-throughput, uniform droplets.

[0156] The main advantage of the device described in this application is that it can obtain high-density arrayed droplets in a short time without the need for any external equipment such as precision pumps. In addition, it offers advantages such as distortion-free and undistorted bacterial imaging, long-term live cell culture, and avoidance of the effects of surfactants on bacterial growth.

[0157] Another advantage of the device in this application is that the droplets prepared by the device have a long storage time (7-10 days), and can be used as a high-density array storage system for droplets, and can be directly used as a site for cell separation, culture, screening and other operations.

[0158] This application provides a method for single-cell droplet culture, comprising the following steps:

[0159] Step 1: Provide the aforementioned micro-liquid dispensing micro-dish array device;

[0160] Step 2: Add the aqueous phase and oil phase sequentially to the reservoir of the sample introduction component;

[0161] Step 3: Hold the sample introduction component perpendicular to the chip substrate so that the two come into contact, and the aqueous phase and oil phase seep out from the end of the reservoir.

[0162] Step 4: The sample introduction component slides slowly along a specific direction, allowing the aqueous phase to enter the microcruise under the action of surface tension and be covered by the subsequent oil phase;

[0163] Step 5: After the sample introduction component leaves the chip, the aqueous phase enters the micro-dish and forms droplets, while the oil phase covers the surface of the droplets to prevent them from evaporating.

[0164] Step Six: Perform microscopic photography to obtain image information inside the micropores for subsequent analysis.

[0165] In this application, the aqueous phase is a suspension containing bacteria or cells.

[0166] Furthermore, the aqueous phase is a hydrogel solution that solidifies into a coagulated state in a microplate at a specific temperature to support the fixation and growth of bacteria or cells into cell clusters or microcolonies within the gel.

[0167] In this application, the micro dish is pre-filled with a solidified culture medium hydrogel that does not contain cells or bacteria, and a suspension containing bacteria or cells is introduced again by the coating method to achieve the growth of bacteria or cells on the surface of the hydrogel.

[0168] The method described in this application overcomes the shortcomings of existing droplet arrays and provides a method for rapid formation of aggregated droplets, variable droplet composition, and online positioning and extraction of droplets. This will improve the practicality and operability of microfluidic droplet arrays in fields such as biology, chemistry, clinical diagnosis, and inspection and quarantine.

[0169] This application realizes a simple, fast, variable, controllable, and locatable online extraction and monitoring high-density arrayed chip substrate, which is a simple and fast array generation method with high operability and practicality.

[0170] This application eliminates the need for any surface treatment of the chip substrate, avoiding additional operational costs and difficulties. Furthermore, the device does not require any external instruments or equipment, resulting in low cost and suitability for various applications.

[0171] This application provides the application of the aforementioned microfluidic dispensing microplate array device in the preparation of droplet sensor arrays, optical lens arrays, large-scale single-cell culture, bacterial strain isolation, digital PCR quantitative analysis, as an arrayed storage system for droplet microfluidics, or as an arrayed screening system for droplet microfluidics.

[0172] Example

[0173] The materials and test methods used in the embodiments of this application are described in a general and / or specific manner. In the following embodiments, unless otherwise specified, % means wt%, i.e., weight percentage. Reagents or instruments used, unless otherwise specified, are all commercially available conventional reagent products.

