Gene sequencing device, consumable assembly and method

Abstract

The invention discloses a gene sequencing device. The gene sequencing device comprises a bearing element and a rotating element which are coaxially arranged, the sequencing chip comprises a sequencing surface for sequencing reaction; a covering element covering the sequencing surface to form a chamber; the liquid storage tank comprises a reaction reagent storage container and a recycled reagent storage container; wherein the covering element comprises a sample adding channel; a recovery channel; and an exhaust passage, in which the sample injection passage has a smaller linear velocity than the recovery passage, and the inner diameter of the recovery passage is smaller than the inner diameter of the sample injection passage. The invention also provides a gene sequencing consumable assembly and a corresponding gene sequencing method. According to the device, the reaction reagent can be repeatedly used in the sequencing cycle until reactants in the reaction reagent are completely consumed, the reagent dosage is greatly saved, and the device is suitable for sample sequencing of any scale.

Description

Technical Field

[0001] The present disclosure relates to a device for gene sequencing, in particular to a gene sequencing consumable component capable of recycling reagents, a device including the component, and a method for performing gene sequencing using the component or the device. Background Art

[0002] Traditional gene sequencing cycles use flow cell design. The reaction reagents in this type of "sample fixed, reagent flow" reaction scheme are all disposable, with extremely low reagent utilization and serious waste (>99%). In addition, due to the small size of the flow channel, when the remaining reagents in the flow channel are pushed out by air, bubbles will remain in the flow channel; when the remaining reagents are pushed out of the flow channel by oil, the oil will have fluorescence, which will interfere with subsequent detection and analysis; the remaining reagents can only be pushed out of the flow channel by water, but this will cause the remaining reagents to be diluted and can only be treated as waste liquid, and the remaining reagents cannot be reused. The entire sequencing process requires about 300 cycles, and the required amount of reagents is 300x the amount required for each cycle. This leads to a large amount of reagents being wasted and the detection cost remains high. Moreover, in order to prevent bubbles from existing in the flow channel, the reagents must be able to fill the entire flow channel (the reagent thickness of the flow cell is 50-100μm), which also increases the amount of reagents required for each cycle, which makes the reagent application rate of each cycle also low. If the amount of reagent is reduced by reducing the thickness of the flow channel, when the reagent is driven to fill the flow channel, the viscous resistance is large, making it difficult for the reagent to fill the entire flow channel, and bubbles are easily generated in the flow channel, affecting the detection.

[0003] In contrast, the "immersion reaction" technology uses a biochemical reaction scheme of "fixed reagents and mobile samples", in which sequencing slides without surface covers are immersed in different reaction containers in turn to complete specific biochemical reactions. The biggest advantage of the "immersion reaction" technology is that it can achieve the reuse of sequencing reagents. It is said that the reaction pool loaded with polymerization reaction raw materials such as DNA polymerase and nucleotide analogs can be reused at least 10 times, and the reaction pool loaded with fluorescent group removal and 3'-Block reduction reagents can be reused at least 20 times. However, when using this technology, the consumables need to be completely immersed, and the amount of reagents needs to be able to immerse the consumables. The purpose of saving reagents can only be achieved when testing large quantities of samples. It is not suitable for individual or single sample testing, and the bubbles in the consumables cannot be eliminated, affecting the detection.

[0004] Recently, a new sequencing reagent supply solution called "surface coating technology (SCT)" was reported. SCT uses "slit coating technology", which means that under a certain pressure, various reagents required for sequencing reactions are pressed out along multiple mold gaps and applied to the surface of a moving substrate (PET Strip) to form a reagent layer of uniform thickness (20μm). Then the substrate with reagents passes over the surface of the sequencing slide to complete a specific biochemical reaction. SCT technology greatly reduces the amount of reagents used in sequencing reactions and improves the efficiency of reagent utilization to a certain extent, thereby greatly reducing sequencing costs. However, the reagents are laid flat on the consumables through the spin coating process. Since the detection requires a heating step, the heating step will cause some reagents to evaporate. In order to enable normal detection, the amount of reagents needs to be increased during spin coating to avoid the inability to perform the detection due to reagent evaporation. In addition, it is impossible to ensure that the reagents are evenly distributed on the consumables, and uneven thickness will lead to deviations in the test results. At the same time, this technology cannot recycle reagents, and the reagents can only be used once and cannot be reused.

