Digital PCR chip

Abstract

The utility model discloses a kind of digital PCR chips, including droplet detection area and anchoring component, anchoring component is spaced in the opposite ends of droplet detection area, anchoring component is used to fix droplet in droplet detection area.The digital PCR chip of the utility model embodiment, by setting anchoring component in the opposite ends of droplet detection area, to facilitate the droplet stably stays in droplet detection area, so that droplet is retained in droplet detection area after heating, can effectively reduce the loss caused by flow in heating process, benefit to carry out PCR reaction and fluorescence detection to droplet, realize on-chip droplet generation and detection integration design, avoid the pollution of reagent, simplify the operation steps of user, to improve the quantitative detection accuracy of digital PCR chip to some extent.

Description

Technical Field

[0001] This utility model relates to the field of microfluidic chip technology, and in particular to a digital PCR chip. Background Technology

[0002] Digital PCR (Polymerase Chain Reaction) chips achieve absolute quantification by counting individual molecules, effectively distinguishing samples with minute concentration differences. They can be used to accurately determine the relative expression of target genes and analyze gene copy number variations.

[0003] Among them, the droplet digital PCR chip forms water-in-oil (W / O) or oil-in-water (O / W) droplets by shearing the discrete phase from the continuous phase. The droplets react with chemical substances in the PCR detection area. By measuring the fluorescence value of the droplets after the reaction in the PCR detection area, the biological copy number and initial concentration can be accurately measured. At the same time, by setting a cooling and heating platform below the digital PCR chip, the digital PCR chip is heated by repeated high and low temperature heating to realize the PCR reaction of the droplets.

[0004] However, during the PCR thermal cycling process, the droplets in the detection area of ​​existing droplet-based digital PCR chips will flow to a certain extent and be lost, resulting in an unstable number of droplets in the detection area and affecting the accuracy of quantitative detection by digital PCR chips. Utility Model Content

[0005] This invention aims to solve at least one of the technical problems existing in the prior art. To this end, this invention proposes a digital PCR chip that can effectively reduce droplet loss due to flow during heating, improve the stability of the heating process, and facilitate PCR reactions on the droplets. This, in turn, improves the accuracy of quantitative detection by the digital PCR chip to a certain extent, solving the technical problem of droplet loss caused by movement during thermal cycling in the detection area of ​​the prior art.

[0006] A digital PCR chip according to an embodiment of the present invention includes: a droplet detection area; and anchoring components, wherein the anchoring components are spaced apart at opposite ends of the droplet detection area, and the anchoring components are used to fix the droplets within the droplet detection area.

[0007] According to the embodiments of the present invention, the digital PCR chip has anchoring components at both ends of the droplet detection area to stably keep the droplets in the detection area. This allows the droplets to remain in the detection area after heating, effectively reducing droplet loss due to flow during heating and thus minimizing droplet volume loss. This facilitates PCR reaction and fluorescence detection of the droplets, achieving an integrated design of on-chip droplet generation and detection. It avoids reagent contamination, simplifies user operation, and improves the quantitative detection accuracy of the digital PCR chip to a certain extent.

[0008] In some embodiments, the digital PCR chip further includes a droplet generation area and a waste liquid discharge area. The droplet detection area includes a first detection area, a second detection area, and a third detection area arranged sequentially and interconnected along a first direction. The first detection area is connected to the droplet generation area, the third detection area is connected to the waste liquid discharge area, and the anchoring components are spaced apart at opposite ends of the second detection area.

[0009] In some embodiments, the anchoring component includes a plurality of anchoring structures arranged along a second direction, the anchoring structures being used to limit the displacement of the droplet, the second direction intersecting the first direction; adjacent two anchoring structures are spaced apart to form a flow channel, the minimum width of the flow channel being W, and the diameter of the droplet being D, wherein W = 0.1D~2D.

[0010] In some embodiments, the width of the flow channel gradually decreases in the direction toward the second detection area.

[0011] In some embodiments, the anchoring structure has an anchoring recess on the side facing the second detection area, the anchoring recess being recessed in a direction away from the second detection area, and in a second direction, the width of the anchoring recess is greater than or equal to the diameter of the droplet, the anchoring recess being used to limit the displacement of the droplet.

[0012] In some embodiments, in the second direction, the width of the anchoring component is equal to or less than the width of the second detection area, and the second direction intersects with the first direction; and / or, in the third direction, the height of the anchoring component is equal to the height of the second detection area, the height of the second detection area is H, and the diameter of the droplet is D, where H = 1.0D~1.5D, and the third direction, the second direction, and the first direction intersect each other.

[0013] In some embodiments, multiple rows of the anchoring components are respectively provided at opposite ends of the second detection area, and the multiple rows of the anchoring components are arranged along the first direction.

[0014] In some embodiments, the digital PCR chip further includes a sealing element that covers the droplet detection area to seal the droplet detection area, and the thickness of the sealing element is in the range of 0.1mm-1mm; a support column is provided in the second detection area, and the support column is fixedly connected to the sealing element.

[0015] In some embodiments, the droplet generation region has an oil phase flow channel and a sample flow channel, and the waste liquid discharge region has a discharge flow channel. At least one of the oil phase flow channel, the sample flow channel, and the discharge flow channel has multiple bends. The oil phase flow channel includes a first oil phase flow channel and a second oil phase flow channel. The first end of the first oil phase flow channel and the first end of the second oil phase flow channel are connected to an oil phase inlet. The first end of the sample flow channel is connected to a sample inlet. The droplet generation region also has a main flow channel. The second ends of the first oil phase flow channel, the second end of the second oil phase flow channel, and the second end of the sample flow channel are all connected to the main flow channel. The extension direction of the main flow channel is consistent with the extension direction of the second end of the sample flow channel and perpendicular to the extension directions of the second ends of the first oil phase flow channel and the second end of the second oil phase flow channel. A filter element is provided at the outlet of the oil phase inlet and / or the sample inlet.

