Micro-fluidic chip structure for generating PCR (Polymerase Chain Reaction) liquid drops

Abstract

The utility model relates to a micro-fluidic chip structure for generating PCR (Polymerase Chain Reaction) liquid drops, which comprises an oil phase injection port, a water phase injection port, a liquid drop size adjusting flow channel and a liquid drop collecting chamber with a supporting structure, the oil phase injection port is communicated with the liquid drop size adjusting flow channel through two channels, and the water phase injection port is communicated with the liquid drop size adjusting flow channel through the supporting structure. The oil phase injection port is communicated with the water phase injection port through a channel, the water phase injection port is communicated with the liquid drop size adjusting flow channel through a channel, the liquid drop size adjusting flow channel is communicated with the liquid drop collecting chamber, the oil phase injection port is used for injecting silicone oil, the water phase injection port is used for injecting a mixed solution of a PCR sample and a reaction reagent, and the liquid drop size adjusting flow channel is used for adjusting the size of generated water-in-oil liquid drops. And the liquid drop collecting chamber is used for collecting and containing the liquid drops. Compared with the prior art, the reaction device has the advantages that the influence on a reaction system in a liquid generation process can be reduced, and the uniformity and the stability of generated liquid drops are ensured.

Description

TECHNICAL FIELD

[0001] The utility model relates to PCR droplet generation technical field, especially a kind of microfluidic chip structure for generating PCR droplet. BACKGROUND

[0002] Polymerase Chain Reaction (PCR) is an important in vitro nucleic acid amplification technique, since the mid-1980s, with its high specificity, sensitivity and easy operation, in clinical diagnosis and biotechnology and other fields play an important role, it can rapidly amplify target DNA fragments by millions of times. With the progress of technology, Digital Polymerase Chain Reaction (dPCR) came into being, further solved the limitations of traditional PCR in gene copy number and mutation analysis. dPCR by distributing samples to tens of thousands of independent reaction units, each unit may contain zero or one DNA template, realize single molecule level PCR amplification and absolute quantification. This method provides a precise nucleic acid quantification method independent of cycle threshold by counting fluorescent signals.

[0003] At present, dPCR equipment mainly uses micropore or droplet technology to divide samples and carry out single molecule PCR reaction. dPCR technology divides samples into thousands of nanoliter droplets, each droplet contains zero or more target molecules, and determines the presence or absence of target sequences through PCR amplification and fluorescence detection. In a sense, droplet generation is crucial for PCR analysis.

[0004] The existing material selection for the structure for droplet generation, patent CN 209619360 U proposes a structure with one unit or two or more identical units, which is used to collect the generated droplets and carry out PCR amplification reaction, and the polycarbonate (PC) plastic or cycloolefin copolymer (COC) material of the structure is injection molded; Patent CN103451088A proposes a structure with multiple injection ports to realize digital PCR reaction generation. However, the above schemes do not consider the influence of droplet size difference on digital PCR structure recognition, which is not conducive to reliable generation of stable droplets, and it is difficult to ensure the accuracy of subsequent droplet recognition. UTILITY MODEL CONTENT

[0005] The utility model aims at overcoming the defects of the prior art and provides a microfluidic chip structure for generating PCR droplets, which can improve the uniformity and stability of the generated droplets.

[0006] The utility model discloses a micro -fluidic chip structure for generating PCR droplet, including oil phase injection entrance, water phase injection entrance, droplet size adjustment flow passage and with support structure's droplet collection chamber, oil phase injection entrance is communicated to droplet size adjustment flow passage through two -way channel, water phase injection entrance is communicated to droplet size adjustment flow passage through a channel, droplet size adjustment flow passage is linked together with droplet collection chamber, oil phase injection entrance is used for injecting silicon oil, water phase injection entrance is used for injecting the mixed solution of PCR sample and reaction reagent, droplet size adjustment flow passage is used for adjusting the size of the water -in -oil droplet of generation, and the droplet collection chamber is used for collecting the liquid drop.

[0007] Further, the oil phase injection entrance, water phase injection entrance, droplet size adjustment flow passage are all provided with micro column array, the micro column array in the oil phase injection entrance and water phase injection entrance is used for eliminating the air bubble in the injected liquid, and the micro column array in the droplet size adjustment channel is used for adjusting the size of the plurality of liquid drops, so that the sizes of the liquid drops are consistent.

