Reagent transport method and surface fluidics system for genetic sequencing

Abstract

The application discloses a reagent transmission method and a surface liquid channel system for gene sequencing. The reagent transmission method adopts a coating die to coat a reagent on the surface of a sequencing chip; when the reagent on the sequencing chip is displaced, the coating die coats a new reagent on the sequencing chip, so that the newly coated reagent flows on the sequencing chip and displaces the original reagent on the sequencing chip.

Description

Technical Field

[0001] This application relates to the field of gene sequencing technology, such as a reagent delivery method and a surface liquid circuit system for gene sequencing. Background Technology

[0002] In currently widely used next-generation sequencing methods, sample loading and fluorescent labeling biochemical reactions are both completed within the sequencing chip. The sequencing chip consists of a chip and a cover plate assembled vertically, with the chip and cover plate forming a flow cell chamber to hold reagents. Base sequence identification is achieved through multiple rounds of imaging.

[0003] Gene sequencing requires the use of various reagents and up to 200 reaction-photograph cycles. The thickness of the reagent in each cycle is the gap between the chip and the cover plate. Due to assembly and manufacturing difficulties, the smallest gap that can be achieved at present is 50 micrometers, while the reagent layer that reacts is only about 1 micrometer close to the chip. More than 95% of the reagents are wasted.

[0004] Furthermore, the liquid circuit system in the related technology includes tubing that introduces reagents into the flow cell chamber, where the reagent components remain on the inner walls of the flow cell chamber and the tubing. However, according to sequencing requirements, each cycle requires new reagents to replace 99.9% of the previous reagent before the reaction can begin. This necessitates a prolonged introduction of new reagents to replace the existing reagents on the tubing and the inner walls of the flow cell chamber.

[0005] The performance, time, and cost of gene sequencing in related technologies are limited for the reasons mentioned above. Summary of the Invention

[0006] This application provides a reagent transport method and a surface liquid circuit system for gene sequencing, in order to avoid the serious waste of reagents, long time and high cost in the gene sequencing process of related technologies.

[0007] This application provides a reagent delivery method, which involves coating a sequencing chip surface with a coating head;

[0008] When replacing reagents on the sequencing chip, the coating head applies new reagents to the sequencing chip, causing the newly applied reagents to flow on the sequencing chip and replace the original reagents on the sequencing chip.

[0009] This application provides a surface liquid path system for gene sequencing, comprising:

[0010] A sequencing chip, wherein one side surface of the sequencing chip is a coated surface;

[0011] A coating apparatus includes a coating die and an angle adjustment assembly. The coating die is located above the coating surface, and the coating die and the sequencing chip are movable relative to each other in a direction parallel to the coating surface. The coating die is configured to coat the coating surface with reagent to form a reagent coating layer or to displace the existing reagent on the coating surface and form a new reagent coating layer. The angle adjustment assembly is configured to adjust the coating angle of the coating die, which is the angle between the reagent flow direction within the coating die and the coating surface. Attached Figure Description

[0012] Figure 1 This is a schematic diagram of the surface liquid path system provided in this application during reagent coating;

[0013] Figure 2 This is a schematic diagram of the surface liquid circuit system provided in this application during reagent displacement.

[0014] In the picture:

[0015] 10. Sequencing chip; 20. Coating head; 21. Flow channel; 22. Soft flow tank; 30. Reagent; 30a. Displacement reagent; 30b. Original reagent. Detailed Implementation

[0016] In the description of this application, unless otherwise expressly specified and limited, the terms "connected," "linked," and "fixed" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral part; 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; they can refer to the internal communication of two components or the interaction between two components. Those skilled in the art can understand the specific meaning of the above terms in this application based on the specific circumstances.

[0017] In this application, unless otherwise expressly specified and limited, "above" or "below" the second feature can include direct contact between the first and second features, or contact between the first and second features through another feature between them. Furthermore, "above," "over," and "on top" of the second feature includes the first feature being directly above or diagonally above the second feature, or simply indicates that the first feature is at a higher horizontal level than the second feature. "Below," "below," and "under" the second feature includes the first feature being directly below or diagonally below the second feature, or simply indicates that the first feature is at a lower horizontal level than the second feature.

[0018] In the description of this embodiment, the terms "upper," "lower," "right," etc., refer to the orientation or positional relationship shown in the accompanying drawings. They are used only for ease of description and simplification of operation, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. In addition, the terms "first" and "second" are used only for distinction in description and have no special meaning.