[0016] In some embodiments, the flow resistance of the droplet generation zone is equal to the flow resistance of the waste liquid discharge zone.

[0017] Additional aspects and advantages of this invention will become apparent from the description which follows, or may be learned by practice of this invention. Attached Figure Description

[0018] The above and / or additional aspects and advantages of this utility model will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which: Figure 1 This is a schematic diagram of a digital PCR chip according to some embodiments of the present invention; Figure 2 for Figure 1 A magnified view of region I in the middle; Figure 3 This is a schematic diagram of the anchoring structure of some embodiments of the present invention; Figure 4 The model diagrams for the flow resistance of the oil phase flow channel and sample flow channel in some embodiments of this utility model are established based on the circuit equivalence method. Figure 5 This is a schematic diagram of the droplet generation region in some embodiments of the present invention.

[0019] Figure label: 1000, Digital PCR Chip; 100. Droplet detection area; 110. First Testing Area; 120. Second inspection area; 121. Support column; 130. Third Testing Area; 200. Anchoring components; 210. Anchoring structure; 211. Anchoring recess; 500. Flow diversion channel; 300. Droplet generation region; 310. Oil phase flow channel; 311. First oil phase flow channel; 312. Second oil phase flow channel; 320. Sample flow channel; 330. Main flow channel; 400 Waste liquid discharge area; 410 Discharge channel; 420 Waste liquid discharge outlet; 321. Bending section; 600. Oil phase inlet; 700. Sample inlet; 610. Filter element. Detailed Implementation

[0020] The embodiments of this utility model are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain this utility model, and should not be construed as limiting this utility model.

[0021] In the description of this utility model, it should be understood that the terms "center", "longitudinal", "transverse", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", "clockwise", "counterclockwise", "axial", "radial", "circumferential", etc., indicating the orientation or positional relationship are based on the orientation or positional relationship shown in the accompanying drawings, and are only for the convenience of describing this utility model and simplifying the description, and are not intended to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of this utility model.

[0022] The digital PCR chip 1000 of this utility model is described below with reference to the accompanying drawings.

[0023] like Figure 1 As shown, a digital PCR chip 1000 according to an embodiment of the present invention includes: a droplet detection area 100 and an anchoring component 200.

[0024] Among them, such as Figure 1 As shown, the droplet detection area 100 provides a certain area space for the droplet, concentrating the droplet in the droplet detection area 100 to facilitate subsequent PCR reaction of the droplet.

[0025] like Figure 1 As shown, anchoring components 200 are spaced apart at opposite ends of the droplet detection area 100, and are used to fix the droplets within the droplet detection area 100. This ensures that the droplets remain within the droplet detection area 100 to the greatest extent possible after heating, effectively reducing droplet loss due to flow during heating. This facilitates subsequent PCR reactions and fluorescence detection, achieving an integrated design of on-chip droplet generation and detection. It avoids reagent contamination, simplifies user operation, and thus improves the quantitative detection accuracy of the digital PCR chip 1000 to a certain extent.

[0026] It should be noted that by realizing the integrated design of on-chip droplet generation and detection, the digital PCR chip 1000 of this application does not require the generated droplets to be transferred to a small tube for heating, and has highly integrated and easy-to-operate functions.

[0027] It is worth noting that, in existing methods of fixing droplets using micropits, the number of micropits needs to be consistent with the number of droplets, resulting in a limited number of droplets that the detection area can accommodate. Furthermore, the micropits are difficult to manufacture and have a complex structure. Compared with the prior art of fixing droplets using micropits, this application uses at least two rows of anchoring components 200 arranged in opposite directions to fix the droplets, which not only reduces the difficulty of fixing but also simplifies the structure of the digital PCR chip 1000 and allows the droplet detection area 100 to accommodate a larger number of droplets.

[0028] Meanwhile, this application provides anchoring components 200 at both ends of the droplet detection area 100. At least two rows of anchoring components 200 work together to improve the fixation effect on the droplets, so that the droplets can remain stably in the droplet detection area 100. This allows the droplets to remain in the droplet detection area 100 after heating, effectively reducing the loss of droplets due to flow during heating, and thus facilitating the PCR reaction of the droplets.

[0029] As can be seen from the above structure, the digital PCR chip 1000 of this utility model, by setting anchoring components 200 at intervals at both ends of the droplet detection area 100, can easily keep the droplets stably in the droplet detection area 100, so that the droplets can remain in the droplet detection area 100 after heating. This can effectively reduce the loss of droplets due to flow during heating, thereby reducing the loss of droplet volume. This facilitates PCR reaction and fluorescence detection of droplets, realizes the integrated design of on-chip droplet generation and detection, avoids reagent contamination, simplifies the user's operation steps, and thus improves the quantitative detection accuracy of the digital PCR chip 1000 to a certain extent.

[0030] Meanwhile, by realizing the integrated design of on-chip droplet generation and detection, the droplets do not need to be transferred after generation, avoiding contamination of reagents used in PCR reactions and detection, and simplifying the user's operation steps.

[0031] Understandably, compared to the existing technology that uses micropits to fix droplets, this application not only reduces the difficulty of fixing droplets but also simplifies the structure of the digital PCR chip 1000. Furthermore, it allows the droplet detection area 100 to accommodate a larger number of droplets. In addition, this application provides anchoring components 200 at both ends of the droplet detection area 100. The cooperation of at least two rows of anchoring components 200 can improve the fixation effect on droplets, effectively reduce droplet loss due to flow during heating, and thus facilitate PCR reaction of droplets. This, in turn, improves the quantitative detection accuracy of the digital PCR chip 1000 to a certain extent.

