Single cell capturing method and micropore coupling micro-cavity micro-fluidic chip

Abstract

The invention relates to a single cell capturing method, which comprises the following steps that a single cell to be captured is injected into a micro-fluidic chip containing a micropore coupling micro-cavity through fluid, the chip comprises a base material and a main channel arranged on the base material, the surface of the main channel is provided with a plurality of micropores and micro-cavities corresponding to the micropores, and the micro-cavities are communicated with the main channel; after the main channel is filled with the fluid, the fluid in the main channel is kept static, so that the single cells fall into the micropores under the action of gravity; after the single cells are captured by the micropores, the fluid is introduced again to discharge the uncaptured single cells; and the chip is overturned and inclined after being discharged, so that the single cells enter the micro-cavity. The technical problems that in the prior art, single cell capture efficiency is low, capture precision is insufficient, complex external equipment is needed for control, and single cell capture, culture and the like cannot be completed in the same chip are solved.

Description

Technical Field

[0001] The invention belongs to the technical field of single cell capture, and in particular relates to a single cell capture method and a micropore-coupled microchamber microfluidic chip. Background Art

[0002] In the field of single-cell research, microfluidic chip technology, as an important research tool, has the advantage of precisely manipulating trace amounts of liquid. However, existing single-cell capture and analysis technologies, such as droplet microfluidics and microchamber-based capture methods, have certain limitations. Droplet microfluidics generates tiny droplets by controlling the flow of two immiscible liquids, but because it conforms to the Poisson distribution law, the single-cell capture efficiency is low, with only about 10%-30% of the droplets containing a single cell. It is also easy to generate multi-cell droplets, affecting the accuracy of the experiment.

[0003] Although the single-cell capture technology based on microchambers can improve some of the defects of droplet capture technology, it also faces the problem of low capture rate caused by Poisson distribution. In order to improve the capture efficiency, researchers have developed microporous chip technology, which overcomes the Poisson distribution and improves the single-cell capture flux and efficiency. However, in order to ensure the normal growth and proliferation of cells, especially adherent cells, the increase in micropore size leads to a decrease in single-cell encapsulation rate and even the capture of multiple cells.

[0004] In order to further improve the accuracy of single-cell manipulation, existing technologies use photoelectric positioning technology (such as the Beacon single-cell photoconductive platform) to transfer single cells to microchambers for cultivation. Although this method can achieve precise manipulation of single cells, it relies on complex microelectromechanical system (MEMS) processing technology and external control circuits, which increases the cost and complexity of the equipment, making it unaffordable for most laboratories and companies.

[0005] Therefore, existing technologies have obvious limitations in single-cell capture efficiency, accuracy of the culture process, and equipment cost, which restricts their application in extensive research. Summary of the invention

[0006] The present invention discloses a single cell capture method, comprising a chip, and comprising the following steps:

[0007] S1, injecting a single cell to be captured into a chip through a fluid, wherein the chip is a micropore-coupled microchamber microfluidic chip, and the chip comprises: a substrate and a main channel arranged on the substrate, a plurality of micropores and microchambers corresponding to the micropores are arranged on the surface of the main channel, wherein the microchamber is connected to the main channel;

[0008] S2, after the fluid fills the main channel, the fluid in the main channel is kept still, so that the single cell falls into a number of micro-holes arranged on the main channel under the action of gravity;

[0009] S3, after the micropores capture the single cells, the fluid is introduced again to discharge the uncaptured single cells in the main channel;

[0010] S4, after the single cells in the main channel are discharged, the chip is flipped and tilted so that the microwells of the chip are located above the microchambers, so that the single cells captured in the microwells enter the microchambers located below the microwells from the microwells.

[0011] In some embodiments, it further comprises:

[0012] S5, single cell export method, after the single cell is introduced into the microchamber, the fluid in the microchamber is stationary, and a laser is used to irradiate the position of the microchamber away from the micropore to form bubbles, and the target cells in the microchamber are introduced into the main channel by the laser-induced bubble method. At this time, fluid is passed into the main channel, and driven by the fluid, the target cells are exported from the chip and collected.

[0013] In some embodiments, it further comprises:

[0014] S6, a single cell culture method, after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is stationary, and the culture medium is introduced into the main channel again, at which time the culture medium replaces the fluid in the main channel and the microchamber, and the microchamber is filled with the culture medium, completing the culture of the single cell in the microchamber;

[0015] The single-cell marking detection method is as follows: after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is stationary, and the dye is introduced into the main channel again. At this time, the dye replaces the fluid in the main channel and the microchamber. After the microchamber is filled with the dye, the single cell in the microchamber is stained and marked by the dye. The state of the single cell in the microchamber can be detected in combination with the microscopic image, and the target single cell can be obtained by laser-induced bubble sorting.

[0016] In some embodiments, it further comprises:

[0017] S7, a method for interaction of multiple different single cells, firstly, the first single cell is introduced into the main channel through the fluid, so that the first single cell falls into the micropore under the action of gravity, and after the micropore captures the first single cell, the first single cell that is not captured is discharged; the chip is flipped downward from the micropore to the microchamber and tilted so that the first single cell is transferred to the corresponding microchamber by gravity; the second single cell is introduced into the main channel again through the fluid; the second single cell falls into the micropore under the action of gravity, and after the micropore captures the second single cell, the second single cell that is not captured is discharged; the chip is flipped downward from the micropore to the microchamber and tilted so that the second single cell is transferred to the same microchamber as the first single cell by gravity, thereby completing the interaction of multiple different single cells in the microchamber.

[0018] In some embodiments, in step S3, the uncaptured single cells in the main channel are discharged by controlling the fluid flow rate of the injection port to ensure that the single cells in the micropores are retained in the main channel, and the fluid flow rate of the injection port is 1uL / min to 40uL / min.

