Reagent transmission method and surface liquid path system for gene sequencing

Abstract

The invention discloses a reagent transmission method and a surface liquid path system for gene sequencing. According to the reagent transmission method, a coating die head is adopted to coat a reagent on the surface of a sequencing chip; when the reagent on the sequencing chip is displaced, the coating die head 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

Reagent delivery method and surface liquid path system for gene sequencing Technical Field

[0001] The present application relates to the field of gene sequencing technology, for example, to a reagent delivery method and a surface liquid path system for gene sequencing. Background Art

[0002] In the widely used second-generation gene sequencing method, sample loading and fluorescent labeling biochemical reactions are both performed within the gene sequencing chip. The sequencing chip consists of a chip and a cover plate assembled vertically. The space between the chip and the cover plate forms a flow cell chamber for holding reagents. The base sequence is determined by multiple rounds of imaging.

[0003] The gene sequencing process requires the use of multiple reagents and up to 200 "reaction-photography" cycles. The thickness of the reagents during each cycle is the gap between the chip and the cover plate. Due to assembly and processing difficulties, the current minimum achievable gap is 50 microns. The reagent layer that reacts only covers about 1 micron near the chip, and over 95% of the reagents in the remaining space are wasted.

[0004] Furthermore, the fluidic systems used in related technologies include pipelines that feed reagents into the flow cell chamber, where the reagent components accumulate on the inner walls of the flow cell chamber and the pipelines. However, sequencing requirements dictate that each cycle requires new reagents to replace 99.9% of the previous reagent before the reaction can begin. This results in a long period of time before the new reagents are introduced to replace the existing reagents on the pipelines and inner walls of the flow cell chamber.

[0005] Due to the above reasons, the performance, time and cost of gene sequencing in related technologies are limited.

[0006] Application Contents

[0007] The embodiments of the present application provide a reagent transfer method and a surface liquid path system for gene sequencing, so as to avoid the serious waste of reagents, long time and high cost in the gene sequencing process in related technologies.

[0008] The embodiment of the present application provides a reagent delivery method, which uses a coating die to coat the reagent on the surface of a sequencing chip;

[0009] When replacing the reagent on the sequencing chip, the coating die head coats new reagent onto the sequencing chip, so that the newly coated reagent flows on the sequencing chip and replaces the original reagent on the sequencing chip.

[0010] The present invention provides a surface liquid path system for gene sequencing, comprising:

[0011] A sequencing chip, wherein one side surface of the sequencing chip is a coated surface;

[0012] A coating device, comprising a coating die and an angle adjustment component, wherein the coating die is located above the coating surface, and the coating die and the sequencing chip can move relative to each other in a direction parallel to the coating surface; the coating die is configured to apply a reagent to the coating surface to form a reagent coating layer or to displace the original reagent 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, wherein the coating angle is the angle between the flow direction of the reagent in the coating die and the coating surface. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] FIG1 is a schematic diagram of a surface liquid path system provided by the present application when applying a reagent;

[0014] FIG2 is a schematic diagram of the surface fluid path system provided in the present application when displacing reagents.

[0015] In the picture:

[0016] 10. Sequencing chip; 20. Coating die; 21. Flow channel; 22. Slow flow trough; 30. Reagent; 30a. Displacing reagent; 30b. Original reagent. DETAILED DESCRIPTION

[0017] In the description of this application, unless otherwise expressly specified or limited, the terms "connected," "connected," and "fixed" should be understood in a broad sense. For example, they can refer to fixed connections, detachable connections, or integration; mechanical connections or electrical connections; direct connections or indirect connections through an intermediate medium; and can refer to internal communication between two components or interaction between two components. Those skilled in the art will understand the specific meanings of the above terms in this application based on the specific circumstances.

[0018] In this application, unless otherwise expressly specified or limited, a first feature being "above" or "below" a second feature may include the first and second features being in direct contact, or may include the first and second features being in contact not directly but through another feature between them. Moreover, a first feature being "above," "above," and "above" a second feature may include the first feature being directly above or obliquely above the second feature, or may simply mean that the first feature is higher in level than the second feature. A first feature being "below," "below," and "below" a second feature may include the first feature being directly below or obliquely below the second feature, or may simply mean that the first feature is lower in level than the second feature.

