A microfluidic chip, kit and device for tightly arranging solid gel microspheres

Abstract

The utility model relates to the micro -fluidic field especially relates to a micro -fluidic chip, kit and device that make solid gel microspheres compact arrangement. The utility model discloses through increasing the liquid discharge channel on one side or both sides of solid gel microspheres phase, effectively reduces even eliminates the buffer solution gap between solid gel microspheres, makes solid gel microspheres produce one -to -one arrangement effect, then is dispersed by cell phase, is sheared by oil phase and forms the water -in -oil droplet that only wraps one solid gel microspheres, to improve the capture efficiency of cell. It is found in the experiment that the micro -fluidic chip containing branch channel improves 20% of the proportion of wrapping single gel microspheres compared with the micro -fluidic chip not containing branch channel. Therefore, the micro -fluidic chip provided by the utility model can improve the use rate of cell sample.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, and in particular to a microfluidic chip, reagent kit, and device for tightly arranging solid gel microspheres. Background Technology

[0002] Selective gene expression is a common phenomenon within different tissues of an individual and among different cells within the same tissue, characterizing different cellular functions. Single-cell sequencing technology utilizes solid gel microspheres modified with differentially encoded molecules to encapsulate individual cells into independent droplets. The differentially encoded molecules modified on the solid gel microspheres capture the selectively expressed mRNAs of the cells. After reverse transcription, amplification, fragmentation, ligation of sequencing tags, and sequencing, selective expression data of the cells are finally obtained, which is of great significance for studying and analyzing cellular expression heterogeneity.

[0003] Single-cell sequencing utilizes elastically deformable solid gel microspheres as carriers of differentially encoded molecules. These microspheres interact with the cell sample within a microfluidic chip, passing through cross-shaped or T-shaped channels and encountering the dispersed oil phase. The microspheres are then sheared by the oil phase, forming monodisperse water-in-oil droplets. Within these independent droplet environments, the solid gel microspheres lyse cells and capture mRNA. While cells follow a Poisson distribution during droplet dispersion, the elastically deformable solid gel microspheres can achieve a denser arrangement within the microfluidic chip channels, exceeding the Poisson distribution during droplet dispersion. Therefore, cell utilization is the product of the proportion of droplets containing only a single cell and the proportion of droplets containing only a single solid gel microsphere. However, since cells consistently follow a Poisson distribution under these conditions, improving cell utilization requires increasing the proportion of droplets containing only a single solid gel microsphere.

[0004] However, under the current microfluidic chip design and fabrication conditions, there will still be gaps filled with buffer solution between solid gel microspheres. When these gaps are sheared by the oil phase, they will form water-in-oil droplets that do not contain solid gel microspheres. This will reduce the single-encapsulation rate of solid gel microspheres when forming monodisperse droplets, and thus reduce the cell capture efficiency.

[0005] Existing single-cell droplet generation systems have not improved this parameter accordingly, which is a problem that urgently needs to be solved in this field. Utility Model Content

[0006] In view of this, the present invention provides a microfluidic chip, reagent kit, and device for achieving close packing of solid gel microspheres. The present invention reduces or even eliminates the buffer gaps between solid gel microspheres by adding branch channels to one or both sides of the original solid gel microsphere phase channels, thereby achieving close packing of solid gel microspheres and ultimately increasing the single-packing yield of solid gel microspheres.

[0007] To achieve the above-mentioned objectives, this utility model provides the following technical solution:

[0008] In the first aspect, this utility model provides a microfluidic chip, including a main channel, a flow channel for the solid gel microsphere phase, a flow channel for the cell phase, a flow channel for the oil phase, an injection port, and a collection port;

[0009] The main channel is connected to the flow channels of the solid gel microsphere phase, the flow channels of the cell phase, and the flow channels of the oil phase, respectively.

[0010] The flow channel of the cell phase is perpendicularly connected to the main channel through the first junction.

[0011] The flow channel of the oil phase is perpendicularly connected to the main channel through a second junction.

[0012] The flow channels of the oil phase and the flow channels of the solid gel microsphere phase are located on both sides of the flow channels of the cell phase.

[0013] The microfluidic chip also includes a buffer discharge channel.

[0014] In some specific embodiments of this utility model, the buffer discharge channel includes a main buffer discharge channel and a buffer discharge branch channel.

[0015] In some specific embodiments of this utility model, one side of the buffer discharge branch channel is connected to the main buffer discharge channel; the other side of the buffer discharge branch channel is perpendicularly connected to the main channel through a third junction.