[0019] This embodiment provides a reagent delivery method for gene sequencing, such as... Figure 1 As shown, a coating head 20 is used to coat the surface of the sequencing chip 10 with reagent 30. For example, the coating head 20 moves in a direction parallel to the sequencing chip 10 to coat the reagent 30 directly onto the surface of the sequencing chip 10. No flow chamber is needed, allowing the reagent 30 to flow on the surface of the sequencing chip 10. The flow area of ​​the reagent 30 is an open space, which reduces the thickness of the reagent 30. The thickness of the coated reagent 30 only needs to meet the requirements of gene sequencing, avoiding the situation in related technologies where the assembly difficulty limits the thickness of the reagent 30 flowing between the cover plate and the sequencing chip 10, resulting in significant reagent waste. This helps to save costs.

[0020] For example, the thickness L of the coated reagent 30 can be 5μm-50μm, such as 5μm, 15μm, 20μm, 25μm, 30μm, 35μm, 40μm, 45μm or 50μm.

[0021] In related technologies, due to the difficulty of assembling and sealing the cover plate and the chip, the distance between the cover plate and the chip cannot be less than 50 μm. That is, the thickness of reagent 30 in related technologies is not less than 50 μm. However, the actual thickness of reagent 30 participating in the reaction is only about 1 μm close to the chip, resulting in more than 95% of reagent 30 being wasted. In this embodiment, the coating space is an open space, and the thickness of reagent 30 can be reduced to less than 50 micrometers, greatly reducing the waste of reagent 30.

[0022] In related technologies, because the sequencing chip 10 and the cover plate form a flow cell chamber, the flow rate of the reagent 30 within the flow cell chamber cannot be too fast. Excessive flow of the reagent 30 generates significant pressure within the flow cell chamber, which can easily lead to the breakage of the sequencing chip 10 or the cover plate. In this embodiment, the flow area of ​​the reagent 30 is an open space, resulting in less pressure exerted on the sequencing chip 10 by the flow of the reagent 30, thus preventing excessive pressure from causing the sequencing chip 10 to break.

[0023] To meet gene sequencing needs, when it is necessary to replace the original reagent 30b on sequencing chip 10, such as Figure 2As shown, new displacing reagent 30a is applied through the coating head 20. When the displacing reagent 30a flows on the sequencing chip 10, it displaces the original reagent 30b on the sequencing chip 10. Because the reagent 30 is in an open space, it only contacts the surface of the sequencing chip 10. Compared to related technologies that use a flow cell chamber, the contact area is smaller, which helps to shorten the time required to displace the original reagent 30b. Furthermore, there is no need to install pipes to introduce the reagent 30 into the flow cell chamber, meaning that when replacing the original reagent 30b, only the original reagent 30b on the sequencing chip 10 needs to be replaced. This reduces the amount of displacing reagent 30a required to replace the original reagent 30b, saving costs and avoiding waste of reagent 30.

[0024] To improve the displacement effect of the original reagent 30b, combined with Figure 1 and Figure 2 As shown, when the reagent 30 on the sequencing chip 10 is replaced, the coating angle B of the coating head 20 is smaller than the coating angle A when the reagent 30 is first coated onto the surface of the sequencing chip 10. The coating angle is the angle between the flow direction of the reagent 30 in the coating head 20 and the sequencing chip 10.

[0025] Understandably, the smaller the coating angle, the greater the driving pressure of the reagent 30 provided by the coating head 20 flowing along the surface of the sequencing chip 10, while the driving pressure of the reagent 30 in the direction perpendicular to the sequencing chip 10 decreases. A greater driving pressure of the reagent 30 flowing along the surface of the sequencing chip 10 results in a better displacement effect of the displacing reagent 30a on the original reagent 30b.

[0026] like Figure 1 As shown, when the reagent 30 is initially coated onto the surface of the sequencing chip 10, the sequencing chip 10 remains stationary, while the coating head 20 can move along the X direction parallel to the top surface of the sequencing chip 10 (hereinafter referred to as the coating surface) to perform coating and form the first reagent layer. Figure 1 The flow direction of reagent 30 within the coating die 20 is the Z-direction, which is perpendicular to the X-direction. Therefore... Figure 1 The coating angle of the coating die 20 shown is A, for example, 90°. When the coating angle is 90°, the coating of reagent 30 conforms to the characteristics of Couette flow between parallel plates, so that the driving pressure of the liquid outlet of the coating die 20 on reagent 30 is evenly distributed and perpendicular to the coating surface, ensuring the uniformity of the coating of reagent 30.

[0027] When it is necessary to replace reagent 30 on the surface of sequencing chip 10, such as Figure 2As shown, the sequencing chip 10 remains stationary, and the coating head 20 moves along the X direction, parallel to the coating surface, to coat and form the second reagent layer. At this time, the coating head 20 is tilted, and the flow direction of the displacing reagent 30a within the coating head 20 is the Y direction. The Y direction forms an angle with both the X and Z directions, and the coating angle of the coating head 20 is B, for example, 30°. When the coating angle is 30°, it achieves both good displacing of the original reagent 30b and ensures that the surface of the sequencing chip 10 remains moist.