[0019] In the description of this embodiment, the terms "upper," "lower," "right," and other orientations or positional relationships are based on the orientations or positional relationships shown in the accompanying drawings and are intended solely for ease of description and simplified operation. They do not indicate or imply that the devices or components referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on this application. Furthermore, the terms "first" and "second" are used solely for descriptive purposes and have no special meanings.

[0020] This embodiment provides a reagent transfer method for gene sequencing. As shown in FIG1 , a coating die 20 is used to coat a reagent 30 onto the surface of a sequencing chip 10. For example, the coating die 20 moves parallel to the sequencing chip 10 to coat the reagent 30. The reagent 30 is directly coated on the surface of the sequencing chip 10, eliminating the need for a flow cell chamber. This allows the reagent 30 to flow across the surface of the sequencing chip 10. The flow area of ​​the reagent 30 is an open space, which reduces the thickness of the coated reagent 30. The thickness of the coated reagent 30 only needs to meet the requirements of gene sequencing. This avoids the problem in related technologies where the reagent 30 flowing between the cover plate and the sequencing chip 10 is thick and wasteful due to assembly difficulties, thereby saving costs.

[0021] For example, the thickness L of the coated agent 30 may be 5 μm to 50 μm, such as 5 μm, 15 μm, 20 μm, 25 μm, 30 μm, 35 μm, 40 μm, 45 μm or 50 μm.

[0022] In the prior art, the distance between the cover plate and the chip cannot be less than 50 μm due to the difficulty of assembly and sealing between them. This means that the thickness of the reagent 30 in the prior art is no less than 50 μm, while the thickness of the reagent 30 actually involved in the reaction is only about 1 μm near the chip, resulting in more than 95% of the reagent 30 being wasted. In this embodiment, the coating space is open, and the thickness of the reagent 30 can be reduced to less than 50 μm, significantly reducing reagent 30 waste.

[0023] In the prior art, because the flow cell chamber is enclosed between the sequencing chip 10 and the cover plate, the flow rate of the reagent 30 within the flow cell chamber cannot be too fast. Excessive flow of the reagent 30 generates high pressure within the flow cell chamber, which can easily cause the sequencing chip 10 or the cover plate to rupture. In this embodiment, the flow area of ​​the reagent 30 is an open space, so the pressure exerted by the flow of the reagent 30 on the sequencing chip 10 is relatively small, and the sequencing chip 10 will not rupture due to excessive pressure.

[0024] To meet the needs of gene sequencing, when the original reagent 30b on the sequencing chip 10 needs to be replaced, as shown in FIG2 , a new displacing reagent 30a is applied through the coating die 20. When the displacing reagent 30a flows on the sequencing chip 10, it can displace the original reagent 30b on the sequencing chip 10. Because the reagent 30 is in an open space, the reagent 30 only contacts the surface of the sequencing chip 10. Compared with the flow cell chamber provided in the related art, the contact area is smaller, which helps to shorten the time to displace the original reagent 30b. Furthermore, there is no need to set up a pipeline to pass the reagent 30 into the flow cell chamber. Therefore, when replacing the original reagent 30b, only the original reagent 30b on the sequencing chip 10 needs to be displaced. This reduces the amount of displacing reagent 30a required to displace the original reagent 30b, which helps save costs and avoids waste of reagent 30.

[0025] To improve the displacement effect of the original reagent 30b, as shown in Figures 1 and 2, when replacing the reagent 30 on the sequencing chip 10, the coating angle B of the coating die 20 is smaller than the coating angle A when the reagent 30 is first coated on the surface of the sequencing chip 10. The coating angle is the angle between the flow direction of the reagent 30 in the coating die 20 and the sequencing chip 10.

[0026] It is understood that the smaller the coating angle, the greater the driving pressure provided by the coating die 20 to cause the reagent 30 to flow along the surface of the sequencing chip 10, while the driving pressure of the reagent 30 in a direction perpendicular to the sequencing chip 10 decreases. The greater the driving pressure for the reagent 30 to flow along the surface of the sequencing chip 10, the better the displacement effect of the displacing reagent 30a on the original reagent 30b.

[0027] As shown in Figure 1 , when the reagent 30 is initially applied to the surface of the sequencing chip 10, the sequencing chip 10 remains stationary, and the coating die 20 can move in the X direction, parallel to the top surface of the sequencing chip 10 (hereinafter referred to as the coating surface), to apply the reagent 30 and form a first reagent layer. In Figure 1 , the flow direction of the reagent 30 within the coating die 20 is the Z direction, which is perpendicular to the X direction. Therefore, the coating angle of the coating die 20 shown in Figure 1 is A, for example, 90°. When the coating angle is 90°, the coating of the 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 the reagent 30 is evenly distributed and perpendicular to the coating surface, ensuring the uniformity of the coating of the reagent 30.