[0032] In some embodiments, such as Figure 1 As shown, the digital PCR chip 1000 also includes a droplet generation area 300 and a waste liquid discharge area 400. The droplet detection area 100 includes a first detection area 110, a second detection area 120, and a third detection area 130 arranged sequentially and interconnected along a first direction. The first detection area 110 is connected to the droplet generation area 300, and the third detection area 130 is connected to the waste liquid discharge area 400. Anchoring components 200 are spaced at opposite ends of the second detection area 120. It should be noted that the first direction mentioned here can be understood as... Figure 1 As shown in the X direction, by setting the first detection area 110 to connect with the droplet generation area 300, it can be ensured that the droplets generated in the droplet generation area 300 can effectively enter the droplet detection area 100 for detection.

[0033] The droplet generation region 300 can divide the sample liquid into tens of thousands or even millions of tiny droplets, forming independent reaction units, thereby achieving single-molecule amplification and absolute quantification, so as to facilitate the subsequent PCR reaction of droplets in the droplet detection region 100.

[0034] Meanwhile, by setting the third detection zone 130 to connect to the waste liquid discharge zone 400, it is convenient to use the waste liquid discharge zone 400 to discharge excess liquid, residual reaction liquid or cleaning liquid that failed to form droplets in a timely manner, so as to avoid liquid accumulation affecting the efficiency and uniformity of droplet formation, and at the same time, it can also prevent this part of the waste liquid from leaking and polluting the external environment.

[0035] Furthermore, by spaced the anchoring components 200 at opposite ends of the second detection area 120, the anchoring components 200 are spaced at opposite ends of the droplet detection area 100, which facilitates the stable retention of the droplets in the second detection area 120. This allows the droplets to remain in the second detection area 120 after heating, effectively reducing the loss of droplets due to flow towards the waste liquid discharge area 400 during heating. This, in turn, facilitates PCR reaction and fluorescence detection of the droplets, realizing an integrated design of on-chip droplet generation and detection, and to a certain extent improving the quantitative detection accuracy of the digital PCR chip 1000.

[0036] In summary, the design of the digital PCR chip 1000 in this application integrates droplet generation, PCR reaction, and fluorescence detection into one unit. That is, droplet generation, PCR reaction, and fluorescence detection all occur on the digital PCR chip 1000 without the need for transfer, thus avoiding reagent contamination and simplifying the user's operation steps.

[0037] In a specific example, the first detection zone 110 is used to receive droplets generated in the droplet generation zone 300. As the droplets flow, they flow from the first detection zone 110 to the second detection zone 120, which is the main area for the droplets to perform PCR reactions. The third detection zone 130 is used to receive excess liquid, residual reaction liquid, or washing liquid that failed to form droplets, and transfer this waste liquid to the waste liquid discharge zone 400 to avoid liquid accumulation affecting the efficiency and uniformity of droplet generation.

[0038] It should be noted that the anchoring components 200 are spaced apart at opposite ends of the second detection zone 120, so that anchoring components 200 are provided at both the inlet and outlet near the droplet detection zone 100. The anchoring components 200 near the inlet of the droplet detection zone 100 can reduce the flow of droplets to the droplet generation zone 300 during thermal motion, and the anchoring components 200 near the outlet of the droplet detection zone 100 can prevent droplets from flowing to the waste liquid discharge zone 400 due to thermal motion. This achieves stable retention of droplets in the second detection zone 120, avoids droplet loss, and facilitates PCR reaction and detection of droplets.

[0039] In the description of this utility model, the features defined as "first", "second" and "third" may explicitly or implicitly include one or more of the features, used to distinguish the descriptive features, without any order or importance.

[0040] In some embodiments, combined with Figure 1 and Figure 2As shown, the anchoring assembly 200 includes multiple anchoring structures 210 arranged along a second direction. The anchoring structures 210 are used to limit the displacement of the droplet, and the second direction intersects with the first direction. It should be noted that the second direction here can be understood as... Figure 2 As shown in the Y direction, by using multiple anchoring structures 210 to restrict the displacement of the droplet, the droplet can be fixed in the second detection area 120, so that the droplet remains in the second detection area 120 after heating, effectively reducing the loss of the droplet due to flow during the heating process.

[0041] In the description of this utility model, unless otherwise stated, "a plurality of" means two or more.

[0042] It should be noted that the number of multiple anchoring structures 210 in the anchoring component 200 is not limited in this application and can be set according to actual needs. Multiple anchoring structures 210 can cooperate to cover the full width or part of the width of the second detection area 120.

[0043] In some embodiments, combined with Figure 1 and Figure 2 As shown, two adjacent anchoring structures 210 are spaced apart to form a flow channel 500. The minimum width of the flow channel 500 is W, and the diameter of the droplet is D, where W = 0.1D~2D. It should be noted that when the minimum width W of the flow channel 500 is too large compared to the droplet diameter D, the droplet will drift or move randomly during PCR thermal cycling, preventing it from staying in the second detection area 120 and affecting the anchoring structure 210's ability to restrict droplet displacement. Conversely, when the minimum width W of the flow channel 500 is too small compared to the droplet diameter D, the droplet will not be able to pass through the flow channel 500 normally, affecting the PCR reaction and detection of the droplet, thereby reducing the quantitative detection accuracy of the digital PCR chip 1000.

[0044] In summary, this application sets the minimum width W of the flow channel 500 and the diameter D of the droplet to satisfy: W=0.1D~2D. This not only allows the droplet to pass through the flow channel 500 normally, but also avoids the droplet from drifting or moving randomly during the PCR thermal cycling process to a certain extent, so that the droplet stays in the second detection area 120 to the maximum extent, thereby ensuring the ability of the anchoring structure 210 to restrict the displacement of the droplet.

