A microfluidic chip for single-cell high-throughput biological and mechanical signal loading and high-temporal and spatial resolution manipulation and its application method

Abstract

A microfluidic chip for high-throughput biological and mechanical signal loading and high-temporal-spatial resolution manipulation of single cells and its application method, relating to the field of microfluidic chip technology. The present invention utilizes flow focusing theory and micro-droplet generation theory to achieve high-throughput single-cell droplet encapsulation, encapsulates single cells in an independent and stable external environment, achieves precise loading of biochemical signals, and ensures the effectiveness and accuracy of single-cell manipulation; at the same time, it further utilizes micro-droplet retention areas and micro-suction arrays, through precise flow / pressure negative feedback control, to precisely control the loading of biochemical and mechanical signals from both time and space dimensions, and achieve high-"time-space" resolution single-cell manipulation. The present invention can obtain a microfluidic chip for high-throughput biological and mechanical signal loading and high-temporal-spatial resolution manipulation of single cells and its application method.

Description

Technical Field

[0001] The present invention relates to the technical field of microfluidic chips, and in particular to a microfluidic chip for loading single-cell high-throughput biological and mechanical signals and high-temporal-spatial resolution manipulation, and an application method thereof. Background Art

[0002] Cells are the fundamental structural and functional units of the human body, forming a complex mediator system comprised of the cell membrane, cytoplasm, cytoskeleton, nucleus, intracellular second messengers, genes, and signaling proteins. Cells in the body exist within a complex dynamic flow microenvironment comprised of surrounding tissue cells, intercellular matrix, and body fluids. They are stimulated not only by mechanical force signals within this microenvironment but also by the co-stimulation of concentration signals from biochemical factors such as hormones and neurotransmitters. Through interactions between signaling molecules, they regulate a series of downstream cellular biological events, exhibiting multi-scale temporal and spatial nonlinear dynamic responses. These signal dynamic responses are closely related to functions and behaviors such as cell division, differentiation, proliferation, and apoptosis.

[0003] The scientific issues of cell biomechanics and cell mechanobiology involved in the above processes are crucial for understanding the mechanisms of the occurrence and development of major human diseases such as malignant tumors. In the field of cytological research, existing technical methods mostly use samples obtained after cell destruction for detection and analysis. These methods only obtain static information about the genome and proteome at a specific moment, and cannot fully reflect the nonlinear interactions between molecules in living cells and their dynamic information that is constantly evolving due to the influence of the external and internal environments. They also cannot simultaneously observe the impact of internal and external environmental factors on cell structure, function, and overall behavior in a single experiment in real time. Summary of the Invention

[0004] The purpose of the present invention is to solve the above technical problems and to provide a microfluidic chip and its application method for single-cell high-throughput biological signal and mechanical signal loading and high spatiotemporal resolution manipulation.

[0005] A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal-resolution manipulation, comprising a substrate and a PDMS complete structure 2, wherein the PDMS complete structure 2 is disposed on the upper surface of the substrate and is a rectangular open box structure comprising an external phase liquid channel 8, a cell buffer channel 9, a cell culture fluid channel 10, a buffer channel 5, a cell droplet retention area 6, and a suction channel;

[0006] One end of the external phase liquid channel 8 is provided with an external phase liquid injection hole 3-1, one end of the cell buffer channel 9 is provided with a cell buffer injection hole 3-2, and one end of the cell culture fluid channel 10 is provided with a cell culture fluid injection hole 3-3; the other ends of the external phase liquid channel 8, the cell buffer channel 9 and the cell culture fluid channel 10 are connected to the water inlet of the buffer channel 5 through the cross droplet shear 4, and the water outlet of the buffer channel 5 is connected to the water inlet of the cell droplet retention area 6 through a pipeline; the head end of the cell droplet retention area 6 is provided with a plurality of pressure-driven holes, and the tail end of the cell droplet retention area 6 is provided with a waste liquid hole 3-6, and the sucking channel is vertically arranged in the middle section of the cell droplet retention area 6, and the two ends of the sucking channel are connected to the inner wall of the cell droplet retention area 6.