[0174] Example 1

[0175] The microfluidic liquid dispensing micro-dish array device of this embodiment includes a microfluidic array chip and a sample introduction component. The chip includes a chip substrate, on which micro-dishes are arranged in an array and recessed into the chip substrate on a first surface. The gap between adjacent micro-dishes is 1300 μm. Each micro-dish includes a flow channel 1 and a main channel 2. The flow channel 1 has a first end and a second end. The first end of the flow channel 1 is flush with the first surface of the chip substrate, and the height difference of the flow channel 1 relative to the first surface is from... The flow channel 1 increases in size from its first end to its second end. The bottom of the second end of the flow channel 1 is connected to the bottom of the main channel 2. The angle θ between the extension direction of the flow channel 1 from its first end to its second end and the first surface is 20°. The bottom of the flow channel 1 is an arc-shaped surface recessed into the chip substrate. The maximum width d of the arc-shaped surface is 300 μm. The height of the sidewall of the flow channel 1 is 150 μm. The sidewall of the flow channel 1 is directly connected to the bottom of the flow channel 1 and is perpendicular to the first surface. The angle α between the sidewall and the bottom of the main channel 2 is 90°. The bottom of the main channel 2 is a circular plane with a diameter of 600 μm and a depth h of 150 μm.

[0176] The sample introduction assembly includes a holding part 4 and a liquid storage part. The holding part 4 includes an integrally formed holding part 4 and a connecting part 5. The liquid storage part is connected to the holding part 4 via the connecting part 5. The connecting part 5 has two sets of connecting components, one set for engaging the liquid storage part for storing the oil phase and the other set for engaging the liquid storage part for storing the aqueous phase. The distance between adjacent sets of connecting components is 9 mm. Each set of connecting components includes a first connecting part 6 and a second connecting part 7. The first extending direction of both the first connecting part 6 and the second connecting part 7 extends from the end of the connecting part 5 near the holding part 4 to the end away from the holding part 4, and their second extending direction extends from the first side surface to the second side surface of the connecting part 5. The first connecting part 6 and the second connecting part 7 are continuously distributed in their first and second extending directions. Both the first connecting part 6 and the second connecting part 7 are arc-shaped grooves, and the shape formed by the longitudinal sections of the first connecting part 6 and the second connecting part 7 is "()". The liquid storage section includes a first liquid storage plate 8 and a second liquid storage plate 9. A portion of the first liquid storage plate 8 extends into the first connecting portion 6, and a portion of the second liquid storage plate 9 extends into the second connecting portion 7. The included angle α between the first liquid storage plate 8 and the second liquid storage plate 9 is 15°, and the minimum distance L between the first liquid storage plate 8 and the second liquid storage plate 9 is 0.5 mm. A circular injection hole 10 with a diameter of 4 mm is provided on the first liquid storage plate 8.

[0177] A fluorescein solution was used as the aqueous phase, and FC-40 was selected as the oil phase to prevent droplet evaporation. Multi-channel scanning imaging of the entire chip substrate was performed using a fluorescence microscope to obtain the droplet formation rate in the entire droplet array. The results are shown in [Figure Number]. Figure 6 .

[0178] The difference between Example 2 and Example 1 is that in this example, the maximum width d at the bottom of the flow channel 1 is proposed to be 0, 200 μm, 300 μm, 450 μm, or 600 μm, and the angle θ of the flow channel 1 is 10°, 20°, or 30°. This example freely combines these two factors into 12 design structures. A fluorescein solution is used as the aqueous phase, and FC-40 is selected as the oil phase to prevent droplet evaporation. A fluorescence microscope is used to perform multi-channel scanning imaging of the entire chip substrate to obtain the droplet formation rate in the entire droplet array. The results are shown in [Figure number missing]. Figure 6 .Depend on Figure 6 It is known that the device of this application can rapidly form single-cell droplets.

[0179] Example 3 is used to achieve the isolation of microorganisms and large-scale single-cell purification culture.