[0045] In a specific example, when the anchoring component 200 is positioned near the inlet of the droplet detection area 100, by setting W=0.1D~2D, the minimum width between each adjacent anchoring structure 210 allows the droplets to enter the second detection area 120 without breaking, thereby enabling the droplets to flow into the second detection area 120 in a dispersed and uniform manner during generation, thus achieving the flattening of the droplets in the second detection area 120. When the anchoring component 200 is positioned near the outlet of the droplet detection area 100, by setting W=0.1D~2D, the minimum width between each adjacent anchoring structure 210 allows the discharge of waste liquid during droplet generation, while effectively reducing the speed of the droplets at the outlet position, reducing the loss during droplet generation, and improving sample utilization.

[0046] Specifically, the minimum width W of the flow channel 500 and the diameter D of the droplet satisfy: W=0.1D, W=0.5D, W=1D, W=1.5D or W=2D, etc.

[0047] In some embodiments, combined with Figure 1 and Figure 2 As shown, the width of the guide channel 500 gradually decreases in the direction towards the second detection area 120. Specifically, when the anchoring component 200 is positioned near the inlet of the droplet detection area 100, this configuration helps to increase the droplet flow rate, allowing the droplets to enter the second detection area 120 and flow evenly and dispersed within it, achieving a flat distribution of droplets within the second detection area 120. Furthermore, it prevents droplets from exiting the second detection area 120 through the guide channel 500 into the first detection area 110. When the anchoring component 200 is positioned near the outlet of the droplet detection area 100, this configuration effectively reduces the droplet flow rate at the outlet of the second detection area 120, preventing droplets from exiting the second detection area 120 through the guide channel 500 into the third detection area 130, reducing loss during droplet generation, and improving sample utilization.

[0048] In summary, by gradually reducing the width of the flow channel 500 in the direction toward the second detection area 120, the droplets can be stably held in the second detection area 120 to the maximum extent possible, so as to facilitate the PCR reaction of the droplets.

[0049] In some embodiments, combined with Figure 1 , Figure 2 and Figure 3As shown, the anchoring structure 210 has an anchoring recess 211 formed on the side facing the second detection area 120. The anchoring recess 211 is recessed in the direction away from the second detection area 120. In the second direction, the width of the anchoring recess 211 is greater than or equal to the diameter of the droplet. The anchoring recess 211 is used to limit the displacement of the droplet. By setting the anchoring recess 211 to be recessed in the direction away from the second detection area 120, the anchoring surface of the anchoring structure 210 facing the second detection area 120 can be formed as a concave surface, and the width of the anchoring recess 211 is set to be greater than or equal to the diameter of the droplet, so that the anchoring recess 211 can anchor at least a single droplet, thereby using the anchoring recess 211 to limit the displacement of the droplet, so as to fix the droplet in the second detection area 120 and improve the anchoring effect of the anchoring assembly 200.

[0050] In some embodiments, combined with Figure 1 and Figure 2 As shown, in the second direction, the width of the anchoring component 200 is equal to or less than the width of the second detection area 120, and the second direction intersects with the first direction. This allows the anchoring component 200 to confine a greater number of droplets within the second detection area 120 to a certain extent, ensuring the PCR reaction of the droplets.

[0051] It should be noted that the width of the anchoring component 200 can be set according to actual needs, and this application does not impose any restrictions here. When the width of the anchoring component 200 is less than the width of the second detection area 120, the anchoring component 200 is at least directly opposite the inlet of the first detection area 110 and the outlet of the third detection area 130, so as to prevent the droplets from being discharged through the inlet of the first detection area 110 and the outlet of the third detection area 130, thereby achieving the purpose of fixing the droplets within the droplet detection area 100.

[0052] In specific examples, such as Figure 1 As shown, the width of the anchoring component 200 can be set to be equal to the width of the second detection area 120, so as to achieve the flat laying of the anchoring component 200 in the width direction of the second detection area 120 and improve the anchoring effect of the anchoring component 200.

[0053] In some embodiments, in the third direction, the height of the anchoring component 200 is equal to the height of the second detection area 120, the height of the second detection area 120 is H, and the diameter of the droplet is D, where H = 1.0D~1.5D. The third direction, the second direction, and the first direction intersect each other. It should be noted that the third direction mentioned here can be understood as... Figure 3 The Z direction is shown in the figure. By setting the height of the anchoring component 200 to be equal to the height of the second detection area 120, the anchoring component 200 can cover the entire second detection area 120 in the height direction, thereby facilitating the fixation of the droplet within the second detection area 120 using the anchoring component 200.

[0054] Meanwhile, when the height H of the second detection area 120 is too high compared to the diameter D of the droplet, the droplets in the second detection area 120 may overlap in the Z direction, resulting in a smaller number of droplets actually performing PCR reactions. When the height H of the second detection area 120 is too low compared to the diameter D of the droplet, the droplet may be squeezed into a flat or elliptical shape, or even squeezed and broken, causing the droplet to deform, which is not conducive to the PCR reaction of the droplet, and thus results in low quantitative detection efficiency of the digital PCR chip 1000.

[0055] In summary, this application sets the height H of the second detection area 120 and the diameter D of the droplet to satisfy H=1.0D~1.5D. This not only avoids the overlap of droplets in the second detection area 120 and ensures the actual number of droplets performing PCR reactions, but also prevents the droplets in the second detection area 120 from deforming, thereby preventing the droplets from being squeezed into a flat or elliptical shape or even breaking, which is beneficial to the PCR reaction of the droplets. This, to a certain extent, ensures the quantitative detection efficiency of the digital PCR chip 1000.

[0056] In specific examples, H = 1.0D, 1.1D, 1.2D, 1.3D, 1.4D, or 1.5D, etc.

[0057] In some embodiments, combined with Figure 1 and Figure 2 As shown, multiple rows of anchoring components 200 are respectively provided at both ends of the second detection area 120, and the multiple rows of anchoring components 200 are arranged along the first direction. The multiple rows of anchoring components 200, in combination, can improve their ability to restrict droplet displacement to a certain extent, making it easier to keep the droplets stably in the second detection area 120, which is beneficial to the PCR reaction of the droplets and ensures the quantitative detection accuracy of the digital PCR chip 1000.