[0007] The above-mentioned application method of the microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation is carried out according to the following steps:

[0008] Step S1: Connect the container containing silicone oil to the external phase liquid injection port 3-1, connect the container containing buffer to the cell buffer injection port 3-2, and connect the container containing cell culture fluid to the cell culture fluid injection port 3-3; at the same time, connect the air outlet of the pressure pump to the No. 1 pressure drive port 3-4 and the No. 2 pressure drive port 3-5, and connect the waste liquid port 3-6 to the water inlet of the waste liquid container;

[0009] Step S2: injecting silicone oil through the external phase liquid injection port 3-1, injecting a buffer through the cell buffer injection port 3-2, and injecting a cell culture fluid containing cells through the cell culture fluid injection port 3-3 by a pump, and adjusting the flow rate of the silicone oil to 100-3000 μL / min, the flow rate of the buffer to 10-1000 μL / min, and the flow rate of the cell culture fluid to 0.5-200 μL / min;

[0010] Step S3: Silicone oil, buffer solution and cell culture fluid converge at the cross droplet shearing opening 4 to form a single-cell encapsulated droplet; the single-cell encapsulated droplet enters the cell droplet retention area 6 through the buffer channel 5, and a pressure pump is used to apply a positive pressure of 0.2 to 8 kPa to the pressure driving hole 3-4 No. 1 and the pressure driving hole 3-5 No. 2, and a negative pressure of 0.2 to 8 kPa to the waste liquid hole 3-6, respectively, to control the single-cell encapsulated droplet to pass through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3, and finally flow out through the waste liquid hole 3-6 and be collected by the waste liquid container.

[0011] Principle of the present invention:

[0012] During operation, the high-throughput single-cell encapsulation device achieves high-throughput single-cell droplet encapsulation through flow shear force and fluid solubility differences. The flow / pressure driver is used to inject corresponding liquids into the external phase liquid injection hole, cell buffer injection hole and cell culture fluid injection hole respectively. When the flow rate matches, under the action of flow shear force and fluid solubility differences, the multiphase liquid will form oil-in-water droplets and encapsulate the single cell inside, providing an independent and stable external environment for the single cell. The principle is as follows Figure 3 As shown, in this process, gradient concentration signal loading and various biochemical stimulus loading can be achieved through an external gradient concentration generator or flow rate control. After the single-cell droplet is generated, it will flow through a serpentine channel of a specific length, allowing the biochemical stimulus to fully take effect. It then flows into a single-cell controller with high "temporal-spatial" resolution. The controller contains two pressure-driven holes and one waste liquid hole. During operation, each hole is connected to a flow / pressure controller. The positive and negative pressures of multiple holes are synergistically used to achieve precise control of single-cell movement and precise loading of mechanical signals. The controller can not only manipulate cells to reside in the cell droplet retention area to accurately control the reaction time, but also accurately control the movement of single-cell droplets within the cell droplet retention area, causing them to flow through specific suction channels, thereby applying specific mechanical signals to the single cell. Therefore, it is a single-cell controller with high "temporal-spatial" resolution.

[0013] Innovations of the present invention:

[0014] The innovation of this invention lies in providing a microfluidic chip for high-throughput loading of biological and mechanical signals and high-temporal and spatial resolution manipulation of single cells. The high throughput is reflected in the number of single-cell encapsulated droplets passing through the micro-suction channel array, which is 80 to 150 per second. This throughput has not yet been reported. Biological signal loading is reflected in the precise control of the concentration of biochemical factors applied to single cells by regulating the flow rate of each phase. Mechanical signal loading is reflected in the application of different mechanical stimuli to cells using different suction channels. High spatiotemporal resolution, of which temporal resolution is reflected in the precise control of the residence time of single-cell droplets in the droplet retention zone by adjusting the pressure signal of the pressure control hole, providing reaction time for biochemical stimuli. Spatial resolution is reflected in the precise application of different mechanical stimuli by utilizing the morphological differences of the suction channels. Using a high-speed camera, the response of single cells to different mechanical stimuli can be captured.