[0019] In some embodiments, in step S3, after the fluid fills the main channel, the fluid in the main channel is kept still, and the single cells fall into a plurality of micropores provided on the main channel under the action of gravity. At this time, after 1 to 10 minutes, the fluid is introduced again to expel the single cells that have not been captured in the main channel.

[0020] The present invention also discloses a micropore-coupled microchamber microfluidic chip, the chip comprising:

[0021] Base material;

[0022] A main channel is provided on the substrate and penetrates the substrate;

[0023] A plurality of micropores are arranged on the main channel and used for capturing single cells;

[0024] A plurality of micro-chambers, corresponding to the micro-holes one by one and connected to the main channel;

[0025] Wherein, the diameter of the micropore is d1, the depth of the micropore is t1, the diameter of the single cell is d2, d1:t1=1:2-4:5, d1:d2=1:1-4:3;

[0026] The width of the microchamber is x, the length of the microchamber is y, x:y=1:1-1:3, x>d1.

[0027] In some embodiments, a distance between two adjacent micro-holes and a distance between two adjacent micro-chambers are both L, and 20 μm<L<1000 μm.

[0028] In some embodiments, the volume above each of the microwells is V, V = πr 2 h, wherein r is the radius of the micropore and h is the height of the main channel;

[0029] The single cell concentration above each microwell is C, C=1000 / V×λ, λ refers to the theoretical average number of cells in the volume above each microwell, wherein λ=1, so that the number of cells above each microwell is greater than or equal to 1 cell.

[0030] In some embodiments, the chip further includes a shunt channel, which is disposed between the micropore and the microchamber, wherein the width of the shunt channel is smaller than the width of the microchamber, so that the shunt channel and the microchamber are combined to form a b-shaped structure, and the shunt channel is used to allow the single cell in the micropore to fall into the microchamber, and the shunt channel is interconnected with the main channel.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] 1. The single-cell capture method disclosed in the present invention can achieve efficient capture and processing of single cells by using a micropore-coupled microchamber microfluidic chip. The method first utilizes the micropore design in the microfluidic chip to allow single cells to naturally fall into the micropores by gravity under static fluid conditions, thereby achieving accurate capture of single cells; thereafter, by reintroducing the fluid, excess single cells that have not been captured are excluded, thereby further improving capture efficiency and accuracy. Finally, the flipping and tilting design of the chip allows the single cells captured in the micropores to smoothly enter the microchamber, ensuring that the captured single cells can be effectively transferred to the area for subsequent processing. This design greatly simplifies the operational process of single-cell capture and improves the repeatability and efficiency of the experiment. It is particularly suitable for scenarios that require large-scale single-cell capture, such as cell biology research and single-cell genomics analysis.

[0033] 2. The present invention can also realize the export operation of single cells. The microchamber design allows single cells to be exported at a fixed point after entering the microchamber, and this operation is achieved through laser-induced bubble technology. Specifically, the laser is used to irradiate the position of the microchamber away from the micropore to form bubbles, and the single cells in each microchamber are introduced into the main channel through the pushing effect of the bubbles. This non-contact export method not only reduces the physical damage to single cells, but also improves the accuracy and efficiency of single-cell operations, which is conducive to the subsequent analysis and processing of single cells, such as single-cell gene sequencing or functional testing.

[0034] 3. The present invention also specially designs the culture and staining operation of single cells. After introducing the single cell into the microchamber, the culture medium or stain is injected into the main channel to achieve the replacement of the fluid in the microchamber, and complete the culture or staining of the single cell respectively. This method effectively maintains the activity of the single cell in the microenvironment, and marks the single cell by staining technology, so that the state of the single cell can be quickly distinguished, which is suitable for subsequent research applications such as cell imaging and functional testing. This design not only improves the processing efficiency of single cells, but also ensures the reliability of single cell operations, and is particularly suitable for experimental needs for long-term culture and analysis of single cells.

[0035] 4. The chip of the present invention also supports the study of the interaction between multiple different types of single cells. By sequentially introducing different types of single cells, using gravity capture and chip flipping technology, different types of single cells are introduced into the same microchamber, so as to observe the interaction process between cells at a microscopic scale. This method can achieve precise control of cell-to-cell interactions and is suitable for research in the fields of cell communication, cellular immune response, and the interaction between cancer cells and immune cells, providing a powerful tool support for the analysis of complex biological systems.

[0036] 5. The present invention realizes multiple operation functions such as single cell capture, export, culture, staining and cell interaction through the innovative design of microporous coupled microchamber microfluidic chip. The design of this chip not only improves the automation and accuracy of single cell operation, but also reduces the damage to single cells in traditional methods. It is suitable for a wide range of single cell research application scenarios, such as single cell sequencing, drug screening, cell function analysis and other fields, and has great application potential and practical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The drawings described herein are used to provide further understanding of the present invention and constitute a part of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0038] Figure 1 This is a schematic diagram of the structure of the micropore-coupled microchamber microfluidic chip provided by the present invention.

[0039] Figure 2 This is a schematic structural diagram of a template for a micropore-coupled microchamber microfluidic chip provided by the present invention.

[0040] Figure 3 This is a COMSOL fluid dynamics simulation diagram of the micropore cross section provided by the present invention.

[0041] Figure 4 This is a simulation diagram of the fluid flow rate in the main channel and micropores provided by the present invention.

[0042] 1-substrate; 2-main channel; 3-micropore; 4-microchamber; 5-dividing channel; 6-carrier; 7-first structural unit; 8-second structural unit. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is described and illustrated below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Based on the embodiments provided by the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work belong to the scope of protection of the present invention.

[0044] Obviously, the drawings described below are only some examples or embodiments of the present invention. For ordinary technicians in this field, the present invention can also be applied to other similar scenarios based on these drawings without creative work. In addition, it can also be understood that although the efforts made in this development process may be complicated and lengthy, for ordinary technicians in this field related to the content disclosed by the present invention, some changes in design, manufacturing or production based on the technical content disclosed by the present invention are just conventional technical means, and should not be understood as insufficient content disclosed by the present invention.