[0005] In view of this, the present invention provides a sequencing device capable of recycling reaction reagents, which can reuse reaction reagents in one sequencing cycle until the reactants in the reaction reagents are completely consumed, thereby greatly saving reagent usage, and the device is suitable for sequencing samples of any scale. Summary of the invention

[0006] One aspect of the present invention provides a gene sequencing device, comprising: (a) a coaxially arranged carrying and rotating element, including a carrying platform and a driver; (b) a sequencing chip, including a sequencing surface for sequencing reaction, the sequencing chip being detachably carried on the carrying platform; (c) a covering element, which covers the sequencing surface to form a chamber, and (d) a liquid storage tank, which includes a reaction reagent storage container and a recovery reagent storage container; wherein the covering element includes (i) a loading channel for fluid communication between the chamber and the reaction reagent storage container; (ii) a recovery channel for fluid communication between the chamber and the recovery reagent storage container; and (iii) an exhaust channel for gas communication between the chamber and the atmosphere, wherein, when the chamber is driven to rotate, the loading channel has a linear velocity smaller than that of the recovery channel, and the inner diameter of the recovery channel is smaller than the inner diameter of the loading channel.

[0007] In some embodiments, the sequencing surface of the sequencing chip includes an exhaust sample loading area, a sequencing reaction area, and a sealing area from the center to the periphery. In a preferred embodiment, a buffer isolation area is further provided between the sequencing reaction area and the sealing area.

[0008] In some embodiments, the covering element includes a cover portion corresponding to a sequencing surface of the sequencing chip and a wall portion extending from the periphery of the cover portion toward the sequencing surface, wherein the wall portion abuts against a sealing area of ​​the sequencing chip and is sealingly connected to the sealing area.

[0009] In some embodiments, the covering element further comprises a recovery portion extending from the wall portion toward the sequencing reaction area, and the contour of the recovery portion toward the center is expanded in an involute manner relative to the center. In a preferred embodiment, one end of the recovery channel is located at the farthest point from the center of the contour of the recovery portion.

[0010] In some embodiments, the sample loading channel and / or the exhaust channel corresponds to the exhaust loading area. In a preferred embodiment, the central axis of the exhaust channel passes through the center.

[0011] In some embodiments, the inner diameter of the recovery channel is 1 / 5 to 1 / 100 of the inner diameter of the sample addition channel. In a preferred embodiment, the recovery channel is a capillary tube.

[0012] In some embodiments, there is no valve between the recycling channel and the recycling reagent storage container. In some embodiments, there is no valve between the sample addition channel and the reaction reagent storage container. In some embodiments, there is no valve between the recycling channel and the recycling reagent storage container, and there is no valve between the sample addition channel and the reaction reagent storage container.

[0013] In some embodiments, the sequencing surface of the sequencing chip is circular, and includes an exhaust and sample loading area, a sequencing reaction area, and a sealing area in sequence from the center to the circumference.

[0014] In some embodiments, the covering element is disc-shaped. In some embodiments, the sequencing surface of the sequencing chip is circular and the covering element is disc-shaped.

[0015] In some embodiments, the liquid reservoir includes a vent hole, and the vent hole is in gas communication with the exhaust channel.

[0016] In some embodiments, the recovery reagent storage container is in fluid communication with the reaction reagent storage container.

[0017] In some embodiments, the reservoir comprises at least two reaction reagent storage containers and at least two recovery reagent storage containers. Preferably, each reaction reagent storage container is in fluid communication with one recovery reagent storage container.

[0018] In some embodiments, the fluid reservoir comprises a second rotation driver for driving the fluid reservoir to rotate.

[0019] In some embodiments, the reaction reagent storage container and / or the recovery reagent storage container sequentially include a first inner diameter, a second inner diameter, a third inner diameter and a fourth inner diameter in the sample loading direction, the first inner diameter and the third inner diameter are respectively smaller than the second inner diameter, and the fourth inner diameter is smaller than the third inner diameter. In a preferred embodiment, the fourth inner diameter of the reaction reagent storage container is greater than or equal to the inner diameter of the sample loading channel. In a preferred embodiment, the fourth inner diameter of the recovery reagent storage container is greater than or equal to the inner diameter of the recovery channel.

[0020] In some embodiments, the liquid storage tank and the covering element are detachably sealed.

[0021] Another aspect of the present invention provides a gene sequencing consumables assembly, comprising: (a) a sequencing chip, comprising a sequencing surface for sequencing reaction; and (b) a covering element, which covers the sequencing surface to form a chamber; wherein the covering element comprises (i) a loading channel connected to the chamber fluid; (ii) a recovery channel connected to the chamber fluid; and (iii) an exhaust channel connected to atmospheric gas, wherein, when the chamber is driven to rotate, the loading channel has a linear velocity smaller than that of the recovery channel, and the inner diameter of the recovery channel is smaller than the inner diameter of the loading channel.

[0022] In some embodiments, the sequencing surface of the sequencing chip includes an exhaust sample loading area, a sequencing reaction area, and a sealing area from the center to the periphery. In a preferred embodiment, a buffer isolation area is further provided between the sequencing reaction area and the sealing area.

[0023] In some embodiments, the covering element includes a cover portion corresponding to a sequencing surface of the sequencing chip and a wall portion extending from the periphery of the cover portion toward the sequencing surface, wherein the wall portion abuts against a sealing area of ​​the sequencing chip and is sealingly connected to the sealing area.