[0008] Further, the micro column array in the oil phase injection entrance and water phase injection entrance includes a plurality of cylinders arranged in a trapezoidal shape, the diameters of the cylinders in the same row are consistent, the diameters of the cylinders at the bottom edge of the trapezoid to the cylinders at the top edge of the trapezoid are in a decreasing relationship from row to row, and the center distances between the cylinders in the same row are 3 times the radius of the cylinders.

[0009] Further, in the micro column array in the oil phase injection entrance and water phase injection entrance, the diameters of the cylinders at the bottom edge of the trapezoid to the cylinders at the top edge of the trapezoid are in a decreasing relationship of 5 μm from row to row.

[0010] Further, in the micro column array in the oil phase injection entrance and water phase injection entrance, the diameters of the cylinders at the bottom edge of the trapezoid are 50-70 μm, and the diameters of the cylinders at the top edge of the trapezoid are 20-45 μm.

[0011] Further, in the micro column array in the oil phase injection entrance and water phase injection entrance, the diameters of the cylinders at the bottom edge of the trapezoid are 60 μm, and the diameters of the cylinders at the top edge of the trapezoid are 30 μm.

[0012] Further, the micro column array in the droplet size adjustment channel includes a plurality of cylinders arranged in a rectangular shape, the diameters of the plurality of cylinders are consistent, and the center distances between the cylinders are consistent.

[0013] Further, in the micro column array in the droplet size adjustment channel, the diameters of the cylinders are 20-80 μm.

[0014] Further, in the micro-column array in the droplet size adjustment channel, the diameter of the cylinder is 50 μm.

[0015] Further, in the micro-column array in the droplet size adjustment channel, the center distance between each cylinder is 75 μm.

[0016] Compared with the prior art, the utility model has the advantages of:

[0017] The utility model discloses a micro -fluidic chip includes oil phase note entry, water phase note entry, droplet size adjustment flow passage, droplet collection chamber, wherein, oil phase note entry, water phase note entry, droplet size adjustment flow passage all are provided with micro -column array, and the micro -column array in oil phase note entry and water phase note entry is used for eliminating the air bubble in the injection liquid, and the micro -column array in the droplet size adjustment channel is used for making the size of multiple droplets adjust, makes the size between the droplet be consistent. Thus through the mode of introducing micro -column array accurate control droplet's generation and size adjustment, make the size between the droplet of final generation have certain consistency.

[0018] The utility model discloses a micro -fluidic chip includes oil phase note entry, water phase note entry, droplet size adjustment flow passage, droplet collection chamber, wherein, oil phase note entry, water phase note entry, droplet size adjustment flow passage all are provided with micro -column array, and the micro -column array in oil phase note entry and water phase note entry is used for eliminating the air bubble in the injection liquid, and the micro -column array in the droplet size adjustment channel is used for making the size of multiple droplets adjust, makes the size between the droplet be consistent. Thus through the mode of introducing micro -column array accurate control droplet's generation and size adjustment, make the size between the droplet of final generation have certain consistency. BRIEF DESCRIPTION OF DRAWINGS

[0019] Figure 1 It is the structural schematic diagram of micro -fluidic chip in the utility model;

[0020] Figure 2 It is the micro -column array schematic diagram of oil phase note entry in micro -fluidic chip;

[0021] Figure 3 It is the micro -column array schematic diagram of water phase note entry in micro -fluidic chip;

[0022] Figure 4 It is the micro -column array schematic diagram of droplet size adjustment flow passage in micro -fluidic chip.

[0023] Marking in the drawing: 1, oil phase note entry, 2, water phase note entry, 3, droplet size adjustment flow passage, 4, droplet collection chamber. DETAILED DESCRIPTION

[0024] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0025] Example

[0026] like Figure 1 As shown, a microfluidic chip structure for generating PCR droplets includes an oil-phase injection port 1, an aqueous-phase injection port 2, a droplet size adjustment channel 3, and a droplet collection chamber 4 with a support structure. The oil-phase injection port 1 is used to inject silicone oil, the aqueous-phase injection port 2 is used to inject a mixture of PCR sample and reaction reagents, the droplet size adjustment channel 3 is used to adjust the size of the generated water-in-oil droplets to obtain droplets of uniform size, and the droplet collection chamber 4 is used to collect and hold the droplets. The oil-phase injection port 1, the aqueous-phase injection port 2, and the droplet size adjustment channel 3 are all equipped with micropillar arrays. The micropillar arrays within the oil-phase injection port 1 and the aqueous-phase injection port 2 are used to eliminate bubbles in the injected liquid, and the micropillar array within the droplet size adjustment channel 3 is used to adjust the size of multiple droplets to achieve consistent size.