[0058] It should be noted that the provision of multiple rows of anchoring components 200 at each of the opposite ends of the second detection area 120 means that each end of the second detection area 120 is provided with two or more rows of anchoring components 200.

[0059] Of course, in some other embodiments, a row of anchoring components 200 may be provided at both ends of the second detection area 120 to simplify the structure of the digital PCR chip 1000. The specific number of anchoring components 200 is not limited in this application.

[0060] In specific examples, such as Figure 1 As shown, a row of anchoring components 200 is arranged at each of the opposite ends of the second detection area 120, and the number of anchoring structures 210 in the anchoring components 200 exactly fills the entire width of the second detection area 120.

[0061] In some embodiments, the digital PCR chip 1000 further includes a sealing element (not shown in the figure), which covers the droplet detection area 100 to seal the droplet detection area 100. The thickness of the sealing element ranges from 0.1 mm to 1 mm. By covering and sealing the droplet detection area 100 with the sealing element, a closed space is formed between the sealing element and the droplet detection area 100. This prevents droplets from overflowing from the droplet detection area 100 due to external pressure fluctuations or external interference. Furthermore, the sealing element isolates the droplet detection area 100 from external air, moisture, or impurities, preventing environmental changes (such as humidity or temperature fluctuations) from affecting the droplet properties (such as volume or composition), thereby facilitating the PCR reaction of the droplets.

[0062] It should be noted that when the thickness of the seal is too thick, it will increase the thermal resistance, which will reduce the heating rate of the droplet detection area 100, affecting the heating of the droplet detection area 100. This will result in a long PCR thermal cycle time and low quantitative detection efficiency of the digital PCR chip 1000. When the thickness of the seal is too thin, the sealing effect of the seal on the droplet detection area 100 will be poor, which may cause the droplets to overflow from the droplet detection area 100 due to external pressure fluctuations or external interference, which is not conducive to the PCR reaction of the droplets.

[0063] In summary, this application sets the thickness of the sealing element to a range of 0.1mm-1mm, which not only reduces thermal resistance to a certain extent, ensuring a certain heating rate for the droplet detection area 100, thus facilitating heating of the droplet detection area 100 and avoiding long PCR thermal cycling time, thereby ensuring the quantitative detection efficiency of the digital PCR chip 1000, but also ensures the sealing effect of the sealing element on the droplet detection area 100, preventing droplets from overflowing from the droplet detection area 100 due to external pressure fluctuations or external interference, thus facilitating the PCR reaction of the droplets.

[0064] Specifically, the thickness of the seal is 0.1mm, 0.2mm, 0.3mm, 0.4mm, 0.5mm, 0.6mm, 0.7mm, 0.8mm, 0.9mm or 1mm, etc.

[0065] In some embodiments, such as Figure 1 As shown, a support column 121 is provided in the second detection area 120, and the support column 121 is fixedly connected to the sealing element. This allows the support column 121 to stably support the sealing element, evenly distribute external forces, and ensure that the sealing element remains flat during the packaging, transportation, or experimental operation of the digital PCR chip 1000. This facilitates the detection and heating of droplets to a certain extent, and helps to improve the quantitative detection accuracy of the digital PCR chip 1000.

[0066] In some embodiments, during the connection between the support column 121 and the seal, the seal can be placed on the support column 121, and then the support column 121 and the seal can be bonded together by hot pressing to achieve a fixed connection between the support column 121 and the seal.

[0067] Of course, in other embodiments, a double-layer force-adhesive film or epoxy resin, polyurethane, acrylate or special plastic adhesive can be applied to the top of the support column 121 or the position of the seal corresponding to the support column 121 to achieve a fixed connection between the support column 121 and the seal.

[0068] Meanwhile, the support column 121 can also block the droplets in the second detection area 120 to a certain extent. On the one hand, it reduces the flow speed of the droplets toward the waste liquid discharge area 400, making it easier to keep the droplets stably in the second detection area 120, reducing the loss of droplets due to flow during heating, and improving the quantitative detection accuracy of the digital PCR chip 1000. On the other hand, it can also disperse the droplets, making it easier to spread the droplets evenly in the second detection area 120, improving the detection of droplets and the heating effect.

[0069] In some embodiments, the support column 121 may be a square, round, or rhomboid column shape.

[0070] In specific examples, such as Figure 1 As shown, the support column 121 is a rhomboid column.

[0071] In some embodiments, such as Figure 1 As shown, the support column 121 includes multiple columns. The cooperation of multiple support columns 121 can improve the support effect on the seal to a certain extent and ensure the stability of the seal.

[0072] In some embodiments, the height of the support column 121 is the same as the height of the second detection area 120. This allows the support column 121 to be effectively fixedly connected to the seal, providing support for the seal and preventing the second detection area 120 from collapsing during use of the digital PCR chip 1000, thereby ensuring the size of the droplets and their arrangement in the second detection area 120 to a certain extent.

[0073] In some embodiments, such as Figure 1As shown, the droplet generation area 300 has an oil phase flow channel 310 and a sample flow channel 320, and the waste liquid discharge area 400 has a discharge flow channel 410. At least one of the oil phase flow channel 310, sample flow channel 320 and discharge flow channel 410 has multiple bends 321. The oil phase flow channel 310 is responsible for conveying a hydrophobic oil phase (such as mineral oil or silicone oil), and the sample flow channel 320 is responsible for conveying an aqueous reaction mixture containing DNA (Deoxyribonucleic Acid) / RNA (Ribonucleic Acid) template, PCR primers, DNA polymerase and buffer. The hydrophobic oil phase material in the oil phase flow channel 310 can encapsulate the aqueous reaction mixture to form a "water-in-oil" droplet system.