[0045] Reference to "embodiments" in the present invention means that a particular feature, structure, or characteristic described in conjunction with the embodiments may be included in at least one embodiment of the present invention. The appearance of the phrase in various places in the specification does not necessarily refer to the same embodiment, nor is it an independent or alternative embodiment that is mutually exclusive with other embodiments. It is explicitly and implicitly understood by those of ordinary skill in the art that the embodiments described in the present invention may be combined with other embodiments without conflict.

[0046] The present invention discloses a single cell capture method, comprising the following steps: injecting a single cell to be captured into a chip through a fluid, wherein the chip is a micropore coupled microchamber microfluidic chip, and the chip comprises: a substrate 1 and a main channel 2 arranged on the substrate 1, and a plurality of micropores 3 and a microchamber 4 corresponding to the micropores 3 are arranged on the surface of the main channel 2, wherein the microchamber 4 is connected to the main channel 2; after the fluid fills the main channel 2, the fluid in the main channel 2 is kept stationary, so that the single cell falls into the plurality of micropores 3 arranged on the main channel 2 under the action of gravity; after the micropores 3 capture the single cell, the fluid is introduced again to discharge the single cell not captured in the main channel 2; after the single cell in the main channel 2 is discharged, the chip is flipped and tilted so that the micropores 3 of the chip are located above the microchamber 4, so that the single cell captured in the micropore 3 enters the microchamber 4 located below the micropore 3 from the micropore 3. The method first controls the fluid to introduce the single cell into the main channel 2, and uses gravity to capture the single cell in the micropore 3. After the single cell is captured, the remaining uncaptured single cells are discharged from the main channel 2 through the fluid. Then, the chip is flipped and tilted so that the captured single cell falls from the micropore 3 into the microchamber 4 below. This method is based on the micropore-coupled microchamber structure for single cell capture. First, the fluid is introduced through the main channel 2 in the microfluidic chip to suspend the single cell therein. When the fluid fills the main channel 2, the static fluid cooperates with the gravity of the single cell to sink it into the micropore 3, completing the capture. Subsequently, the excess single cells that are not captured by the micropore 3 are discharged by injecting the fluid again, and finally, the captured single cells are introduced into the corresponding microchamber 4 by gravity through the flipping and tilting action of the chip. This method uses microfluidic technology and gravity to achieve efficient capture and transfer of single cells. Through the precise design of the micropore 3 and the microchamber 4, it is ensured that the single cell can be effectively captured and can be quickly transferred to the microchamber 4, effectively avoiding the influence of fluid interference on the capture and ensuring the convenience of subsequent operations.

[0047] In some embodiments, the micropore-coupled microchamber microfluidic chip of the present invention adopts Figure 2 The microfluidic chip of the present invention is made by integrally forming a chip template through photolithography and micromachining technology, wherein the template mainly includes a carrier 6, a first structural unit 7 and a second structural unit 8.

[0048] In some embodiments, during the single cell capture process, the fluid flow rate and introduction time can be adjusted according to the experimental requirements. An automated chip flipping device can also be introduced to further improve the operating efficiency and accuracy, and achieve higher precision operations. The flip angle and flip time can be accurately set according to specific needs. If the flip angle is small, the single cell may not fall off from the microwell. If the flip angle is large, the single cell may not be transferred to the microchamber 4 and directly fall out of the microchamber 4.

[0049] The flip angle is crucial for the transfer of single cells from microwell 3 to microchamber 4. In experiments, the flip angle can usually be set in the range of 30° to 90°, and can be precisely adjusted according to the physical properties of the single cell and the viscosity of the fluid:

[0050] Small-angle flipping (30°-45°): suitable for cells that are lighter and easier to slide off, and helps to reduce the failure of cells to detach from the microwell 3. However, if the angle is too small, a single cell may not be able to detach from the microwell 3 smoothly.

[0051] Medium-angle flip (45°-60°): suitable for routine experimental operations, which can ensure that single cells fall off and enter the microchamber4.

[0052] Large-angle flipping (60°-90°): suitable for heavier cells or cells that are difficult to slide off. However, if the angle is too large, the single cell may fall directly out of the microchamber 4 due to excessive acceleration due to gravity, causing the experiment to fail.

[0053] Control of flipping speed: A flipping speed that is too fast may cause fluid disturbance, resulting in the inaccurate transfer of captured single cells; a flipping that is too slow will affect the efficiency of the experiment. Combined with an automated device, the flipping time can be precisely controlled. It is usually recommended that the flipping time be controlled within 1 second to 5 seconds to ensure that the single cell can slide smoothly into the microchamber 4.

[0054] Furthermore, S5 is a single cell export method. After the single cell is introduced into the microchamber 4, the fluid in the microchamber 4 is stationary, and a laser is used to irradiate the position of the microchamber 4 away from the micropore 3 to form bubbles. The target cells in the microchamber 4 are introduced into the main channel 2 by the laser-induced bubble method. At this time, the fluid is passed into the main channel 2. Driven by the fluid, the target cells are exported out of the chip and collected. The laser-induced bubble technology uses high-energy lasers to form local bubbles in the microchamber. The volume expansion and movement of the bubbles can generate liquid flow, thereby pushing the single cells in the microchamber 4 to move toward the main channel 2. Finally, by passing the fluid, the single cells in the main channel 2 are discharged and the export is completed. The application of laser-induced bubble technology realizes the precise movement and export of single cells, avoids direct contact of physical tools with cells, reduces damage to cells, and maintains the integrity and activity of single cells.