[0027] like Figure 2 and Figure 3 As shown, the microcolumn array in the oil phase injection port 1 and the water phase injection port 2 includes a plurality of cylinders arranged in a trapezoidal shape. The diameters of the cylinders in the same row are consistent, and the diameters of the cylinders at the bottom edge of the trapezoid to the cylinders at the top edge of the trapezoid decrease row by row. The center-to-center spacing between the cylinders in the same row is 3 times the radius of the cylinder.

[0028] like Figure 4 As shown, the micro-column array in the droplet size adjustment channel 3 includes a plurality of cylinders arranged in a rectangular shape, the diameters of the plurality of cylinders are consistent, and the distances between the centers of the cylinders are consistent.

[0029] In practical applications, the fabrication process of microfluidic chips primarily relies on computer-aided design (CAD) tools for graphic design. Semiconductor processing techniques then involve sequential steps such as coating (SU8 3050), photolithography, and development on a four-inch silicon wafer to create a chip template. Subsequently, PDMS (Polydimethylsiloxane) is used for casting to create the microfluidic structure. The prepared PDMS structure is bonded to a glass slide to form a closed fluid system. Furthermore, the oil phase injection port 1 and the water phase injection port 2 are both punched using a hole punch.

[0030] When using this solution to generate PCR droplets, it is necessary to use a precision instrument such as an injection pump (i.e., an injection device) to inject silicone oil as the oil phase and the aqueous phase containing reagents such as the PCR template reaction solution into the corresponding injection ports (i.e., oil phase injection port 1 and aqueous phase injection port 2). The purpose of this step is to form stable droplets in the microfluidic chip and lay the foundation for subsequent PCR reactions. When it is observed through a microscope that the droplet collection end is full of droplets, the entire microfluidic chip is placed in a commercial PCR device for PCR reaction cycles. After the PCR cycle is completed, the microfluidic chip can be placed under a microscope for observation.

[0031] The generation of microfluidic droplets is mainly based on the surface tension of oil and water. When the two oil phases intersect with the water phase, more oil phase will be injected, resulting in the presence of water droplets in the oil system. Such "oil-in-water" droplets contain samples, enzymes and other components used to realize PCR reactions.

[0032] In terms of droplet generation and processing, this solution utilizes an innovative microfluidic structure, introducing a micropillar array to precisely control droplet generation and size adjustment. In practical applications, the micropillar array can be cylindrical or irregular, but this embodiment uses cylindrical structures. Specifically, two micropillar arrays are located at the injection port. These two locations primarily support the microfluidic chamber and, to a certain extent, remove bubbles. In terms of dimensional design, the diameter of these array structures decreases by 5 μm from the injection port toward the interior of the channel. The outermost diameter ranges from 50 to 70 μm, and in this embodiment, it is designed to be 60 μm. The innermost diameter ranges from 20 to 45 μm, and in this embodiment, it is designed to be 30 μm. The cylinders are arranged in a trapezoidal array, with the diameter of the columns in each row being the same, and the distance between the centers of the columns in the same row is three times the radius. The diameters vary between rows. The number of columns at the injection port is approximately 5 to 6 columns, and the number of columns in each row is adjusted according to the size of the channel.

[0033] In the micro-pillar array at the droplet generation site, the main role is to adjust the size of the droplet in addition to supporting the microfluidic structure. First, a cylinder with a bottom diameter of 20-80 μm is designed, and in this embodiment, the bottom diameter is 50 μm. A rectangular array is used to generate, and the spacing between the outer walls of the micro-pillars is 25-140 μm, and in this embodiment, it is designed to be 50 μm (i.e. the center-to-center spacing between the cylinders is 75 μm). During the generation of the droplet, its size will change with the difference in flow rate between the water phase and the oil phase, and the droplet with a diameter greater than 50 μm will deform after passing through the carefully designed array, and the size of multiple droplets will be adjusted to ensure the consistency of the size between the droplets. The size of the water-in-oil droplet generated by the micro-pillar array is adjusted, so that the amount of template and reaction reagent contained in the PCR reaction system is more uniform, thereby reducing the influence of the liquid generation process on the reaction system, and ensuring the uniformity and stability of the droplet, which is crucial for improving the accuracy of dPCR. This structural design takes into account the influence of the size difference of the droplet on the recognition efficiency, thereby overcoming some limitations in the prior art.