[0074] Meanwhile, after the digital PCR chip 1000 completes droplet generation, amplification and detection, there will be unreacted reagents, broken droplet fragments or oil phase waste liquid remaining in the digital PCR chip 1000 that need to be discharged. By setting a discharge channel 410 in the waste liquid discharge area 400, the discharge channel 410 can be driven by negative pressure or pressure to export the above-mentioned waste from the digital PCR chip 1000, which can prevent residual substances from depositing in the droplet detection area 100, thereby preventing the guide channel 500 from becoming narrow or blocked.

[0075] Furthermore, having multiple bends 321 in at least one of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 means that one of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 has multiple bends 321; or, two of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 have multiple bends 321; or, all of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 have multiple bends 321. The multiple bends 321 are beneficial for increasing the extension length of the flow channel, allowing the flow channel with multiple bends 321 to have a longer buffer zone for stable droplet generation.

[0076] In some embodiments, such as Figure 1 As shown, the oil phase flow channel 310, sample flow channel 320 and discharge flow channel 410 all have multiple bends 321, so that the oil phase flow channel 310, sample flow channel 320 and discharge flow channel 410 all have a relatively long buffer zone.

[0077] In some embodiments, at least a portion of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 are formed as rectangular flow channels, S-shaped arc flow channels, or spiral flow channels, such that the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 each have multiple bends 321.

[0078] In specific examples, such as Figure 1As shown, at least a portion of the oil phase flow channel 310, sample flow channel 320, and discharge flow channel 410 are formed as S-shaped arc flow channels. The S-shaped arc flow channels serve as buffer flow channel structures to further optimize the stability of droplet generation.

[0079] In some embodiments, combined with Figure 1 and Figure 5 As shown, the oil phase flow channel 310 includes a first oil phase flow channel 311 and a second oil phase flow channel 312. The first end of the first oil phase flow channel 311 and the first end of the second oil phase flow channel 312 are connected to the oil phase inlet 600. The first end of the sample flow channel 320 is connected to the sample inlet 700. The droplet generation region 300 also has a total flow channel 330. The second ends of the first oil phase flow channel 311, the second end of the second oil phase flow channel 312, and the second end of the sample flow channel 320 are all connected to the total flow channel 330. The extension direction of the total flow channel 330 is consistent with the extension direction of the second end of the sample flow channel 320 and perpendicular to the extension direction of the second end of the first oil phase flow channel 311 and the second end of the second oil phase flow channel 312. Specifically, by setting the extension direction of the main flow channel 330 to be consistent with the extension direction of the second end of the sample flow channel 320 and perpendicular to the extension directions of the second end of the first oil phase flow channel 311 and the second end of the second oil phase flow channel 312, the connection between the first oil phase flow channel 311, the second oil phase flow channel 312, the sample flow channel 320 and the main flow channel 330 can form a "cross-focusing structure". The "cross-focusing structure" can also be called a cross-cutting opening. This allows the oil phase medium of the first oil phase flow channel 311 and the second oil phase flow channel 312 to encapsulate and cut the sample droplets of the sample flow channel 320, forming "water-in-oil" droplets. This also facilitates the generation of uniformly sized droplets. The droplets flow through the main flow channel 330 to the droplet detection area 100 for subsequent PCR reactions.

[0080] It should be noted that at the cross-shaped shearing opening in the droplet generation zone 300, the sample flow channel 320 (aqueous phase) and the oil flow channel 310 (oil phase) intersect perpendicularly. The oil phase shears the aqueous phase at a higher flow rate, causing the sample droplets in the aqueous phase to break into microdroplets.

[0081] It should also be noted that the size of the droplets can be adjusted by controlling the flow rates of the oil and water phases and their ratio, which is beneficial for generating droplets of uniform size.

[0082] In some embodiments, such as Figure 1 As shown, the waste liquid discharge area 400 has a waste liquid discharge outlet 420, and the discharge channel 410 is connected to the waste liquid discharge outlet 420, which facilitates the discharge of waste liquid.

[0083] In some embodiments, such as Figure 1 and Figure 5As shown, a filter element 610 is provided at the outlet of the oil phase inlet 600 and / or the sample inlet 700. This means that a filter element 610 is provided at the outlet of the oil phase inlet 600, or at the outlet of the sample inlet 700, or at the outlets of both the oil phase inlet 600 and the sample inlet 700. The filter element 610 can filter impurities in the oil phase and the aqueous phase, to a certain extent preventing clogging of the oil phase flow channel 310 and the sample flow channel 320, and facilitating droplet formation.

[0084] In some embodiments, the filter element 610 includes multiple elements. The combination of multiple filter elements 610 can improve the ability to filter impurities in the oil phase and water phase to a certain extent, avoid clogging of the oil phase flow channel 310 and the sample flow channel 320, and thus facilitate the formation of droplets.

[0085] In a specific example, the filter element 610 is formed into a cylindrical structure, and multiple filter elements 610 are distributed at the outlets of the oil phase inlet 600 and / or the sample inlet 700, so as to achieve a cylindrical array of a certain diameter distributed at the outlets of the oil phase inlet 600 and / or the sample inlet 700.

[0086] Specifically, the height of the filter element 610 is consistent with the height of the oil phase flow channel 310 and / or the sample flow channel 320, and the diameter of the filter element 610 is 10 micrometers to 100 micrometers, so that the filter element 610 can effectively filter impurities in the oil phase and the water phase.

[0087] In one embodiment, the flow resistance of the droplet generation region 300 is equal to the flow resistance of the waste liquid discharge region 400. This reduces the movement of droplets during the PCR thermal cycling process and improves the stability of the number of droplets in the droplet detection region 100.

[0088] Of course, in some other embodiments, the flow resistance of the droplet generation region 300 and the flow resistance of the waste liquid discharge region 400 may not be exactly equal, as long as the flow resistance of the droplet generation region 300 and the flow resistance of the waste liquid discharge region 400 are basically the same.