[0016] In some specific embodiments of this utility model, the buffer solution discharge branch channel is disposed between the flow channel of the solid gel microsphere phase and the flow channel of the cell phase;

[0017] and / or

[0018] The buffer solution discharge branch channel is located between the injection ports of the solid gel microsphere phase.

[0019] In some specific embodiments of this invention, the cross-sectional width of the buffer solution discharge branch channel is smaller than the diameter of the solid gel microspheres.

[0020] Secondly, this invention also provides a reagent kit, including the microfluidic chip and acceptable reagents.

[0021] Thirdly, this utility model also provides an apparatus, including the microfluidic chip.

[0022] This invention effectively reduces or even eliminates the buffer gaps between solid gel microspheres by adding drainage channels on one or both sides of the solid gel microsphere phase, resulting in a one-to-one arrangement of solid gel microspheres. Subsequently, the microspheres are dispersed by the cell phase and sheared by the oil phase to form water-in-oil droplets that encapsulate only one solid gel microsphere, thereby improving cell capture efficiency.

[0023] Experiments revealed that microfluidic chips containing branch channels increased the proportion of encapsulated individual gel microspheres by 20% compared to microfluidic chips without branch channels. Therefore, the microfluidic chip provided by this invention can improve the utilization rate of cell samples. Attached Figure Description

[0024] To more clearly illustrate the technical solutions in the embodiments of this utility model or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0025] Figure 1 This invention illustrates a microfluidic chip channel design that enables the tight arrangement of solid gel microspheres; wherein, channel C10 is the flow channel of the solid gel microsphere phase, channels C12, C13, and C14 are the buffer solution discharge channels of the solid gel microsphere phase, channel C11 is the connection port between the solid gel microsphere phase flow channel and the solid gel microsphere phase buffer solution discharge channel, and channels 1-6, 1-7, 1-8, and 1-9 represent the flow direction of the solid gel microsphere buffer solution;

[0026] Figure 2 This invention illustrates the preparation of monodisperse droplets using a single-cell microfluidic system; wherein, (A) shows the flow state of each phase fluid at an earlier moment during the generation of a single encapsulated droplet in the microfluidic chip; and (B) shows the flow state of each phase fluid at a later moment during the generation of a single encapsulated droplet in the microfluidic chip.

[0027] Among them, channel C1 is the flow channel of the solid gel microsphere phase, and direction 1-1 is the flow direction of the solid gel microsphere phase in its channel; channel C2 is the flow channel of the cell phase, and direction 1-2 is the flow direction of the cell phase in its channel; channel C3 is the junction and mixing point of the solid gel microsphere phase and the cell phase; channel C4 is the mixed flow channel of the solid gel microsphere phase and the cell phase, and direction 1-3 is the flow direction of the mixed phase of the solid gel microsphere phase and the cell phase; channel C5 is the flow channel of the oil phase as the continuous phase, and direction 1-4 is the flow direction of the oil phase; channel C6 is the junction and shear dispersion point of the mixed phase of the solid gel microsphere phase and the cell phase and the oil phase, where the mixed phase meets and is sheared by the oil phase to form water-in-oil droplets; channel C7 is the flow channel of the oil phase shearing the mixed phase to form droplets, and direction 1-4 is the flow direction of the water-in-oil emulsion; G1, G2, G3 and G4 are four solid gel microspheres, among which... Microsphere G1 is a solid gel microsphere that enters the mixing flow channel C4 slightly earlier in the solid gel microsphere flow channel C1, and microsphere G2 is a solid gel microsphere that enters the mixing flow channel C4 slightly later in the solid gel microsphere flow channel C4. During the process of oil phase shearing aqueous phase to form droplets, solid gel microsphere G1 first enters the junction of channel C3, followed by solid gel microsphere G2 at a distance L1. The length L1 is the distance between microspheres G1 and G2 in channel C1, and the space between the two microspheres is filled with the buffer solution of the solid gel microsphere phase. The length L2 is the distance between microspheres G1 and G2 when they flow into channel C4 after being dispersed by the cell phase. The space between the two microspheres is filled with the mixture of the solid gel microsphere phase buffer solution and the cell phase. When the solid gel microspheres flow in channel C1 with a distance of 0, the distance between them when they are dispersed by the cell phase and flow into channel C4 in pairs is L3.

[0028] Figure 3 The optimized single-cell microfluidic system is used to prepare monodisperse droplets; (A) shows the flow state at an earlier time during the generation of monodisperse droplets in the microfluidic system; (B) shows the flow state at a later time during the generation of monodisperse droplets in the microfluidic system.