[0055] Furthermore, the single cell capture method also includes S6: single cell culture and single cell labeling detection method. After the single cell is introduced into the microchamber 4, the fluid in the main channel 2 and the microchamber 4 is stationary, and the culture medium is introduced into the main channel 2 again. The culture medium replaces the original fluid, and the microchamber 4 is filled with the culture medium to complete the single cell culture. Alternatively, by introducing a dye into the main channel 2 again, the single cell in the microchamber 4 is stained and marked by the dye, and the state of the single cell in the microchamber 4 can be detected in combination with the microscopic image, and the target single cell can be obtained by laser-induced bubble sorting. After the single cell is introduced into the microchamber 4, the static fluid helps to keep the single cell stable and avoid physical stress caused by fluid flow. Subsequently, by introducing fresh culture medium, the original fluid is gradually replaced to ensure that the single cell can be in a suitable culture environment. The culture medium provides the nutrients required for the growth of single cells and supports their long-term culture and observation in the microchamber 4. The static environment enables the single cell to stay stably in the microchamber 4, avoiding the influence of external fluid interference on the single cell. Through the precise operation of the microfluidic device, the original fluid can be effectively replaced to ensure that the single cell is always in fresh culture medium, which is conducive to the long-term survival and growth of the single cell. The culture environment in the microchamber 4 is precisely designed and regulated to support the long-term growth and metabolic activities of the single cell, which is suitable for further experimental operations.

[0056] In some embodiments, the culture medium can be selected according to the single cell type and the purpose of the experiment, such as DMEM, RPMI-1640, FBS, etc. Different types of culture medium can be prepared according to the growth requirements of different cells. The introduction speed of the culture medium can be adjusted according to the experimental requirements to ensure the adequacy and stability of the fluid replacement. Under certain special experimental conditions, the fluid replacement time can be adjusted as needed to ensure that the fluid replacement operation is completed at the appropriate time.

[0057] In some embodiments, the method can also utilize the chemical properties of the stain to interact with biomarkers related to the membrane integrity or activity of single cells, and then distinguish live cells from dead cells through staining reactions. For example, common live-death stains include trypan blue, propidium iodide (PI) and fluorescein ester dyes. Live cells have intact cell membranes that can exclude certain stains, while dead cell membranes are damaged, and stains can enter the interior of the cells and react with cell components, thereby producing a visible staining effect. Through live-death stains, live and dead cells in the microchamber can be quickly distinguished, which is convenient for subsequent experimental operations and data analysis. During the staining process, the fluid is stationary, which reduces the physical impact of the stain on the single cell and ensures the stability and consistency of the staining process. The automated fluid injection and replacement are achieved through a microfluidic system, which simplifies the live-death staining operation steps and reduces the errors of manual intervention.

[0058] In some embodiments, different experiments can choose different stains. Common stains such as trypan blue are suitable for rapid determination of dead cells, while fluorescent dyes such as propidium iodide (PI) and calcein-AM are suitable for more accurate differentiation of live and dead cells by fluorescence microscopy. Depending on the type of stain, the staining concentration and action time can be adjusted appropriately. For example, some dyes may require lower concentrations and longer staining times, while some fast dyes can complete staining in a short time. In order to further enhance the differentiation effect, two or more stains can be used simultaneously to mark live cells and dead cells, respectively. For example, the combination of propidium iodide and calcein-AM can simultaneously display different fluorescent signals of live cells and dead cells.

[0059] Further, the single cell capture method also includes S7, a method for interaction of multiple different single cells, firstly, the first single cell is introduced into the main channel 2 through the fluid, so that the first single cell falls into the micropore 3 under the action of gravity, and after the micropore 3 captures the first single cell, the first single cell that is not captured is discharged; the chip is flipped downward from the micropore 3 to the microchamber 4 and tilted so that the first single cell is transferred to the corresponding microchamber 4 by gravity; the second single cell is introduced into the main channel 2 again through the fluid; the second single cell falls into the micropore 3 under the action of gravity, and after the micropore 3 captures the second single cell, the second single cell that is not captured is discharged; the chip is flipped downward from the micropore 3 to the microchamber 4 and tilted so that the second single cell is transferred to the same microchamber 4 as the first single cell by gravity, and the interaction of multiple different single cells in the microchamber 4 is completed. This method is based on the precise operation of the microfluidic chip, and realizes the introduction of multiple single cells into the same microchamber 4 through the capture and transfer of different single cells twice. Gravity plays a core role in the whole process, and single cells rely on gravity to transfer from the micropores 3 of the main channel 2 to the microchamber 4. Each time the chip is flipped, the directional movement of the single cell can be controlled to ensure that it enters the microchamber 4, thereby creating conditions for the interaction between different single cells. This method uses a microfluidic chip to accurately control the capture, transfer and interaction of different single cells, providing an efficient and repeatable platform for studying the interaction between single cells. By introducing different types of single cells one by one, mutual interference between different single cells during the capture process is avoided, thereby improving the accuracy and success rate of single cell interactions. Through this method, the interaction of multiple different single cells can be easily realized in the same microchamber 4, which is suitable for analyzing the mutual influence between cells, such as signal transmission, cell communication, etc.

[0060] This microfluidic chip provides an efficient and precise experimental platform for multi-cell interaction research. Capturing and culturing different types of single cells in the same microchamber 4 can simulate the real cell microenvironment, making it easier to study interactions such as communication, cooperation or competition between multiple different single cells. At the same time, through the repeated operation function of the chip, experimenters can easily control the type and number of captured single cells, further improving the flexibility and controllability of the experiment.

[0061] In some embodiments, two or more types of single cells can be captured as needed, and more complex multi-cell interaction phenomena can be studied by adjusting the capture order and quantity. At the same time, the size and shape of the microchamber 4 can also be adjusted according to the single cell type and experimental objectives. In addition, the system can also be combined with a sensor module to monitor chemical signals or physical changes in the process of cell interaction in real time to improve the accuracy and visualization of the experiment.