[0015] Beneficial effects of the present invention:

[0016] The present invention discloses a microfluidic chip for high-throughput loading of biological and mechanical signals and high-temporal-space resolution manipulation of single cells. It provides a biological sample loading system with functions such as gradient concentration generation, single-cell droplet encapsulation, precise droplet / cell manipulation, and microfluidic dynamics loading, thereby achieving high-throughput single-cell manipulation and non-destructive precise loading of biochemical and mechanical signals for single cells, for high-temporal-space resolution detection of single cells.

[0017] The present invention utilizes flow focusing theory and micro-droplet generation theory to achieve high-throughput single-cell droplet encapsulation, encapsulating single cells in an independent and stable external environment, realizing precise loading of biochemical signals, and ensuring the effectiveness and accuracy of single-cell manipulation; at the same time, it further utilizes micro-droplet retention areas and micro-suction arrays (including but not limited to straight suction channels, single-arc suction channels, multi-arc suction channels, etc.), through precise flow / pressure negative feedback control, to precisely control the loading of biochemical and mechanical signals in both time and space dimensions, thereby achieving high "time-space" resolution single-cell manipulation.

[0018] The present invention combines the advantages of droplet microfluidics such as miniaturization, compartmentalization, and parallelization to design a microfluidic chip system with functions such as biochemical signal gradient concentration generation, single-cell droplet encapsulation, precise droplet / cell manipulation, and microfluidic dynamics loading. It achieves non-destructive and precise loading of biochemical and mechanical signals to single cells and high "time-space" resolution detection of single cells. It is a feasible and scientifically important research work.

[0019] Because the microfluidic chip is colorless and transparent, it exhibits excellent compatibility with various optical and detection systems, making it ideal for applications such as flow cytometry, cell biology research, and biomedical testing. Based on negative feedback flow / pressure control, the system enables high-throughput single-cell droplet encapsulation and high-resolution spatiotemporal manipulation of single cells. This enables high-precision loading of biochemical and mechanical signals for qualitative and quantitative studies of cellular biochemical reactions, effectively improving analytical efficiency and measurement accuracy.

[0020] The present invention can obtain a microfluidic chip for single-cell high-throughput biological signal and mechanical signal loading and high-temporal and spatial resolution manipulation and an application method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a schematic structural diagram of a microfluidic chip for high-throughput single-cell biological signal and mechanical signal loading and high-temporal-space resolution manipulation of the present invention. 1 represents a glass substrate, 2 represents a complete PDMS structure, 3-1 represents an external phase liquid injection hole, 3-2 represents a cell buffer injection hole, 3-3 represents a cell culture fluid injection hole, 3-4 represents a pressure-driven hole No. 1, 3-5 represents a pressure-driven hole No. 2, 3-6 represents a waste liquid hole, 4 represents a cross droplet shearing opening, 5 represents a buffer channel, 6 represents a cell droplet retention area, 7-1 represents a straight suction channel, 7-2 represents a single-arc suction channel, 7-3 represents a multi-arc suction channel, 8 represents an external phase liquid channel, 9 represents a cell buffer channel, and 10 represents a cell culture fluid channel.