[0029] Wherein, C8 is a "T"-shaped drainage structure, and C9 is a drainage channel; G5 is a solid gel microsphere that flows through the C8 "T"-shaped drainage structure and enters the mixing flow channel slightly earlier in the solid gel microsphere flow channel, and G6 is a solid gel microsphere that flows through the C8 "T"-shaped drainage structure and enters the mixing flow channel slightly later in the solid gel microsphere flow channel; L4 is the distance between G5 and G6 solid gel microspheres when flowing in the gel microsphere channel, and L5 is the distance between G5 and G6 solid gel microspheres when flowing in the mixing channel; 1-5 represent the flow direction of drainage through the drainage channel;

[0030] Figure 4The experimental results of Example 3 are shown; wherein, (A) shows the experimental results of a microfluidic chip with branch channel design; and (B) shows the experimental results of a microfluidic chip without branch channel design. Detailed Implementation

[0031] This invention discloses a microfluidic chip, reagent kit, and device for tightly packed solid gel microspheres. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired results. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0032] This invention utilizes a unique design of branch channels on both sides of the main channel of solid gel microspheres to drain the buffer solution present between the microspheres, thereby eliminating the gaps between them. This improves the single-encapsulation rate of droplets formed during continuous phase shearing in the microfluidic droplet generation chip. The method of improving the single-encapsulation rate of solid gel microspheres through its elastic deformation properties is a key aspect that needs to be protected. However, this solution should not be limited to the field of gel beads, but can be extended to solid microspheres and similar microspheres with solid properties. Furthermore, this solution should not be limited to the application scenario of gel microspheres paired with cells, but can also be applied to applications of gel microspheres with other gel microspheres, bacteria, etc., as well as applications that do not require pairing but only require improving the single-encapsulation rate.

[0033] In summary, this utility model mainly protects the method of reducing or even eliminating the gaps between solid gel beads by utilizing branch channels. The applicable scenarios should not be limited to the single-cell field, nor should the objects paired with it be limited.

[0034] The raw materials and reagents used in the microfluidic chip, reagent kit, and device that enable the close arrangement of solid gel microspheres provided by this invention are all commercially available.

[0035] The present invention will be further illustrated below with reference to the embodiments:

[0036] Example 1: Structural Description of a Microfluidic Chip Channel Design for Closely Aligned Solid Gel Microspheres

[0037] like Figure 1This design presents a branch channel drainage scheme for gel microspheres. The white C10 channel is the flow channel for the solid gel microsphere phase; C12, C13, and C14 are buffer solution drainage channels for the solid gel microsphere phase; C11 is the connection point between the solid gel microsphere phase flow channels and the buffer solution drainage channels; and 1-6, 1-7, 1-8, and 1-9 represent the flow directions of the solid gel microsphere buffer solution. Solid gel microspheres flow into the microfluidic chip along the main channel C10 in direction 1-6. When buffer solution gaps exist between microspheres, the buffer solution flows from the junction of the branch channel and the main channel C11 along direction 1-7 into the branch channel C12. Multiple branch channels converge into channel C14 via channel C13 along direction 1-8, and the buffer solution flowing into channel C14 flows out of the microfluidic chip through direction 1-9. This branch channel array can reduce and eliminate long gaps between solid gel microspheres, achieving a denser packing of solid gel microspheres and thus improving the encapsulation rate of individual solid gel microspheres during droplet formation. The cross-sectional design of the branch channels should not be limited to being the same as the main channel, nor should it be limited to being a long rectangle. Other methods of discharging liquid phase through branch channels with different cross-sectional shapes and sizes should be included in this patent. At the same time, the white C10 channel and the gray C12-C14 channel can be set to different channel heights during the design. C10 is a height that matches the diameter of the solid gel microspheres, and C12-C14 is a height that is smaller than the diameter of the solid gel microspheres, so as to prevent leakage from the C12-C14 channel caused by the elasticity of the solid gel.

[0038] Example 2: Explanation of the principle of the drainage channel in this design

[0039] like Figure 2 For the unimproved microfluidic single-cell droplet fabrication chip design and the process of preparing single-encapsulated droplets for microfluidic single-cell droplet fabrication chips, the optimal preparation result should be that each droplet encapsulates a solid gel microsphere and a cell.