[0062] In some embodiments, the microfluidic chip is used to first capture the first single cell into the microchamber 4 through single cell sampling-capture in micropore 3-transfer to microchamber 4. At this time, there is no single cell in the micropore 3 of the chip. The second single cell is introduced into the main channel 2 again, and the second single cell is captured through the micropore 3. After the capture is successful, the uncaptured single cells in the main channel 2 are discharged through the fluid, and then the chip is turned over and tilted to transfer the second single cell in the micropore 3 to the microchamber 4. At this time, there are two different types of single cells in one microchamber 4, based on which the interaction study of two different types of single cells can be completed.

[0063] Furthermore, in step S3, the uncaptured single cells in the main channel 2 are discharged by controlling the fluid flow rate of the injection port to ensure that the single cells in the micropores 3 are retained in the main channel 2. The preferred fluid flow rate of the injection port is 1uL / min to 40uL / min. By precisely controlling the fluid flow rate, the uncaptured single cells can be effectively discharged while avoiding interference with the captured single cells. The selection of flow rate is crucial to the operational stability and cell capture efficiency of the system; by precisely controlling the fluid flow rate, it is ensured that the uncaptured single cells in the main channel 2 are quickly discharged, while the captured single cells are stably retained in the micropores. This method improves the efficiency and accuracy of the capture process and reduces the impact of fluid disturbances on single-cell capture.

[0064] In some embodiments, the fluid flow rate directly affects the suspension state and capture efficiency of single cells in the main channel 2. In actual operation, the fluid flow rate can be dynamically adjusted according to different experimental requirements:

[0065] Lower flow rate: suitable for capturing lighter or smaller single cells. The flow rate is controlled at 1μL / min to 5μL / min, which helps prevent excessive disturbance of the fluid and ensures that the single cells can fall smoothly into the microwells under the action of gravity.

[0066] Medium flow rate: Suitable for conventional single cell capture, the flow rate range is between 5μL / min and 20μL / min, which can effectively discharge uncaptured single cells and maintain a moderate effect of the fluid on the micropore 3.

[0067] Higher flow rate: suitable for rapid removal of uncaptured single cells, the flow rate can reach 20μL / min to 40μL / min, but it is necessary to avoid excessive flow rate that may cause captured single cells to escape from the micropore 3.

[0068] Furthermore, in step S3, after the fluid fills the main channel 2, the fluid in the main channel 2 is kept still, and the single cells fall into several micropores 3 provided on the main channel 2 under the action of gravity. At this time, after 1min to 10min, the fluid is introduced again to discharge the single cells that have not been captured in the main channel 2. By statically moving the fluid in the main channel 2, the single cells are allowed to naturally settle into the micropores 3 under the action of gravity. Depending on the experimental conditions, the static time can be adjusted between 1 minute and 10 minutes to ensure that the single cells can smoothly fall into the micropores 3. Subsequently, the single cells that have not been captured are discharged by injecting fluid to ensure that the single cells in the micropores 3 are stably retained. The static fluid cooperates with the gravity of the single cells to effectively improve the capture efficiency of the single cells, and the adjustment of the static time also provides a flexible operation time window for different types of single cells. This method reduces the complexity of the operation process and enhances the accuracy of capture.

[0069] In some embodiments, the specific time selection can be optimized according to the requirements of the capture efficiency in the experiment:

[0070] Shorter static time (1-3 minutes): suitable for capturing lighter or smaller single cells. The fluid static time should not be too long to avoid cell accumulation.

[0071] Longer stationary time (4-10 minutes): suitable for capturing larger or heavier single cells to ensure that they can fully settle into the microwell 3.

[0072] Furthermore, a micropore-coupled microchamber microfluidic chip includes a substrate 1, a main channel 2, a plurality of micropores 3 and a plurality of microchambers 4, wherein the main channel 2 is arranged on the substrate 1 and penetrates the substrate 1; the plurality of micropores 3 are arranged on the main channel 2 for capturing single cells; wherein the diameter of the micropore 3 is d1, the depth of the micropore 3 is t1, the diameter of the single cell is d2, d1:t1=1:2-4:5, d1:d2=1:1-4:3; the width of the microchamber 4 is x, the length of the microchamber 4 is y, x:y=1:1-1:3, x>d1. The design basis of the microfluidic chip is to capture single cells within a specific diameter range by utilizing the geometric characteristics of the micropore 3. By setting an appropriate ratio of the diameter and depth of the micropore 3, it can be ensured that the single cell is effectively captured in the micropore 3 without being washed away by the fluid or stuck at the orifice. A reasonable ratio of d1 to t1 ensures that the cells can stay stably in the micropore 3, while ensuring the smoothness of the transfer of single cells to the microchamber 4. The ratio range setting of d1 to d2 helps to capture single cells of different sizes. By adjusting the size ratio of the micropore 3, the present invention can adapt to single cells of different sizes and improve the scope of application of the chip. This flexible size design is conducive to the implementation of different types of single cell experiments and can ensure stability during the capture process. The appropriate ratio setting can also reduce the physical stress on the single cells during the capture and transfer process and reduce single cell damage. The design of the microchamber 4 ensures sufficient space to accommodate and culture single cells. By setting the width x and length y of the microchamber 4 to a ratio of 1:1 to 1:3, a suitable growth space can be provided for single cells. At the same time, x is greater than the diameter d1 of the micropore 3. This design ensures that when a single cell falls from the micropore 3 into the microchamber 4, it will not be stuck due to space limitations, ensuring that the single cell can smoothly enter the microchamber 4. The geometric shape of the microchamber 4 also helps the flow and distribution of culture medium, etc., ensuring that single cells can evenly contact the culture medium. This design of the size ratio of the microchamber 4 can ensure that the single cell has enough space to grow and expand during the culture process, thereby improving the effect of single cell culture. The design in which x is greater than d1 helps the single cell to transfer smoothly and avoids damage or loss of the single cell. In addition, a reasonable size ratio of the microchamber 4 can also improve the fluidity of the culture medium in the microchamber 4, ensure the nutrient supply of the single cell, and promote research on the growth and proliferation of the single cell, secretion detection, environmental stimulus response, cell interaction, genome transcriptome analysis, etc.