[0034] It should be noted that in actual application of the present scheme, the micro-pillar array structure can be designed and adjusted according to the size of the droplet required.

[0035] As can be seen from the above, the present scheme adopts an innovative microfluidic structure design, which precisely controls the generation and size adjustment of the droplet by introducing a micro-pillar array, thereby ensuring the uniformity and stability of the generated droplet.

Claims

1. A microfluidic chip structure for generating PCR droplets, characterized in that: The invention comprises an oil phase injection port (1), a water phase injection port (2), a droplet size adjustment flow channel, and a droplet collection chamber (4) with a support structure, wherein the oil phase injection port (1) is connected to the droplet size adjustment flow channel through two channels, the water phase injection port (2) is connected to the droplet size adjustment flow channel through one channel, the droplet size adjustment flow channel is connected to the droplet collection chamber (4), the oil phase injection port (1) is used to inject silicone oil, the water phase injection port (2) is used to inject a mixture of PCR sample and reaction reagent, the droplet size adjustment flow channel is used to adjust the size of generated oil-water droplets to obtain droplets of uniform size, and the droplet collection chamber (4) is used to collect and contain droplets.

2. A microfluidic chip structure for generating PCR droplets according to claim 1, characterized in that: The oil phase injection port (1), the water phase injection port (2), and the droplet size adjustment channel are all provided with microcolumn arrays. The microcolumn arrays in the oil phase injection port (1) and the water phase injection port (2) are used to eliminate bubbles in the injected liquid, and the microcolumn array in the droplet size adjustment channel is used to adjust the sizes of multiple droplets so that the sizes of the droplets are consistent.

3. A microfluidic chip structure for generating PCR droplets according to claim 2, characterized in that: The microcolumn arrays in the oil phase injection port (1) and the water phase injection port (2) include a plurality of cylinders arranged in a trapezoidal shape, wherein the diameters of the cylinders in the same row are consistent, and the diameters of the cylinders located at the bottom edge of the trapezoid to the cylinders located at the top edge of the trapezoid decrease row by row, and the center spacing between the cylinders in the same row is 3 times the radius of the cylinder.

4. A microfluidic chip structure for generating PCR droplets according to claim 3, characterized in that: In the microcolumn arrays within the oil phase injection port (1) and the water phase injection port (2), the diameters of the cylinders located at the bottom edge of the trapezoid to the cylinders located at the top edge of the trapezoid decrease by 5 μm row by row.

5. A microfluidic chip structure for generating PCR droplets according to claim 4, characterized in that: The diameter of the cylinder located at the bottom of the trapezoid is 50-70 μm, and the diameter of the cylinder located at the top of the trapezoid is 20-45 μm.

6. A microfluidic chip structure for generating PCR droplets according to claim 5, characterized in that: The diameter of the cylinder located at the bottom of the trapezoid is 60 μm, and the diameter of the cylinder located at the top of the trapezoid is 30 μm.

7. A microfluidic chip structure for generating PCR droplets according to claim 2, characterized in that: The micro-column array in the droplet size adjustment channel includes a plurality of cylinders arranged in a rectangular shape, the diameters of the plurality of cylinders are consistent, and the distances between the centers of the cylinders are consistent.

8. A microfluidic chip structure for generating PCR droplets according to claim 7, characterized in that: In the micro-column array in the droplet size adjustment channel, the diameter of the cylinder is 20-80 μm.

9. A microfluidic chip structure for generating PCR droplets according to claim 8, characterized in that: The cylinder has a diameter of 50 μm.

10. The microfluidic chip structure for generating PCR droplets according to claim 7, characterized in that: In the micro-column array in the droplet size adjustment channel, the center spacing between each cylinder is 75 μm.

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