[0040] Figure 2 Neutron diagram A shows the flow states of each phase fluid at an earlier moment during the generation of a single encapsulated droplet in this microfluidic chip. Figure 2Neutron diagram B shows the flow state of each phase fluid at a later moment during the generation of a single encapsulated droplet in this microfluidic chip. Among them, channel C1 is the flow channel of the solid gel microsphere phase, and direction 1-1 is the flow direction of the solid gel microsphere phase in its channel; channel C2 is the flow channel of the cell phase, and direction 1-2 is the flow direction of the cell phase in its channel; channel C3 is the junction and mixing point of the solid gel microsphere phase and the cell phase; channel C4 is the mixed flow channel of the solid gel microsphere phase and the cell phase, and direction 1-3 is the flow direction of the mixed phase of the solid gel microsphere phase and the cell phase; channel C5 is the flow channel of the oil phase as the continuous phase, and direction 1-4 is the flow direction of the oil phase; channel C6 is the junction and shear dispersion point of the mixed phase of the solid gel microsphere phase and the cell phase and the oil phase, where the mixed phase meets and is sheared by the oil phase to form water-in-oil droplets; channel C7 is the flow channel of the oil phase shearing the mixed phase to form droplets, and direction 1-4 is the flow direction of the water-in-oil emulsion; G1, G2, G3 and G4 are four solid gel microspheres, among which... Microsphere G1 is a solid gel microsphere that enters the mixing flow channel C4 slightly earlier in the solid gel microsphere flow channel C1, and microsphere G2 is a solid gel microsphere that enters the mixing flow channel C4 slightly later in the solid gel microsphere flow channel. During the process of oil phase shearing aqueous phase to form droplets, solid gel microsphere G1 first enters the junction of channel C3, followed by solid gel microsphere G2 at a distance L1. The length L1 is the distance between microspheres G1 and G2 in channel C1. The space between the two microspheres is filled with the buffer solution of the solid gel microsphere phase. The length L2 is the distance between microspheres G1 and G2 when they flow into channel C4 after being dispersed by the cell phase. The space between the two microspheres is filled with the mixture of solid gel microsphere phase buffer solution and cell phase. When the solid gel microspheres flow in channel C1 with a distance of 0, the distance between them when they are dispersed by the cell phase and flow into channel C4 in pairs is L3.

[0041] When preparing monodisperse single-cell droplets, the solid gel microsphere phase flows into the microfluidic chip from the C1 channel in the 1-1 direction. The gel microspheres exist in two flow states in the C1 channel: one is the flow state where the distance between the microspheres is 0, and the other is the flow state where there is a certain distance L1 between the microspheres. Meanwhile, the cell phase flows into the microfluidic chip from the C2 channel in the 1-2 direction. The solid gel microsphere phase and the cell phase meet at the C3 "T"-shaped channel. The closely packed solid gel microspheres are dispersed and mixed by the cell phase. The solid gel microspheres flow into the C4 channel with a certain L2 or L3 spacing and a 1-3 flow direction, serving as the dispersion phase for droplet formation. The continuous oil phase flows into the microfluidic chip from the C5 channel in a 1-4 direction, meeting the mixture of the dispersed solid gel microspheres and cell phase at the C6 "T"-shaped channel, dispersing the mixture into independent droplets. Each effective target droplet should contain one cell and one solid gel microsphere. The differentially encoded molecules attached to the microspheres capture the mRNA in the lysed cells for subsequent single-cell reverse transcription, amplification, ligation of sequencing adapters, and sequencing. When a pair of solid gel microspheres flows in the C1 channel without spacing, the pair of microspheres (e.g., ...) will... Figure 2 -After G3 and G4 in -A enter the C4 channel, they are dispersed by the cellular phase and separated by a distance L3, which is filled with the cellular phase; when a pair of solid gel microspheres flows in the C1 channel, with a distance L1 between them, the space between them is filled by the buffer solution of the solid gel microsphere phase, and the pair of microspheres (e.g., G3 and G4 in -A) are separated by a distance L3, which is filled with the cellular phase. Figure 2 In channels -A and 1-B, G1 and G2, upon entering channel C4, are dispersed by the cellular phase and spaced apart by a distance L2. This distance is filled with a buffer solution consisting of the cellular phase and the solid gel microsphere phase. When the solid gel microspheres flow into the C6 "T"-shaped channel at a fixed spacing of L3, they are stably sheared into droplets by the continuous oil phase, with each droplet encapsulating only one solid gel microsphere. However, if the microspheres already have a gap L1 in channel C1, they will be dispersed by the cellular phase to a larger spacing L2 when flowing into channel C4. Upon entering the C6 "T"-shaped channel, they will be stably sheared into droplets by the continuous oil phase, but due to the larger spacing between the microspheres, droplets without encapsulated microspheres will form, which are non-target droplets in single-cell sequencing. Therefore, stabilizing the spacing between microspheres is a method to improve the microsphere encapsulation rate.

[0042] Example 3: The process of forming single-cell solid gel microspheres using the optimized single-cell microfluidic system.