[0073] In some embodiments, the size of the micropore 3 can be adjusted according to specific experimental requirements. In a specific experiment, if it is necessary to capture larger or smaller single cells, the ratio of d1 and t1 can be adjusted within a larger range. In addition, by optimizing the material and selecting low-adhesion materials, the damage to the single cells during the capture and transfer process can be further reduced, thereby improving the cell survival rate.

[0074] In some embodiments, when it is necessary to capture a single cell with a diameter of 15 um, we design the micropore 3 to have a diameter of 20 um and a depth of 25 um. Figure 3 The COMSOL fluid dynamics simulation diagram of the cross section of the micropore 3 shown in the figure shows that when there is fluid flow in the main channel 2, there is flow velocity only at the position about 5um above the micropore 3, and the flow velocity in the micropore 3 is 0, that is, the fluid remains stationary, as shown in the figure. Figure 4 As shown, the fluid in the main channel 2 has a certain flow rate, while the flow rate in the micropore 3 is 0. This means that when a single cell is captured in the micropore 3, the fluid flow in the main channel 2 will not affect the single cell captured at the bottom of the micropore 3. When two single cells fall into the micropore in a stacked manner, the single cell located at the top of the micropore 3 will flow away with the fluid in the main channel 2. Therefore, this size ratio design greatly improves the capture rate of single cells.

[0075] In some embodiments, the size of the microchamber 4 can be adjusted appropriately according to the experimental requirements. If the experimental object is a single cell of a larger volume or a cell colony culture is required, the width and length of the microchamber 4 can be increased accordingly. In addition, the depth of the microchamber 4 can be increased to adapt to specific experimental scenarios to ensure that different types of single cells can grow stably. The shape of the microchamber 4 can also be changed from a rectangle to a circle or other geometric shape to adapt to specific application requirements.

[0076] Furthermore, the spacing between two adjacent micropores 3 and the spacing between two adjacent microchambers 4 are both L, where 20μm<L<1000μm. The spacing design of the micropores 3 and the microchambers 4 not only ensures the efficiency of single cell capture, but also takes into account the overall layout and operational feasibility of the chip. The choice of spacing not only ensures the independence of the micropores 3 and the microchambers 4, but also provides appropriate space to avoid mutual interference between cells during the capture and transfer process. Through reasonable spacing design, the single cells in adjacent micropores 3 or microchambers 4 are effectively prevented from interfering with each other, thereby improving the accuracy of the capture and transfer process. At the same time, a smaller spacing can increase the number of micropores 3 and microchambers 4 on the chip, improve the throughput of single cell operations, and meet the needs of high-throughput experiments. By precisely controlling the spacing, it is ensured that the operation of each micropore 3 and microchamber 4 is carried out independently, reducing interference between cells and experimental errors. For example, when a single cell is transferred from a micropore 3 to a microchamber 4, a reasonable spacing prevents cells in other micropores 3 or microchambers 4 from being disturbed by the fluid, thereby improving the accuracy of the operation.

[0077] Furthermore, the volume above each micropore 3 is V, V = πr 2h, where r is the radius of the micropore 3 and h is the height of the main channel 2. The single cell concentration above each micropore 3 is C, C = 1000 / V × λ, λ represents the theoretical average number of cells in the volume above each micropore 3, and λ = 1, to ensure that the number of single cells above each micropore 3 is greater than or equal to 1. The volume calculation above the micropore 3 is based on its radius and the height of the main channel 2, and the cell concentration calculation ensures that there is at least one single cell in the volume, thereby improving the success rate of capture. By reasonably controlling the concentration of the injected single cell suspension, it is ensured that the single cell concentration above each micropore 3 meets the capture conditions. This method determines the cell concentration above each micropore 3 through theoretical calculation, ensuring the consistency and success rate of single cell capture. The single cell concentration above each micropore 3 is precisely controlled to ensure that there is at least one single cell in each micropore 3, which greatly improves the capture efficiency in the experiment. The volume V above the micropore 3 determines the capture area of ​​the single cell, and the precise calculation of the single cell concentration C ensures that at least one single cell is captured in the area. By controlling the concentration and injection speed of the single cell suspension, it is possible to ensure that the single cells are evenly distributed and have an appropriate concentration in the volume area above each microwell 3. This design significantly improves the efficiency and consistency of single cell capture and reduces randomness in the experiment.

[0078] In some embodiments, the concentration of the injected single cell suspension is reasonably controlled to ensure that the single cell concentration in the area above the micropore 3 meets the capture requirements. By adjusting the concentration of the single cell suspension and combining the geometric design of the micropore 3 and the main channel 2, it can be ensured that: there are enough single cells in the volume area above each micropore 3 to ensure that the micropore 3 can effectively capture single cells; through precise cell concentration control, it is avoided that there are too many or too few single cells above the micropore 3, thereby reducing the situation of capture failure or interference.

[0079] In some embodiments, through theoretical calculation and concentration control, it is ensured that there is at least one single cell above each micropore 3. Such control improves the consistency and success rate of the single cell capture process, ensuring that a single cell can be captured in each micropore 3. Concentration control ensures that the number of single cells distributed in the volume area above each micropore 3 is appropriate, avoiding capture failure due to excessively high or low cell concentrations, thereby significantly improving the capture efficiency of single cells. By accurately controlling the concentration of single cells above the micropore 3, the error caused by uneven cell concentration in the experiment can be reduced, ensuring the repeatability and reliability of each capture experiment result.