[0022] Figure 2 This is a top view of a microfluidic chip for high-throughput single-cell biological signal and mechanical signal loading and high-temporal-resolution manipulation of the present invention. 1 represents a glass substrate, 2 represents a complete PDMS structure, 3-1 represents an external phase liquid injection hole, 3-2 represents a cell buffer injection hole, 3-3 represents a cell culture fluid injection hole, 3-4 represents a No. 1 pressure-driven hole, 3-5 represents a No. 2 pressure-driven hole, 3-6 represents a waste liquid hole, 4 represents a cross droplet shearing opening, 5 represents a buffer channel, 6 represents a cell droplet retention area, 7-1 represents a straight suction channel, 7-2 represents a single-arc suction channel, 7-3 represents a multi-arc suction channel, 8 represents an external phase liquid channel, 9 represents a cell buffer channel, and 10 represents a cell culture fluid channel;

[0023] Figure 3 Schematic diagram of the encapsulation of a single cell encapsulation droplet in the present invention;

[0024] Figure 4 Schematic diagram of a single cell-encapsulated droplet passing through the suction channel in the present invention. DETAILED DESCRIPTION

[0025] Specific embodiment 1: This embodiment is a microfluidic chip for single-cell high-throughput biological signal and mechanical signal loading and high spatiotemporal resolution manipulation, including a substrate and a PDMS complete structure 2, wherein the PDMS complete structure 2 is arranged on the upper surface of the substrate, and the PDMS complete structure 2 is a rectangular open box structure, including an external phase liquid channel 8, a cell buffer channel 9, a cell culture fluid channel 10, a buffer channel 5, a cell droplet retention area 6 and a suction channel;

[0026] One end of the external phase liquid channel 8 is provided with an external phase liquid injection hole 3-1, one end of the cell buffer channel 9 is provided with a cell buffer injection hole 3-2, and one end of the cell culture fluid channel 10 is provided with a cell culture fluid injection hole 3-3; the other ends of the external phase liquid channel 8, the cell buffer channel 9 and the cell culture fluid channel 10 are connected to the water inlet of the buffer channel 5 through the cross droplet shear 4, and the water outlet of the buffer channel 5 is connected to the water inlet of the cell droplet retention area 6 through a pipeline; the head end of the cell droplet retention area 6 is provided with a plurality of pressure-driven holes, and the tail end of the cell droplet retention area 6 is provided with a waste liquid hole 3-6, and the sucking channel is vertically arranged in the middle section of the cell droplet retention area 6, and the two ends of the sucking channel are connected to the inner wall of the cell droplet retention area 6.

[0027] Specific embodiment 2: This embodiment differs from specific embodiment 1 in that the substrate is a glass substrate 1 .

[0028] The other steps are the same as those in the first embodiment.

[0029] Specific embodiment three: This embodiment differs from specific embodiment one or two in that: the external phase liquid channel 8 is a channel composed of two semicircular rings, and two water outlets are provided at the end of the channel.

[0030] The other steps are the same as those in the first or second embodiment.

[0031] Specific embodiment 4: The difference between this embodiment and specific embodiments 1 to 3 is that the cell buffer channel 9 is a channel composed of two semicircular rings, and two water outlets are provided at the end of the channel.

[0032] The other steps are the same as those in Specific Embodiments 1 to 3.

[0033] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the cell culture fluid channel 10 is a spiral channel.

[0034] The other steps are the same as those in Specific Embodiments 1 to 4.

[0035] Specific embodiment 6: The difference between this embodiment and specific embodiments 1 to 5 is that the buffer channel 5 is a pipeline composed of a plurality of U-shaped pipelines and provided with a water inlet and a water outlet.

[0036] The other steps are the same as those in Specific Embodiments 1 to 5.

[0037] Specific embodiment seven: The difference between this embodiment and any one of specific embodiments one to six is ​​that the cell droplet retention area 6 is provided with two pressure-driven holes, namely pressure-driven hole No. 1 3-4 and pressure-driven hole No. 2 3-5, and pressure-driven hole No. 1 3-4 and pressure-driven hole No. 2 3-5 are symmetrically arranged on both sides of the cell droplet retention area 6.

[0038] The other steps are the same as those in Specific Embodiments 1 to 6.