[0028] When the reagent 30 on the surface of sequencing chip 10 needs to be replaced, as shown in Figure 2, the sequencing chip 10 remains stationary, and the coating die 20 moves in the X direction, parallel to the coating surface, to apply the coating and form a second reagent layer. At this point, the coating die 20 is tilted, and the direction of flow of the displaced reagent 30a within the coating die 20 is the Y direction. The Y direction forms an angle with both the X and Z directions. The coating angle of the coating die 20 is B, for example, 30°. A coating angle of 30° effectively displaces the original reagent 30b while ensuring that the surface of the sequencing chip 10 remains moist.

[0029] In other embodiments, the coating angle can be adjusted according to actual needs, and the coating angle can be 0°-90°, for example, 0°, 10°, 20°, 30°, 40°, 50°, 60°, 70°, 80° or 90°.

[0030] To improve coating quality, when the coating die 20 is coating, the distance H between the coating die 20 and the coating surface (hereinafter referred to as the coating height) is 1 to 3 times the thickness L of the reagent 30. According to the Couette flow between parallel plates and the lubrication approximation model of Professor Marcio, the coating height is proportionally correlated with 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 a corrugated coating surface, resulting in an uneven surface of the coated reagent 30. When the coating height is greater than three times the thickness of the reagent 30, the reagent 30 is prone to liquid bridges being broken, resulting in white areas on the coating surface and reduced uniformity of the coated reagent 30.

[0031] For example, the coating height of the coating die 20 during coating can be 5 μm-150 μm, such as 5 μm, 10 μm, 20 μm, 30 μm, 40 μm, 50 μm, 60 μm, 70 μm, 80 μm, 90 μm, 100 μm, 110 μm, 120 μm, 130 μm, 140 μm or 150 μm.

[0032] When the pumping speed of the reagent 30 in the coating die 20 is constant, the coating die 20 applies the reagent 30 at a coating angle perpendicular to the coating plane, and the reagent 30 is evenly coated on the sequencing chip 10 with a certain thickness. For example, the pumping speed of the reagent 30 in the coating die 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.

[0033] The reagent transfer method provided in this embodiment allows reagent 30 to be directly applied to the surface of sequencing chip 10, eliminating the need for a flow cell chamber or piping to transfer reagent 30. This helps conserve reagent 30 usage and avoids reagent 30 waste. The same gene sequencing can be completed using only one-tenth the amount of reagent 30 used in related techniques. Because there is no need to worry about the flow pressure of reagent 30 damaging sequencing chip 10, the X-direction movement speed of coating die 20 during the coating process can be increased from 0.06 m / s to 1 m / s, improving reagent 30 coating efficiency and thus shortening gene sequencing time.

[0034] Furthermore, sequencing chip 10 does not require a tube or cover plate for use; its dimensions can match the width of coating die 20. In related art, coating die 20 can be up to 2 meters wide, meaning it can apply a reagent coating layer up to 2 meters wide in a single application. Therefore, the width of sequencing chip 10 can be increased from 7 cm to 2 meters.

[0035] This embodiment provides a surface liquid path system for gene sequencing, which can adopt the reagent transfer method of the aforementioned embodiment. The surface liquid path system provided by this application adopts the above-mentioned reagent transfer method, which can avoid reagent waste and improve gene sequencing efficiency. As shown in Figure 1, the surface liquid path system includes a sequencing chip 10 and a coating device. One side surface of the sequencing chip 10 is a coating surface, and the coating device is configured to apply a layer of reagent 30 on 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. 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 apply the reagent 30 to the coating surface to form a reagent coating layer or 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. The coating angle is the angle between the flow direction of the reagent 30 in the coating die 20 and the coating surface.

[0036] Compared with the related art in which a cover plate is added to the surface of the sequencing chip 10 to enclose a flow cell chamber, 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 the related art where the thickness of the reagent 30 between the cover plate and the sequencing chip 10 is large due to assembly difficulty limitations, thereby reducing the waste of reagent 30 and saving costs.