[0043] like Figure 3The process of forming single-cell solid gel microspheres in an optimized single-cell microfluidic system is illustrated. Subfigure A shows the flow state at an earlier moment during the formation of monodisperse droplets in the microfluidic system, and subfigure B shows the flow state at a later moment during the formation of monodisperse droplets in the microfluidic system. Compared to the unoptimized microfluidic chip design, a C8 "T"-shaped drainage structure and a C9 drainage channel were added. In the figures, G5 represents a solid gel microsphere that flows through the C8 "T"-shaped drainage structure and enters the mixing flow channel slightly earlier in the solid gel microsphere flow channel, and G6 represents a solid gel microsphere that flows through the C8 "T"-shaped drainage structure and enters the mixing flow channel slightly later in the solid gel microsphere flow channel; L4 represents the distance between G5 and G6 solid gel microspheres when flowing in the gel microsphere channel, and L5 represents the distance between G5 and G6 solid gel microspheres when flowing in the mixing channel; 1-5 represent the flow direction of the drainage channel.

[0044] When solid gel microspheres with an L4 spacing flow in the channel, G5 first passes through the C8 "T"-shaped drainage structure. Since the C9 drainage channel is smaller than the solid gel microspheres, the gel microspheres will not enter the C9 channel. However, the buffer between solid gel microspheres G5 and G6 will flow into the C9 channel through the C8 structure under pressure and be discharged from the solid gel microsphere phase channel in the 1-5 direction, reducing or even eliminating the buffer between solid gel microspheres to achieve a close packing effect, as shown in sub-figure B where the spacing between solid gel microspheres G5 and G6 is reduced to 0. When the solid gel microspheres flow to the mixing channel, they are dispersed by the cell phase to reach an L5 spacing, which is filled with the cell phase buffer. Finally, they are sheared by the continuous oil phase to form droplets. Compared with the design without branch channels, this can increase the proportion of single gel microspheres by 10%.

[0045] Example 4

[0046] Table 1 presents the statistical results of "water-in-oil" droplet preparation using microfluidic chips with and without branch channels within the same batch of solid gel microspheres. This demonstrates the optimization effect of chip optimization on the preparation of single-layer solid gel microsphere droplets. During the preparation process, the aqueous phase is sheared by the continuous oil phase, resulting in droplets containing no gel microspheres, one gel microsphere, and multiple gel microspheres. Droplets containing only one solid gel microsphere are desired. Experiments show that the branch channel design can increase the proportion of droplets containing a single gel microsphere by 20%.

[0047] Table 1. Experimental Comparison Results

[0048] 1 Experiments were conducted on microfluidic chips with branch channel designs. Figure 4 -A) (90.8%)519 (7.1%)41 (2.1%)11 571 2 Experiments were conducted using microfluidic chips without branch channel designs. Figure 4 -B) (67.1%)353 (32.5%)171 (0.4%)2 526

[0049] The above description is only a preferred embodiment of the present utility model. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present utility model, and these improvements and modifications should also be considered within the protection scope of the present utility model.

Claims

1. A microfluidic chip, characterized by, It includes a main channel, a flow channel for the solid gel microsphere phase, a flow channel for the cell phase, a flow channel for the oil phase, an injection port, and a collection port; The main channel is connected to the flow channels of the solid gel microsphere phase, the flow channels of the cell phase, and the flow channels of the oil phase, respectively. The flow channel of the cell phase is perpendicularly connected to the main channel through the first junction. The flow channel of the oil phase is perpendicularly connected to the main channel through a second junction. The flow channels of the oil phase and the flow channels of the solid gel microsphere phase are located on both sides of the flow channels of the cell phase. The microfluidic chip also includes a buffer discharge channel.

2. The microfluidic chip of claim 1, wherein, The buffer discharge channel includes a main buffer discharge channel and a buffer discharge branch channel.

3. The microfluidic chip as described in claim 2, characterized in that, One side of the buffer solution discharge branch channel is connected to the main buffer solution discharge channel; the other side of the buffer solution discharge branch channel is perpendicularly connected to the main channel through a third junction.

4. The microfluidic chip of claim 3, wherein, The buffer solution discharge branch channel is disposed between the flow channels of the solid gel microsphere phase and the flow channels of the cell phase; and / or The buffer solution discharge branch channel is located between the injection ports of the solid gel microsphere phase.

5. The microfluidic chip of claim 4, wherein, The cross-sectional width of the buffer solution discharge branch channel is smaller than the diameter of the solid gel microspheres.

6. A kit characterized in that, Includes the microfluidic chip as described in any one of claims 1 to 5 and acceptable reagents.

7. Device, characterized in that Including the microfluidic chip as described in any one of claims 1 to 5.

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