[0039] Specific embodiment eight: The difference between this embodiment and specific embodiments one to seven is that the sucking channel is composed of a plurality of sucking units, and the sucking unit is composed of a straight sucking channel 7-1, a single-arc sucking channel 7-2 and a multi-arc sucking channel 7-3.

[0040] The other steps are the same as those in Specific Embodiments 1 to 7.

[0041] Specific embodiment 9: This embodiment provides an application method of a microfluidic chip for high-throughput single-cell biological signal and mechanical signal loading and high-temporal and spatial resolution manipulation, which is carried out in the following steps:

[0042] Step S1: Connect the container containing silicone oil to the external phase liquid injection port 3-1, connect the container containing buffer to the cell buffer injection port 3-2, and connect the container containing cell culture fluid to the cell culture fluid injection port 3-3; at the same time, connect the air outlet of the pressure pump to the No. 1 pressure drive port 3-4 and the No. 2 pressure drive port 3-5, and connect the waste liquid port 3-6 to the water inlet of the waste liquid container;

[0043] Step S2: injecting silicone oil through the external phase liquid injection port 3-1, injecting a buffer through the cell buffer injection port 3-2, and injecting a cell culture fluid containing cells through the cell culture fluid injection port 3-3 by a pump, and adjusting the flow rate of the silicone oil to 100-3000 μL / min, the flow rate of the buffer to 10-1000 μL / min, and the flow rate of the cell culture fluid to 0.5-200 μL / min;

[0044] Step S3: Silicone oil, buffer solution and cell culture fluid converge at the cross droplet shearing opening 4 to form a single-cell encapsulated droplet; the single-cell encapsulated droplet enters the cell droplet retention area 6 through the buffer channel 5, and a pressure pump is used to apply a positive pressure of 0.2 to 8 kPa to the pressure driving hole 3-4 No. 1 and the pressure driving hole 3-5 No. 2, and a negative pressure of 0.2 to 8 kPa to the waste liquid hole 3-6, respectively, to control the single-cell encapsulated droplet to pass through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3, and finally flow out through the waste liquid hole 3-6 and be collected by the waste liquid container.

[0045] Specific embodiment ten: This embodiment differs from specific embodiment nine in that in step S3, the total number of single cell-encapsulated droplets passing through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3 is 80 to 150 per second.

[0046] The other steps are the same as those in the ninth embodiment.

[0047] The following examples are used to verify the beneficial effects of the present invention:

[0048] Example 1: A microfluidic chip for high-throughput biological signal and mechanical signal loading and high-temporal-spatial resolution manipulation of single cells, comprising a high-throughput single-cell wrapper and a high-temporal-spatial resolution single-cell controller. The high-throughput single-cell wrapper consists of a substrate, an external phase liquid channel 8, a cell buffer channel 9, a cell culture fluid channel 10, and a buffer channel 5. The high-temporal-spatial resolution single-cell controller consists of a cell droplet retention area 6, pressure drive holes 3-4 No. 1, pressure drive holes 3-5 No. 2, waste liquid holes 3-6, a straight suction channel 7-1, a single-arc suction channel 7-2, and a multi-arc suction channel 7-3. Structurally (e.g., Figure 1-2 As shown), the patterned polydimethylsiloxane (PDMS) complete structure 2 is a rectangular open box structure and is arranged on the upper surface of the substrate;

[0049] One end of the external phase liquid channel 8 is provided with an external phase liquid injection hole 3-1, one end of the cell buffer channel 9 is provided with a cell buffer injection hole 3-2, and one end of the cell culture fluid channel 10 is provided with a cell culture fluid injection hole 3-3; the other ends of the external phase liquid channel 8, the cell buffer channel 9 and the cell culture fluid channel 10 are connected to the water inlet of the buffer channel 5 through the cross droplet shear 4, and the water outlet of the buffer channel 5 is connected to the water inlet of the cell droplet retention area 6 through a pipeline; the head end of the cell droplet retention area 6 is provided with a plurality of pressure-driven holes, and the tail end of the cell droplet retention area 6 is provided with a waste liquid hole 3-6, and the sucking channel is vertically arranged in the middle section of the cell droplet retention area 6, and the two ends of the sucking channel are connected to the inner wall of the cell droplet retention area 6.