[0080] Further, the micropore-coupled microchamber microfluidic chip also includes a shunt channel 5, which is arranged between the micropore 3 and the microchamber 4, and the width of the shunt channel 5 is less than the width of the microchamber 4, so that the shunt channel 5 and the microchamber 4 are combined to form a "b"-shaped structure. The shunt channel 5 is used to drop the single cell in the micropore 3 into the microchamber 4, and the shunt channel 5 is interconnected with the main channel 2. The shunt channel 5 is designed as a transition channel, connecting the micropore 3 and the microchamber 4, and providing a path for the single cell from the micropore 3 to the microchamber 4. The width of the shunt channel 5 is less than the width of the microchamber 4, ensuring that the single cell can pass smoothly and fall into the microchamber 4, while avoiding the interference of the fluid. Through the design of the shunt channel 5, the single cell can smoothly transition to the microchamber 4, reducing the capture error caused by fluid disturbance or gravity change. At the same time, the "b"-shaped structure in the microfluidic chip is designed to optimize the transfer process of the single cell from the micropore 3 to the microchamber 4, and prevent the single cell from falling off during the capture and export process. This structure not only plays an important role in the transfer of single cells from micropore 3 to microchamber 4, but also plays a protective role in the process of exporting single cells from microchamber 4 to micropore 3. Whether in the process of transferring single cells to microchamber 4 or in the process of exporting them, the inner cavity design of the "b"-shaped structure effectively prevents accidental shedding of single cells. This structure ensures that single cells can move stably along the predetermined trajectory, reducing the loss of cells during the operation.

[0081] In some embodiments, the design of the "b"-shaped structure forms a "guardrail"-like structure around the inner cavity of the microchamber 4. When a single cell is transferred from the micropore 3 to the microchamber 4, the walls around the "b"-shaped structure prevent the single cell from deviating from the predetermined track due to gravity or fluid disturbance. When the chip is flipped or tilted, and the single cell moves downward from the micropore 3 and enters the microchamber 4 under the action of gravity, the inner wall of the "b"-shaped structure effectively guides the single cell into the center of the microchamber 4 and prevents it from excessive movement or collision with the wall of the microchamber 4, thereby improving the success rate of single cell transfer. This structure can also reduce the impact of fluid disturbance on single cell transfer, ensuring that the single cell falls smoothly into the microchamber 4 driven by gravity.

[0082] In some embodiments, when a single cell is exported from the microchamber 4 to the micropore 3 by tilting the chip, the "b"-shaped structure can also effectively prevent the single cell from falling off. In this process, the single cell moves from the microchamber 4 to the micropore 3 under the action of the fluid, but due to the transition design of the "b"-shaped structure, the single cell can be slowly transferred along a fixed path, avoiding the detachment of the single cell due to too fast flow or too large an inclination angle. The inner wall of the "b"-shaped structure can also prevent the single cell from directly escaping from the chip due to fluid disturbance or excessive chip tilt during the export process, ensuring that the single cell can be guided back from the microchamber 4 to the micropore 3 along the expected path.

[0083] In some embodiments, the width, depth and shape of the shunt channel 5 can be appropriately adjusted according to different types of experimental requirements. For example, when processing larger cells, the width and depth of the shunt channel 5 can be increased to ensure smooth passage of the cells. For different experimental conditions, a multi-layer shunt channel 5 design can also be used to work in parallel with other fluid channels to improve the processing efficiency of single cells.

[0084] In some embodiments, the depth of the shunt channel 5, the depth of the microchamber 4, and the depth of the main channel 2 are all the same. The present invention ensures the consistency of the internal structure of the entire chip by setting the depths of the shunt channel 5, the microchamber 4, and the main channel 2 to be the same. The same depth design can ensure that the flow of fluid in the chip is more stable, avoiding changes in fluid pressure or uneven flow rate caused by inconsistent depths. At the same time, the single cell will not be hindered by depth changes during the transfer process from the micropore 3 to the microchamber 4, thereby achieving a smooth transfer operation. This consistent depth design helps to simplify the manufacturing process of the chip and reduce the problem of fluid dynamic instability caused by depth differences, thereby improving the overall efficiency of single cell capture and culture. The same depth makes the chip structure more unified, the fluid control more precise, reduces the need for system adjustment during operation, and improves the reliability of the experiment.

[0085] In some embodiments, the depths of the three can be adjusted appropriately according to experimental requirements. For example, the depth of the microchamber 4 can be slightly greater than the depth of the main channel 2 to provide more culture space for single cells; or, the depth of the branch channel 5 can be optimized according to the size of the single cell to ensure that the single cell will not encounter excessive resistance during the transfer process. For different types of single cells or experimental purposes, the depths of each channel and the microchamber 4 can be flexibly designed.

[0086] In order to verify that the single cell capture rate of the micropore-coupled microchamber microfluidic chip of the present invention is greatly improved, a comparative test is conducted with the single cell capture method of droplet microfluidics in the prior art, as follows:

[0087] Example

[0088] The micropore-coupled microchamber microfluidic chip of the present invention is used to capture single cells.

[0089] Comparative Example

[0090] Single cells are captured using the droplet microfluidics method in the prior art.

[0091] The capture rates of single cells were compared according to the above-mentioned embodiments and comparative examples, as shown in Table 1.