[0037] In this embodiment, the coating die 20 can be a slit coating die, and a flow channel 21 is provided in the coating die 20. The flow channel 21 is a flat slit structure. The reagent 30 is pumped through the flow channel 21 in the coating die 20 and transferred to the coating surface to complete the coating.

[0038] To ensure continuous coating of the reagent 30 onto the coating surface of the sequencing chip 10, a slow-flow groove 22 is provided within the coating die 20. The flow channel 21 passes through and communicates with the slow-flow groove 22. By adding the slow-flow groove 22 to the coating die 20, a certain amount of reagent 30 can be buffered within the coating die 20, ensuring that the flow channel 21 can continuously supply the reagent 30 for coating.

[0039] In this embodiment, the slow flow groove 22 is a semicircular groove, and the inner wall of the semicircular groove is smoothly transitioned and has no sharp corners, which can prevent the residue of the reagent 30. In other embodiments, the shape of the slow flow groove 22 can be adjusted as needed, for example, it can be a rectangular groove.

[0040] For example, the coating device further includes a position drive mechanism configured to drive the coating die head 20 to move up and down as well as move in a direction parallel to the coating surface. In this embodiment, the position drive mechanism includes a lifting assembly and a linear translation assembly. The output end of the lifting assembly is connected to the linear translation assembly, which in turn is connected to the angle adjustment assembly. The coating die head 20 is disposed at the output end of the angle adjustment assembly. The lifting assembly is capable of driving the linear translation assembly, the angle adjustment assembly, and the coating die head 20 to move up and down. The linear translation assembly is capable of driving the angle adjustment assembly and the coating die head 20 to reciprocate in the X-direction. The angle adjustment assembly is capable of driving the coating die head 20 to rotate to adjust the coating angle.

[0041] For example, the lifting assembly and the linear translation assembly can each be a screw-nut assembly, a rack-and-pinion assembly, a pneumatic cylinder, a hydraulic cylinder, or a linear motor. The output end of the lifting assembly moves in the vertical direction, and the output end of the linear translation assembly moves in the X direction. The angle adjustment assembly includes a rotary motor, and the output shaft of the rotary motor is perpendicular to the X and Z directions, respectively.

[0042] For example, the coating die head 20 moves at a speed in a direction parallel to the coating surface of 0 m / min to 200 m / min.

[0043] In order to replace different reagents 30, at least two flow channels 21 are provided in the coating die head 20, and each flow channel 21 corresponds to a reagent 30. When the reagent 30 needs to be replaced, the flow channel 21 used is replaced accordingly.

[0044] In some embodiments, a plurality of coating die heads 20 are provided, and each coating die head 20 is configured to accommodate a reagent 30 . When the reagent 30 needs to be replaced, the working coating die head 20 can be replaced.

Claims

1. A reagent transfer method, comprising applying a reagent to the surface of a sequencing chip (10) using a coating die (20); When replacing the reagent on the sequencing chip (10), the coating die head (20) applies new reagent to the sequencing chip (10), so that the newly coated reagent flows on the sequencing chip (10) and replaces the original reagent 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 die (20) is smaller than the coating angle when the reagent is initially coated on the surface of the sequencing chip (10), and the coating angle is the angle between the flow direction of the reagent in the coating die (20) and the sequencing chip (10).

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

4. The method according to claim 2, wherein: The coating angle when the reagent is first coated on the surface of the sequencing chip (10) is 90°; When the reagent on the sequencing chip (10) is displaced, the coating angle of the coating die head (20) is 30°.

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

6. The method according to any one of claims 1 to 4, wherein When the coating die (20) is coating, the distance between the coating die (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 to 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, comprising: A sequencing chip (10), wherein one side surface of the sequencing chip (10) is a coating surface; A coating device, comprising a coating die (20) and an angle adjustment component, wherein 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 a reagent on the coating surface to form a reagent coating layer or to displace the original reagent on the coating surface and form a new reagent coating layer; and the angle adjustment component is configured to adjust the coating angle of the coating die (20), wherein the coating angle is the angle between the flow direction of the reagent in the coating die (20) and the coating surface.

9. The system according to claim 8, wherein: The coating device further comprises 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: At least two flow channels (21) are provided in the coating die head (20), and each flow channel (21) corresponds to a reagent; Alternatively, a plurality of coating die heads (20) are provided, and each coating die head (20) is used to coat a corresponding reagent.

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