[0028] In other embodiments, the coating angle can be adjusted according to actual needs. The coating angle can be 0°-90°, for example, 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80° or 90°.

[0029] To improve coating quality, the distance H (hereinafter referred to as coating height) between the coating die 20 and the coating surface is 1 to 3 times the thickness L of the reagent 30 during coating. According to the approximate lubrication model of Couette flow between parallel plates and Prof. Marcio, the coating height is proportionally related to the thickness of the coated reagent 30. When the coating height is less than the thickness of the reagent 30, the coated reagent 30 is prone to wavy coating surface, resulting in an uneven surface. When the coating height is more than three times the thickness of the reagent 30, the reagent 30 is prone to liquid bridge breakage, resulting in blank areas on the coating surface and a decrease in the uniformity of the coated reagent 30.

[0030] For example, the coating height of the coating die 20 during coating can be 5μm-150μm, such as 5μm, 10μm, 20μm, 30μm, 40μm, 50μm, 60μm, 70μm, 80μm, 90μm, 100μm, 110μm, 120μm, 130μm, 140μm or 150μm.

[0031] When the pumping speed of reagent 30 in the coating head 20 is constant, the coating head 20 coats the reagent 30 at a coating angle perpendicular to the coating plane, uniformly coating the reagent 30 onto the sequencing chip 10 with a certain thickness. For example, the pumping speed of reagent 30 in the coating head 20 is 0.001 ml / s-10 ml / s, such as 0.001 ml / s, 0.03 ml / s, 0.05 ml / s, 0.1 ml / s, 0.5 ml / s, 1 ml / s, 1.5 ml / s, 2 ml / s, 2.5 ml / s, 4 ml / s, 5.5 ml / s, 6.5 ml / s, 8 ml / s, 9.5 ml / s, and 10 ml / s.

[0032] Using the reagent delivery method provided in this embodiment, since the reagent 30 is directly coated on the surface of the sequencing chip 10, there is no need to set up a flow cell chamber or a pipeline for transporting the reagent 30. This helps to save the amount of reagent 30 used and avoid waste. Only one-tenth of the amount of reagent 30 used in related technologies is needed to complete the same gene sequencing. Because there is no need to worry about the flow pressure of the reagent 30 damaging the sequencing chip 10, the moving speed of the coating head 20 in the X direction during the coating process can be increased from 0.06 m / s to 1 m / s, which helps to improve the coating efficiency of the reagent 30 and thus shorten the gene sequencing time.

[0033] Furthermore, the sequencing chip 10 does not require the use of tubing and a cover plate, and its size can match the width of the coating head 20. In related technologies, the width of the coating head 20 can reach 2 μm, meaning that the coating head 20 can coat a reagent layer with a width of 2 μm in one pass. Therefore, the width of the sequencing chip 10 can be increased from 7 cm to 2 μm.

[0034] This embodiment provides a surface liquid path system for gene sequencing, which can employ the reagent transfer method described in the foregoing embodiments. The surface liquid path system provided in this application, using the aforementioned reagent transfer method, can avoid reagent waste and improve gene sequencing efficiency. Figure 1 As shown, the surface liquid path system includes a sequencing chip 10 and a coating device. One side surface of the sequencing chip 10 is the coating surface, and the coating device is configured to coat a layer of reagent 30 onto the coating surface. For example, the coating device includes a coating die 20 and an angle adjustment component. The coating die 20 is located above the coating surface, and the coating die 20 and the sequencing chip 10 can move relative to each other in a direction parallel to the coating surface. The coating die 20 is configured to coat the coating surface with reagent 30 to form a reagent coating layer or to displace the original reagent 30 on the coating surface and form a new reagent coating layer. The angle adjustment component is configured to adjust the coating angle of the coating die 20, which is the angle between the flow direction of the reagent 30 within the coating die 20 and the coating surface.

[0035] Compared to related technologies that use a cover plate to enclose the flow cell chamber on the surface of the sequencing chip 10, the reagent 30 coating space in this embodiment is an open space, which can reduce the thickness of the reagent 30 and avoid the situation in related technologies where the thickness of the reagent 30 between the cover plate and the sequencing chip 10 is large due to assembly difficulty. This reduces reagent 30 waste and helps save costs.

[0036] In this embodiment, the coating die head 20 can be a slit coating die head. The coating die head 20 is provided with a flow channel 21, which is a flat slit structure. The reagent 30 is pumped through the flow channel 21 in the coating die head 20 to be transferred to the coating surface to complete the coating.