[0180] In this embodiment, the sample used was a fluorescently labeled Escherichia coli bacterial suspension, and the apparatus used was the same as in Example 1. Escherichia coli RP437 expressing red fluorescent protein was selected as the isolated sample. The E. coli was cultured under suitable conditions (LB medium, 37°C, 200 rpm) until an OD 600 of 0.6 was reached. After being removed, it was diluted 10,000 times with LB medium and mixed with low-temperature agarose in an equal proportion as the aqueous phase for droplet formation. Simultaneously, FC-40 was selected as the oil phase to prevent droplet evaporation. The bacterial suspension and fluorinated oil were evenly brushed onto the chip substrate using the sample introduction component, forming a high-throughput droplet array. The chip substrate was placed in a sealed container and incubated at 37°C for 18 hours. During the incubation process, the bacterial growth within the droplets was observed using a fluorescence inverted microscope under bright-field and red fluorescence observation conditions. The results are shown below. Figure 7 As shown, by the 6th hour, the single-cell droplets in the microplate had grown into colonies. This image presents a magnified view of a localized fluorescent droplet. In this droplet array, the microbial growth status of each droplet can be observed and recorded in real time. These results demonstrate that the device proposed in this application can achieve large-area, high-throughput droplet array preparation. The prepared droplets can achieve single-cell growth, exhibiting good growth patterns consistent with those of conventional agar plate culture.

[0181] Example 4: Generation of high-throughput arrayed fluorescein droplets

[0182] In this embodiment, the sample used is a fluorescein solution, FC-40 is selected as the oil phase to prevent droplet evaporation, and the apparatus used is the same as in Example 1. A suitable amount of fluorescein solution is pipetted and carefully added to the reservoir of the sample introduction component, keeping the component stable and perpendicular to the chip. By steadily sliding the sample into the component, the liquid in the reservoir falls into the chip's micropits, forming a droplet array. The filling of the micropits on the chip is observed using a microscope. The magnification and focal length of the microscope can be adjusted to clearly observe the fluorescein solution in the micropits and the overall micropit filling rate of the chip. Images under the microscope are captured and saved using a camera or image acquisition software. The microscopic images of different locations on the chip are stitched together to form the entire chip image. The results are shown in [Figure 1]. Figure 8 As shown in the figure. Then, the filling status of the micropits was observed, and the fluorescein solution filling rate of 1000 micropits was statistically analyzed. The experimental results are shown in... Figure 9 As shown, almost all the micropits are filled with fluorescein solution, forming a high-throughput fluorescein droplet array.

[0183] Example 5 is used to achieve single-cell isolation and culture of mixed microorganisms.

[0184] In this embodiment, the samples used were bacterial suspensions of two different fluorescently labeled *E. coli*, and the apparatus used was the same as in Example 1. *E. coli* RP437 expressing red fluorescent protein and *E. coli* RP1616 expressing green fluorescent protein were selected as the isolated samples. The two different *E. coli* strains were cultured under suitable conditions (LB medium, 37°C, 200 rpm) until an OD 600 equaled 0.6. After being removed, they were diluted 10,000 times with LB medium, and the two different *E. coli* strains were mixed in equal proportions, then mixed with low-temperature agarose in equal proportions as the aqueous phase for droplet formation. Simultaneously, FC-40 was selected as the oil phase to prevent droplet evaporation. The bacterial suspension and fluorinated oil were evenly brushed onto the chip substrate using the sample introduction component to form a high-throughput droplet array. The chip substrate was placed in a sealed container and incubated overnight at 37°C. During the incubation process, the bacterial growth within the droplets was observed using a fluorescence inverted microscope under bright-field, red fluorescence, and green fluorescence observation conditions. The results are shown below. Figure 10 As shown, different microplates were filled with different fluorescently labeled E. coli, and each microplate grew into colonies in its own microplate. The growth of E. coli in adjacent microplates did not affect each other.

[0185] Example 6 was used to achieve array culture of colorectal cancer HCT116 cells.