[0089] It should be noted that the digital PCR chip 1000 designed in this application can calculate the flow resistance of the oil phase flow channel 310 of the droplet generation zone 300, the flow resistance of the sample flow channel 320, and the flow resistance of the discharge flow channel 410 of the waste liquid discharge zone 400. By adjusting the parameters to make the flow resistance of the droplet generation zone 300 and the waste liquid discharge zone 400 basically the same, the movement of droplets in the PCR thermal cycling process is reduced, thereby improving the stability of the number of droplets in the droplet detection zone 100 to a certain extent.

[0090] The flow resistance calculation of the droplet generation region 300 is divided into the flow resistance of the oil phase flow channel 310 before the cross-cutting port, the flow resistance of the sample flow channel 320, and the flow resistance of the total flow channel 330 after the cross-cutting port.

[0091] Specifically, for a microchannel with a rectangular cross-section, the formula for calculating the flow resistance under laminar flow conditions is:

[0092] In the formula, R is the flow resistance. w For the flow channel width, h The height of the flow channel. μ The viscosity coefficient of the liquid. l Let be the flow channel length. Based on the circuit equivalent model, the flow resistance of the droplet generation region 300 and the waste liquid discharge region 400 is calculated as follows: First, the flow resistance calculation of the droplet generation region 300 is divided into the flow resistance calculation of the oil phase flow channel 310 and the sample flow channel 320 before the cross-cutting port, and the flow resistance calculation of the total flow channel 330 after the cross-cutting port.

[0093] Step 1: Calculate the flow resistance of the oil phase flow channel 310 and the sample flow channel 320 before the cross-cut inlet, combined with... Figure 1 and Figure 4 As shown, the flow resistance of the oil phase flow channel 310 and the sample flow channel 320 before the cross-cut can be modeled using the circuit equivalence method. The flow resistance of the oil phase flow channel 310 is R1, the flow resistance of the first oil phase flow channel 311 is R2, the flow resistance of the second oil phase flow channel 312 is R3, and the flow resistance of the sample flow channel 320 is R4.

[0094] According to the circuit equivalence method, R1 = (R2 * R3) / (R2 + R3). Since the first oil phase flow channel 311 and the second oil phase flow channel 312 have the same flow width, flow height, liquid viscosity coefficient and flow length, R2 = R3. By calculation, R1 = 1 / 2R2. The flow resistance R2 of the first oil phase flow channel 311 can be calculated by the above flow resistance calculation formula. Substituting R2 into the formula R1 = 1 / 2R2, the flow resistance R1 of the oil phase flow channel 310 is obtained.

[0095] Next, the flow resistance R4 of the sample flow channel 320 is calculated using the above flow resistance calculation formula.

[0096] Similarly, according to the circuit equivalence method, the sum of the flow resistance of the oil phase flow channel 310 and the flow resistance of the sample flow channel 320 is R5 = (R1 * R4) / (R1 + R4).

[0097] In summary, the sum of the flow resistances of the oil phase flow channel 310 and the sample flow channel 320 before the cross-cutting inlet was calculated.

[0098] Step 2: Calculate the flow resistance of the main flow channel 330 after the cross-cut. Define the flow resistance of the main flow channel 330 as R6. Substitute the measured flow channel width, flow channel height, liquid viscosity coefficient and flow channel length of the main flow channel 330 into the flow resistance calculation formula to calculate the flow resistance R6 of the main flow channel 330. Then calculate the flow resistance R7 = R5 + R6 of the droplet generation zone 300.

[0099] Secondly, the flow resistance of the discharge channel 410 in the waste liquid discharge zone 400 is calculated. The flow resistance of the discharge channel 410 is defined as R8. The flow resistance R8 of the discharge channel 410 can be calculated by substituting the measured channel width, channel height, liquid viscosity coefficient and channel length into the flow resistance calculation formula. That is, the flow resistance of the waste liquid discharge zone 400 is calculated as R8. This makes it easier to set the flow resistance of the droplet generation zone 300 and the flow resistance of the waste liquid discharge zone 400 to be basically the same. This can effectively reduce the movement speed of droplets in the droplet detection zone 100 during the PCR thermal cycling process, increase the amount of droplets retained in the droplet detection zone 100, and ensure the stability of the number of droplets in the droplet detection zone 100.

[0100] In summary, the digital PCR chip 1000 of this application uses the shearing action of a focused microfluidic channel to generate droplets. The channel of the digital PCR chip 1000 is divided into three parts: a droplet generation area 300, a droplet detection area 100, and a waste liquid discharge area 400. The droplet generation area 300 is based on focused microfluidics, that is, droplets are generated under the shearing action of the oil phase against the water phase. The droplet size is affected by the flow rate and flow rate ratio of the oil phase and the water phase. A row of anchoring components 200 is provided near the connection between the droplet generation area 300 and the droplet detection area 100. After the droplets are generated from the cross-shaped focusing port, they enter the first detection area 110. Under the action of the anchoring components 200, the droplets can be dispersed and flow evenly to the second detection area 120, achieving droplet flattening. There is a certain width between the adjacent anchoring structures 210 of the anchoring components 200 near the connection between the droplet generation area 300 and the droplet detection area 100. This width allows the droplets to pass through the adjacent anchoring structures 210 during the generation process. The droplet reaches the second detection area 120 without breaking. The anchoring surface of the anchoring structure 210 is set towards the waste liquid discharge area 400. The anchoring surface of the anchoring structure 210 is provided with an anchoring recess 211, which is recessed in the direction away from the second detection area 120, so as to fix the droplet in the second detection area 120 during PCR heating. Near the connection between the droplet detection area 100 and the waste liquid discharge area 400, a row of anchoring components 200 is provided. The anchoring surface of the anchoring structure 210 of the anchoring component 200 is set towards the droplet generation area 300. The anchoring surface of the anchoring structure 210 is provided with an anchoring recess 211, which is recessed in the direction away from the second detection area 120, so as to fix the droplet and prevent the droplet from flowing into the waste liquid discharge area 400. There is a certain distance between the adjacent anchoring structures 210 of the anchoring component 200, so that the corresponding volume of continuous phase can flow out smoothly when the droplet is generated.