[0037] To ensure that reagent 30 is continuously coated onto the coating surface of sequencing chip 10, a buffer groove 22 is also provided inside the coating head 20, and the flow channel 21 passes through and communicates with the buffer groove 22. By adding the buffer groove 22 inside the coating head 20, a certain amount of reagent 30 can be buffered inside the coating head 20 to ensure that the flow channel 21 can continuously supply reagent 30 for coating.

[0038] In this embodiment, the slow-flow channel 22 is a semi-circular channel with a smooth inner wall and no sharp corners, which can prevent reagent 30 residue. In other embodiments, the shape of the slow-flow channel 22 can be adjusted as needed, for example, it can be a rectangular channel.

[0039] For example, the coating apparatus further includes a position driving mechanism, which is configured to drive the coating die 20 to rise and fall and move along a direction parallel to the coating surface. In this embodiment, the position driving mechanism includes a lifting component and a linear translation component. The output end of the lifting component is connected to the linear translation component, and the output end of the linear translation component is connected to an angle adjustment component. The coating die 20 is disposed at the output end of the angle adjustment component. The lifting component can drive the linear translation component, the angle adjustment component, and the coating die 20 to rise and fall. The linear translation component can drive the angle adjustment component and the coating die 20 to reciprocate along the X direction. The angle adjustment component can drive the coating die 20 to rotate to adjust the coating angle.

[0040] For example, both the lifting assembly and the linear translation assembly can be one of a lead screw and nut assembly, a gear and rack assembly, a cylinder, a hydraulic cylinder, or a linear motor. The output end of the lifting assembly moves vertically, and the output end of the linear translation assembly moves along the X-direction. The angle adjustment assembly includes a rotary motor, the output shaft of which is perpendicular to both the X and Z directions.

[0041] For example, the coating die 20 moves at a speed of 0 m / min to 200 m / min in the direction parallel to the coating surface.

[0042] To accommodate different reagents 30, the coating die 20 has at least two flow channels 21, each corresponding to a different reagent 30. When a reagent 30 needs to be replaced, the corresponding flow channel 21 is replaced.

[0043] In some embodiments, multiple coating dies 20 are provided, and each coating die 20 corresponds to a reagent 30. When it is necessary to replace the reagent 30, the working coating die 20 can be replaced.

Claims

1. A reagent transport method, characterized in that, Reagents are applied to the surface of the sequencing chip (10) using a coating head (20); When replacing the reagents on the sequencing chip (10), the coating head (20) coats the sequencing chip (10) with new reagents, so that the newly coated reagents flow on the sequencing chip (10) and replace the original reagents on the sequencing chip (10).

2. The method according to claim 1, wherein, When displacing the reagent on the sequencing chip (10), the coating angle of the coating head (20) is smaller than the coating angle when the reagent is first coated onto the surface of the sequencing chip (10). The coating angle is the angle between the reagent flow direction in the coating head (20) and the sequencing chip (10).

3. The method according to claim 2, wherein, The coating angle of the coating die (20) is 0°-90°.

4. The method according to claim 2, wherein, The initial coating angle when applying reagents to the surface of the sequencing chip (10) is 90°; When displacing the reagents on the sequencing chip (10), the coating angle of the coating head (20) is 30°.

5. The method according to any one of claims 1-4, wherein, The reagent coating on the sequencing chip (10) has a thickness of 5μm-50μm.

6. The method according to any one of claims 1-4, wherein, When the coating head (20) is used for coating, the distance between the coating head (20) and the sequencing chip (10) is 1 to 3 times the thickness of the reagent.

7. The method according to any one of claims 1-4, wherein, The pumping speed of the reagent in the coating die (20) is 0.001 ml / s-10 ml / s.

8. A surface liquid path system for gene sequencing, characterized in that, include: Sequencing chip (10), one side surface of the sequencing chip (10) is a coated surface; A coating apparatus, comprising a coating die (20) and an angle adjustment assembly, wherein the coating die (20) is located above the coating surface, and the coating die (20) and the sequencing chip (10) are movable relative to each other in a direction parallel to the coating surface; the coating die (20) is configured to coat the coating surface with reagents to form a reagent coating layer or to displace the original reagents on the coating surface and form a new reagent coating layer; the angle adjustment assembly is configured to adjust the coating angle of the coating die (20), wherein the coating angle is the angle between the reagent flow direction in the coating die (20) and the coating surface.

9. The system according to claim 8, wherein, The coating apparatus further includes a position driving mechanism, which is configured to drive the coating die head (20) to rise and fall and to move in a direction parallel to the coating surface.

10. The system according to claim 8 or 9, wherein, The coating die (20) is provided with at least two flow channels (21), and each flow channel (21) corresponds to a reagent; Alternatively, multiple coating dies (20) may be provided, with each coating die (20) corresponding to coating a reagent.

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