[0050] The substrate is a glass substrate 1.

[0051] The external phase liquid channel 8 is a channel composed of two semicircular rings, and two water outlets are provided at the end of the channel.

[0052] The cell buffer channel 9 is a channel composed of two semicircular rings, and two water outlets are provided at the end of the channel.

[0053] The cell culture fluid channel 10 is a spiral channel.

[0054] The buffer channel 5 is a pipeline composed of a plurality of U-shaped pipelines and provided with a water inlet and a water outlet.

[0055] The cell droplet retention area 6 is provided with two pressure-driven holes, namely pressure-driven hole No. 1 3-4 and pressure-driven hole No. 2 3-5, and pressure-driven hole No. 1 3-4 and pressure-driven hole No. 2 3-5 are symmetrically arranged on both sides of the cell droplet retention area 6.

[0056] The sucking channel is composed of a plurality of sucking units, and the sucking units are composed of a straight sucking channel 7-1, a single-arc sucking channel 7-2 and a multi-arc sucking channel 7-3.

[0057] The PDMS complete structure 2 is prepared using micro-electromechanical systems (MEMS) technology, and the specific preparation steps are as follows:

[0058] (a) Cleaning the glass substrate: A glass sheet with a length of 70 mm and a width of 50 mm was used as the substrate for making the channel mold. The glass was cleaned in water using detergent and then anhydrous ethanol (to remove organic impurities). The glass was then rinsed repeatedly with deionized water. After rinsing, it was blown dry with nitrogen and placed in a constant temperature drying oven at 80°C for approximately 10 minutes to remove any residual moisture on the glass surface.

[0059] (b) Attaching the channel mold: Remove the glass sheet from the drying oven, wait for the glass sheet to cool, and then use shadowless adhesive to attach the channel mold (brass plate) to the appropriate position on the glass surface;

[0060] (c) Light curing: Rapidly irradiate the bonding area between the flavonoid plate and the glass substrate with a UV light source for 5 seconds to quickly cure the adhesive. Immediately clean the excess adhesive with acetone.

[0061] (d) Casting PDMS: Mix PDMS and curing agent in a mass ratio of 10:1, stir evenly with a glass rod, place in a vacuum kettle and evacuate for 30 minutes, then silanize the PMMA channel structure. The method is as follows: Place the channel mold wrapped in tin foil in a vacuum pump, inject 100 microliters of dimethyldichlorosilane into the vacuum pump with a pipette, evacuate for 3 to 5 minutes, and let it stand for 15 to 20 minutes; the purpose is to deposit a layer of silane on the surface of the channel mold, which helps to prevent PDMS from sticking to the channel mold and easily remove the PDMS channel from the mold. Finally, cast PDMS on the channel mold after silane treatment. Vacuum for another 15 to 30 minutes to ensure that there are no bubbles;

[0062] (e) Curing and molding of PDMS channels: The evacuated PDMS was placed flat in a constant temperature drying oven and cured at 65-115°C for 25 min-1 h. After curing, the PDMS was slowly peeled off from the mold and cut into regular shapes with a blade. Holes were then punched according to the designed structure.

[0063] (f) Bonding of the PDMS channel: Place the PDMS channel and the cleaned glass substrate side by side in the chamber of a plasma machine and perform plasma treatment according to the corresponding steps of the plasma machine. Then take it out and place the PDMS channel on the appropriate position of the glass substrate. Press it firmly and place it on a hot plate at a temperature of 75-90°C for 1-2 hours to ensure a strong bond between the PDMS channel and the glass substrate.