[0101] In a specific example, the digital PCR chip 1000 of this application is suitable for generating droplets with a diameter of 60 μm. The flow channel height of the digital PCR chip 1000 is 60 μm-100 μm. There is a row of anchoring components 200 at the inlet and outlet positions of the second detection area 120. The anchoring components 200 fill the full width of the second detection area 120. The minimum distance between adjacent anchoring structures 210 in each row of the second detection area 120 is 6 μm-120 μm. The second detection area 120 uses rhomboid pillars as support pillars 121, which can generate a certain obstruction when the droplets are generated, reducing the speed of the droplets when they flow to the outlet of the second detection area 120. The height of the support pillars 121 and the height of the second detection area 120 are set to be the same to prevent the second detection area 120 from collapsing during the processing of the digital PCR chip 1000, thereby avoiding affecting the size of the generated droplets and the arrangement of the second detection area 120.

[0102] In the description of this utility model, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "joining" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this utility model based on the specific circumstances.

[0103] Figure 1 The above diagram shows six support columns 121 for illustrative purposes. However, after reading the above technical solution, those skilled in the art will obviously understand that applying this solution to one, two, three, or more support columns 121 would also fall within the protection scope of this utility model.

[0104] Other components of the digital PCR chip 1000 according to embodiments of the present invention, such as the specific structures of the oil phase inlet 600 and the sample inlet 700, are known to those skilled in the art and will not be described in detail here.

[0105] In this specification, the terms "embodiment," "example," etc., refer to specific features, structures, materials, or characteristics described in connection with that embodiment or example, which are included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0106] Although embodiments of the present invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the present invention, the scope of which is defined by the claims and their equivalents.

Claims

1. A digital PCR chip, characterized in that, include: Droplet detection area (100); An anchoring component (200) is spaced at opposite ends of the droplet detection area (100) and is used to fix the droplet within the droplet detection area (100).

2. The digital PCR chip according to claim 1, characterized in that, It also includes a droplet generation area (300) and a waste liquid discharge area (400). The droplet detection area (100) includes a first detection area (110), a second detection area (120) and a third detection area (130) arranged sequentially and interconnected along a first direction. The first detection area (110) is connected to the droplet generation area (300), and the third detection area (130) is connected to the waste liquid discharge area (400). The anchoring components (200) are spaced at opposite ends of the second detection area (120).

3. The digital PCR chip according to claim 2, characterized in that, The anchoring assembly (200) includes a plurality of anchoring structures (210) arranged along a second direction, the anchoring structures (210) being used to limit the displacement of the droplet, the second direction intersecting the first direction; Two adjacent anchoring structures (210) are spaced apart to form a flow channel (500), the minimum width of the flow channel (500) is W, and the diameter of the droplet is D, where W = 0.1D~2D.

4. The digital PCR chip according to claim 3, characterized in that, The width of the flow channel (500) gradually decreases in the direction toward the second detection area (120).

5. The digital PCR chip according to claim 3, characterized in that, The anchoring structure (210) has an anchoring recess (211) on the side facing the second detection area (120). The anchoring recess (211) is recessed in a direction away from the second detection area (120). In the second direction, the width of the anchoring recess (211) is greater than or equal to the diameter of the droplet. The anchoring recess (211) is used to limit the displacement of the droplet.

6. The digital PCR chip according to claim 2, characterized in that, In the second direction, the width of the anchoring component (200) is equal to or less than the width of the second detection area (120), and the second direction intersects with the first direction; And / or, in the third direction, the height of the anchoring component (200) is equal to the height of the second detection area (120), the height of the second detection area (120) is H, the diameter of the droplet is D, where H = 1.0D~1.5D, and the third direction, the second direction and the first direction intersect each other.

7. The digital PCR chip according to claim 2, characterized in that, The second detection area (120) has multiple rows of anchoring components (200) at its opposite ends, and the multiple rows of anchoring components (200) are arranged along the first direction.

8. The digital PCR chip according to claim 2, characterized in that, It also includes a seal that covers the droplet detection area (100) to seal the droplet detection area (100), and the thickness of the seal is in the range of 0.1mm-1mm; The second detection area (120) is provided with a support column (121), and the support column (121) is fixedly connected to the sealing element.

9. The digital PCR chip according to claim 2, characterized in that, The droplet generation area (300) has an oil phase flow channel (310) and a sample flow channel (320), and the waste liquid discharge area (400) has a discharge flow channel (410). At least one of the oil phase flow channel (310), the sample flow channel (320) and the discharge flow channel (410) has a plurality of bends (321). The oil phase flow channel (310) includes a first oil phase flow channel (311) and a second oil phase flow channel (312). The first end of the first oil phase flow channel (311) and the first end of the second oil phase flow channel (312) are connected to the oil phase inlet (600). The first end of the sample flow channel (320) is connected to the sample inlet (700). The droplet generation area (300) also has a total flow channel (330). The second end of the first oil phase flow channel (311), the second end of the second oil phase flow channel (312), and the second end of the sample flow channel (320) are all connected to the total flow channel (330). The extension direction of the total flow channel (330) is consistent with the extension direction of the second end of the sample flow channel (320) and perpendicular to the extension direction of the second end of the first oil phase flow channel (311) and the extension direction of the second end of the second oil phase flow channel (312). A filter element (610) is provided at the outlet of the oil phase inlet (600) and / or the sample inlet (700).

10. The digital PCR chip according to any one of claims 2-9, characterized in that, The flow resistance of the droplet generation zone (300) is equal to the flow resistance of the waste liquid discharge zone (400).

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