[0092] Table 1

[0093] Single cell capture rate Example ＞99％ Comparative Example 10％～30％

[0094] It can be concluded from Table 1 above that the micropore-coupled microchamber microfluidic chip of the present invention is significantly superior to the droplet microfluidic method in the prior art in terms of single-cell capture efficiency. The micropore-coupled microchamber microfluidic chip of the present invention enables single cells to be captured and transferred to the microchamber 4 in an orderly manner due to the precise design of the micropores and the static sedimentation mechanism of the fluid. Experimental data show that the single-cell capture rate of this method reaches >99%, which greatly improves the success rate of single-cell experiments and is particularly suitable for high-throughput and precise single-cell operation scenarios. However, droplet microfluidic technology has significant deficiencies in capture rate and operation accuracy, which is only 10% to 30%. This is because the capture of single cells by droplet microfluidic technology is highly dependent on the stability of the droplet generation process and the randomness of the fluid, which easily leads to low efficiency of cell capture. At the same time, the uneven distribution of single cells in the droplets further reduces the success rate of capture, and there are limitations when processing a large number of single-cell experiments.

[0095] Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent substitutions for some of the technical features therein; and these modifications or substitutions do not make the essence of the corresponding technical solutions deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A single cell capture method, characterized in that: The following steps are involved: S1, injecting a single cell to be captured into a chip through a fluid, wherein the chip is a micropore-coupled microchamber microfluidic chip, and the chip comprises: a substrate and a main channel arranged on the substrate, a plurality of micropores and microchambers corresponding to the micropores are arranged on the surface of the main channel, wherein the microchamber is connected to the main channel; S2, after the fluid fills the main channel, the fluid in the main channel is kept still, so that the single cell falls into a number of micro-holes arranged on the main channel under the action of gravity; S3, after the micropores capture the single cells, the fluid is introduced again to discharge the uncaptured single cells in the main channel; S4, after the single cells in the main channel are discharged, the chip is flipped and tilted so that the microwells of the chip are located above the microchambers, so that the single cells captured in the microwells enter the microchambers paired with the microwells from the microwells.

2. The single cell capture method according to claim 1, characterized in that: Also includes: S5, single cell export method, after the single cell is introduced into the microchamber, the fluid in the microchamber is stationary, and a laser is used to irradiate the position of the microchamber away from the micropore to form bubbles, and the target single cell in the microchamber is introduced into the main channel by the laser-induced bubble method. At this time, fluid is passed into the main channel, and driven by the fluid, the target cell is exported from the chip and collected.

3. The single cell capture method according to claim 1, characterized in that: Also includes: S6, a single cell culture method, after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is stationary, and the culture medium is introduced into the main channel again, at which time the culture medium replaces the fluid in the main channel and the microchamber, and the microchamber is filled with the culture medium, completing the culture of the single cell in the microchamber; The single-cell marking detection method is as follows: after the single cell is introduced into the microchamber, the fluid in the main channel and the microchamber is stationary, and the dye is introduced into the main channel again. At this time, the dye replaces the fluid in the main channel and the microchamber. After the microchamber is filled with the dye, the single cell in the microchamber is stained and marked by the dye. The state of the single cell in the microchamber can be detected in combination with the microscopic image, and the target single cell can be obtained by laser-induced bubble sorting.

4. The single cell capture method according to claim 1, characterized in that: Also includes: S7, a method for interaction of multiple different single cells, firstly, the first single cell is introduced into the main channel through the fluid, so that the first single cell falls into the micropore under the action of gravity, and after the micropore captures the first single cell, the first single cell that is not captured is discharged; the chip is flipped downward from the micropore to the microchamber and tilted so that the first single cell is transferred to the corresponding microchamber by gravity; the second single cell is introduced into the main channel again through the fluid; the second single cell falls into the micropore under the action of gravity, and after the micropore captures the second single cell, the second single cell that is not captured is discharged; the chip is flipped downward from the micropore to the microchamber and tilted so that the second single cell is transferred to the same microchamber as the first single cell by gravity, thereby completing the interaction of multiple different single cells in the microchamber.

5. The method for using the micropore-coupled microchamber microfluidic chip according to claim 1, characterized in that: In step S3, the uncaptured single cells in the main channel are discharged by controlling the fluid flow rate of the injection port to ensure that the single cells in the micropores are retained in the main channel, wherein the fluid flow rate of the injection port is 1uL / min to 40uL / min.

6. The method for using the micropore-coupled microchamber microfluidic chip according to claim 1, characterized in that: In step S3, after the fluid fills the main channel, the fluid in the main channel is kept still, and the single cells fall into several micropores on the main channel under the action of gravity. At this time, after 1 minute to 10 minutes, the fluid is introduced again to expel the single cells that have not been captured in the main channel.

7. A microfluidic chip with micropores coupled to microchambers, characterized in that: include: Base material; A main channel, disposed on the chip and running through the chip; A plurality of micropores are arranged on the main channel for capturing single cells; A plurality of micro-chambers, corresponding to the micro-holes one by one and connected to the main channel; Wherein, the diameter of the micropore is d1, the depth of the micropore is t1, the diameter of the single cell is d2, d1:t1=1:2-4:5, d1:d2=1:1-4:3; The width of the microchamber is x, the length of the microchamber is y, x:y=1:1-1:3, x>d1.

8. The micropore-coupled microchamber microfluidic chip according to claim 7, characterized in that: The distance between two adjacent microholes and the distance between two adjacent microchambers are both L, and 20 μm<L<1000 μm.

9. The micropore-coupled microchamber microfluidic chip according to claim 7, characterized in that: The volume above each micropore is V, V = πr 2 h, wherein r is the radius of the micropore and h is the height of the main channel; The single cell concentration above each microwell is C, C=1000 / V×λ, λ refers to the theoretical average number of cells in the volume above each microwell, wherein λ=1, so that the number of cells above each microwell is greater than or equal to 1 cell.

10. The micropore-coupled microchamber microfluidic chip according to claim 7, characterized in that: It also includes a shunt channel, which is arranged between the micropore and the microchamber. The width of the shunt channel is smaller than the width of the microchamber, so that the shunt channel and the microchamber are combined to form a B-shaped structure. The shunt channel is used to drop the single cell in the micropore into the microchamber, and the shunt channel is interconnected with the main channel.

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