[0064] The above-mentioned application method of the microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation is carried out according to the following steps:

[0065] Step S1: Connect the container containing silicone oil to the external phase liquid injection port 3-1, connect the container containing buffer to the cell buffer injection port 3-2, and connect the container containing cell culture fluid to the cell culture fluid injection port 3-3; at the same time, connect the air outlet of the pressure pump to the No. 1 pressure drive port 3-4 and the No. 2 pressure drive port 3-5, and connect the waste liquid port 3-6 to the water inlet of the waste liquid container;

[0066] Step S2: injecting silicone oil through the external phase liquid injection port 3-1, injecting a buffer through the cell buffer injection port 3-2, and injecting a cell culture fluid containing cells through the cell culture fluid injection port 3-3 by a pump, and adjusting the flow rate of silicone oil to 2100 μL / min, the flow rate of buffer to 90 μL / min, and the flow rate of cell culture fluid to 4 μL / min;

[0067] Step S3: Silicone oil, buffer solution and cell culture medium converge at the cross droplet shearing opening 4 to form a single-cell encapsulated droplet; the single-cell encapsulated droplet enters the cell droplet retention area 6 through the buffer channel 5, and a pressure pump is used to apply a positive pressure of 4 kPa to the pressure drive hole 3-4 No. 1 and the pressure drive hole 3-5 No. 2, and a negative pressure of 4 kPa to the waste liquid hole 3-6, respectively, to control the single-cell encapsulated droplet to pass through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3, and finally flow out through the waste liquid hole 3-6 and be collected by the waste liquid container.

[0068] In step S3, the total number of single cell-encapsulated droplets passing through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3 is 80 to 150 per second.

[0069] Testing process:

[0070] After silicone oil, buffer, and cell culture medium converge at the cross-droplet shearing opening 4, the three elements form single-cell encapsulated droplets under the combined effects of shear force and flow rate. Once generated, the single-cell encapsulated droplets flow through the serpentine buffer channel 5, allowing biochemical stimulation to fully take effect. Subsequently, as the droplets flow into the micro-droplet retention zone 6, changes in cell morphology are observed using a high-speed camera.

[0071] Next, by applying positive pressure to pressure-driven hole No. 1 3-4 and pressure-driven hole No. 2 3-5 and negative pressure (vacuum) to the waste liquid hole 3-6, the single-cell-encapsulated droplet is precisely controlled to pass through the straight suction channel 7-1, the single-arc suction channel 7-2 or the multi-arc suction channel 7-3, thereby applying specific mechanical stimulation (physical pressure) to the single-cell-encapsulated droplet; in this process, the cell morphological changes of the single-cell-encapsulated droplet as it passes through each channel are observed by a high-speed camera.

[0072] In summary, during the entire detection process, a high-speed camera is used to capture the flow of single-cell encapsulated droplets into the micro-droplet retention area 6 and the cell morphology as it passes through each suction channel (e.g. Figure 4 As shown in the figure, the responses of the cells to biochemical and mechanical stimuli were recorded and analyzed using flow cytometry.