[0186] In this embodiment, the sample used was colorectal cancer HCT116 cells, and the apparatus used was the same as in Example 1. Colorectal cancer HCT116 cells were selected as the isolated sample. The HCT116 cells were cultured under suitable conditions (DMEM medium, 37°C) until they covered the entire bottom of the petri dish. After digestion, they were resuspended in 1 mL of DMEM medium. A certain amount of the resuspended solution was centrifuged to collect the precipitate. The precipitate was resuspended in a certain amount of DMEM medium and then mixed thoroughly with matrix gel at a ratio of 3:7 or 4:6. The mixture of colorectal cancer cells and matrix gel was then spread evenly onto the chip substrate using the sample introduction component, forming a high-throughput droplet array. The chip substrate was immersed in DMEM medium for preservation and cultured at 37°C for 3 days. During the culture process, cell growth within the micropits was observed using a conventional stereomicroscope under bright-field observation conditions. The results are shown below. Figure 11 As shown, on the second day, the cells scattered in the micropits began to aggregate and grow in a point-like manner. On the third day, the cells in the micropits connected together. On the fourth day, the cells in the micropits aggregated into a clump, exhibiting a state similar to 3D culture.

[0187] Although the embodiments of this application have been described above in conjunction with the accompanying drawings, this application is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of this application, and these are all within the scope of protection of this application.

Claims

1. A micro-dish array device for dispensing micro-liquids, wherein, The device includes a microfluidic arrayed chip and a sample introduction component. The chip includes a chip substrate, on the first surface of the chip substrate are arrayed microvessels that are recessed into the chip substrate. Each microvessel includes a flow channel and a main channel. The flow channel has a first end and a second end. The first end of the flow channel is flush with the first surface of the chip substrate, and the height difference of the flow channel relative to the first surface increases sequentially from the first end to the second end. The second end of the flow channel communicates with the main channel. The angle between the extension direction of the flow channel from its first end to its second end and the first surface is θ, where 5°≤θ≤45°.

2. The apparatus according to claim 1, wherein, The bottom of the flow channel is an arc-shaped surface that is recessed into the chip substrate. The maximum width of the arc-shaped surface is d, which is 0-8000μm and does not include 0. The length of the bottom is 0-8000μm and does not include 0.

3. The apparatus according to claim 1, wherein, The sidewall of the flow channel is a flat plate structure, and the angle between the sidewall of the flow channel and the first surface is greater than or equal to 90°.

4. The apparatus according to claim 3, wherein, The sidewall of the flow guiding channel is connected to the bottom of the flow guiding channel by an arc-shaped transition structure; or The sidewall of the flow guide channel is directly connected to the bottom of the flow guide channel.

5. The apparatus according to claim 1, wherein, The bottom of the second end of the flow guide channel is connected to the bottom of the main channel, and the sidewall of the second end of the flow guide channel is connected to the sidewall of the main channel.

6. The apparatus according to claim 2, wherein, The angle between the sidewall and bottom of the main channel is α, where α is greater than or equal to 90°; or The angle between the tangent of the sidewall of the main channel and the bottom is α, where α is greater than or equal to 90°.

7. The apparatus according to claim 6, wherein, The bottom of the main channel is a plane or a curved surface, and the outer contour of the bottom of the main channel is a circle or an n-sided polygon, where n is greater than or equal to 3.

8. The apparatus according to claim 7, wherein, When the outer contour of the bottom of the main channel is circular or regular n-gon, its diameter or equivalent diameter is D, and d / D is 0-1, excluding 0.

9. The apparatus according to claim 8, wherein, The value of D is 10-8000 μm, and its depth relative to the first surface is 10-3000 μm.

10. A method for single-cell droplet culture, wherein, Provide a micro-liquid dispensing micro-dish array device according to any one of claims 1-9; The aqueous phase and oil phase are added sequentially to the reservoir of the sample introduction component; Hold the sample introduction component perpendicular to the chip substrate so that the two come into contact, and the aqueous and oil phases seep out from the end of the reservoir. The sample introduction component slides slowly in a specific direction, allowing the aqueous phase to enter the micro-vessel under the action of surface tension and be covered by the subsequent oil phase; After the sample introduction component leaves the chip, the aqueous phase enters the micro-dish and forms droplets, while the oil phase covers the surface of the droplets to prevent them from evaporating. Microscopic imaging is performed to obtain image information inside the micropores for subsequent analysis.

Images