Claims

1. A microfluidic chip for high-throughput loading of biological and mechanical signals and high spatiotemporal resolution manipulation of single cells, characterized by The microfluidic chip comprises a substrate and a PDMS complete structure (2), wherein the PDMS complete structure (2) is arranged on the upper surface of the substrate, and the PDMS complete structure (2) is a rectangular open box structure, comprising an external phase liquid channel (8), a cell buffer channel (9), a cell culture fluid channel (10), a buffer channel (5), a cell droplet retention area (6), and a suction channel; One end of the external phase liquid channel (8) is provided with an external phase liquid injection hole (3-1), one end of the cell buffer channel (9) is provided with a cell buffer injection hole (3-2), and one end of the cell culture fluid channel (10) is provided with a cell culture fluid injection hole (3-3); the other ends of the external phase liquid channel (8), the cell buffer channel (9) and the cell culture fluid channel (10) are all connected to the water inlet of the buffer channel (5) through the cross droplet shearing cut (4), and the water outlet of the buffer channel (5) is connected to the water inlet of the cell droplet retention area (6) through a pipeline; the head end of the cell droplet retention area (6) is provided with a plurality of pressure-driven holes, and the tail end of the cell droplet retention area (6) is provided with a waste liquid hole (3-6); the sucking channel is vertically arranged in the middle section of the cell droplet retention area (6), and the two ends of the sucking channel are connected to the inner wall of the cell droplet retention area (6); The cell droplet retention area (6) is provided with two pressure-driven holes, namely pressure-driven hole No. 1 (3-4) and pressure-driven hole No. 2 (3-5), and pressure-driven hole No. 1 (3-4) and pressure-driven hole No. 2 (3-5) are symmetrically arranged on both sides of the cell droplet retention area (6), and pressure-driven hole No. 1 (3-4) and pressure-driven hole No. 2 (3-5) are both connected to the air outlet of the pressure pump; The sucking channel is composed of a plurality of sucking units, and the sucking units are composed of a straight sucking channel (7-1), a single-arc sucking channel (7-2) and a multi-arc sucking channel (7-3).

2. A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 1, characterized in that The substrate is a glass substrate (1).

3. A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 1, characterized in that The external phase liquid channel (8) is a channel consisting of two semicircular rings, and two water outlets are provided at the end of the channel.

4. A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 1, characterized in that The cell buffer channel (9) is a channel consisting of two semicircular rings, and two water outlets are provided at the end of the channel.

5. A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 1, characterized in that The cell culture fluid channel (10) is a spiral channel.

6. A microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 1, characterized in that The buffer channel (5) is a pipeline composed of a plurality of U-shaped pipelines and provided with a water inlet and a water outlet.

7. The method for using a microfluidic chip for high-throughput loading of biological and mechanical signals and high spatiotemporal resolution manipulation of single cells according to any one of claims 1 to 6, characterized in that The application method is carried out in the following steps: Step S1: Connect the container containing silicone oil to the external phase liquid injection hole (3-1), connect the container containing buffer to the cell buffer injection hole (3-2), and connect the container containing cell culture fluid to the cell culture fluid injection hole (3-3); at the same time, connect the air outlet of the pressure pump to the No. 1 pressure drive hole (3-4) and the No. 2 pressure drive hole (3-5), and connect the waste liquid hole (3-6) to the water inlet of the waste liquid container; Step S2: injecting silicone oil through the external phase liquid injection hole (3-1), injecting buffer through the cell buffer injection hole (3-2), and injecting cell culture fluid containing cells through the cell culture fluid injection hole (3-3) by using a pump, and adjusting the flow rate of silicone oil to 100-3000 μL / min, the flow rate of buffer to 10-1000 μL / min, and the flow rate of cell culture fluid to 0.5-200 μL / min; Step S3: Silicone oil, buffer solution and cell culture medium merge at the cross droplet shear cut (4) to form a single-cell encapsulated droplet; the single-cell encapsulated droplet enters the cell droplet retention area (6) through the buffer channel (5), and a pressure pump is used to apply a positive pressure of 0.2~8 kPa to the pressure drive hole No. 1 (3-4) and the pressure drive hole No. 2 (3-5) and a negative pressure of 0.2~8 kPa to the waste liquid hole (3-6), respectively, to control the single-cell encapsulated droplet to pass through the straight suction channel (7-1), the single arc suction channel (7-2) or the multi-arc suction channel (7-3), and finally flow out through the waste liquid hole (3-6) and be collected by the waste liquid container.

8. The method for applying a microfluidic chip for high-throughput single-cell biological and mechanical signal loading and high-temporal and spatial resolution manipulation according to claim 7, characterized in that In step S3, the total number of single-cell encapsulated droplets passing through the straight suction channel (7-1), the single-arc suction channel (7-2) or the multi-arc suction channel (7-3) is 80 to 150 per second.

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