[0024] In some embodiments, the covering element further comprises a recovery portion extending from the wall portion toward the sequencing reaction area, and the contour of the recovery portion toward the center is expanded in an involute manner relative to the center. In a preferred embodiment, one end of the recovery channel is located at the farthest point from the center of the contour of the recovery portion.

[0025] In some embodiments, the sample loading channel and / or the exhaust channel corresponds to the exhaust loading area. In a preferred embodiment, the central axis of the exhaust channel passes through the center.

[0026] In some embodiments, the inner diameter of the recovery channel is 1 / 5 to 1 / 100 of the inner diameter of the sample addition channel. In a preferred embodiment, the recovery channel is a capillary tube.

[0027] In some embodiments, the sample loading channel and / or the recovery channel does not include a valve.

[0028] In some embodiments, the sequencing surface of the sequencing chip is circular, and includes an exhaust and sample loading area, a sequencing reaction area, and a sealing area in sequence from the center to the circumference.

[0029] In some embodiments, the covering element is disc-shaped. In some embodiments, the sequencing surface of the sequencing chip is circular and the covering element is disc-shaped.

[0030] Another aspect of the present invention provides a gene sequencing method, comprising: (a) providing the gene sequencing device provided by the present invention; (b) driving the carrying and rotating elements to rotate the gene sequencing device as a whole at a first angular velocity, at which the reaction reagent enters the chamber from the reaction reagent storage container via the loading channel and fills the chamber; (c) allowing the reaction reagent to contact the sequencing chip under predetermined conditions for a predetermined time; (d) driving the carrying and rotating elements to rotate the gene sequencing device as a whole at a second angular velocity greater than the first angular velocity, at which the reaction reagent enters the recovery reagent storage container via the recovery channel; (e) transferring the recovery reagent in the recovery reagent storage container to the reaction reagent storage container; and (f) repeating steps (b) to (e) 2 to 300 times until the sequencing reaction is completed.

[0031] In some embodiments, when the liquid storage tank includes at least two reaction reagent storage containers and at least two recovery reagent storage containers, before step (b), the liquid storage tank is driven to rotate separately so that one of the reaction reagent storage containers and one of the recovery reagent storage containers are connected to the sample loading channel and the recovery channel fluid respectively.

[0032] In some embodiments, in step (a), the sequencing chip and the covering element are configured for single use.

[0033] In some embodiments, in step (a), the amount of the reaction reagent in the reaction reagent storage container is set to be equal to the volume of the chamber.

[0034] The present invention sets a cover on the sequencing chip to form a reagent accommodating cavity. While injecting the reagent into the cavity through the sample addition channel, the chip is driven to rotate. On the one hand, the reagent fills the cavity under the action of centrifugal force, and the remaining gas in the cavity converges to the center of the chip; on the other hand, it can also ensure that the reagent is evenly distributed on the entire chip and the thickness is uniform. The present invention can set the central area of ​​the chip as a blank area, not as a detection area, to eliminate the influence of bubbles on the detection. The present invention also sets a reagent recovery channel. After the reaction is completed, the chip is rotated at a larger angular velocity, and the reagent is recovered through the recovery channel. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The present invention will be described in more detail with reference to the accompanying drawings. It should be noted that the illustrated scheme is only a representative example of an embodiment of the present invention, and in order to more clearly explain the details of the exemplary embodiment, the elements in the drawings are not drawn in proportion to the actual size, the number of actual elements may vary, the relative positional relationship of the actual elements is basically consistent with the illustration, and some elements are not shown. In the case of multiple embodiments, when one or more features described in the previous embodiment can also be applied to another embodiment, for the sake of brevity, these reusable features will not be repeated in the following one or more embodiments, and the following one or more embodiments should be understood to have described these reusable features, unless otherwise stated. After reading the present invention, those skilled in the art will realize that one or more features shown in one figure can be combined with one or more features in another figure to construct one or more alternative embodiments not specifically shown in the drawings, and these alternative embodiments also constitute a part of the present invention.

[0036] Figure 1 A sequencing reaction module of a sequencing device according to one embodiment of the present invention is shown.

[0037] Figure 2 A top view of a sequencing chip according to an embodiment of the present invention is shown.

[0038] Figure 3 A bottom view of a cover element according to one embodiment of the present invention is shown.

[0039] Figure 4 A top view of a cover element according to one embodiment of the invention is shown.

[0040] Figure 5 A partial schematic bottom view (A) and a partial top view (B) showing the location of the recovery channel.

[0041] Figure 6 The structure of a liquid storage tank according to an embodiment of the present invention is schematically shown.

[0042] Figure 7 The partial cross-sectional views of the recovery channel and the recovery reagent storage container in the assembled state are shown from different perspectives (A and B).

[0043] Figure 8 A schematic diagram showing the switching of reagent addition and recovery when the liquid storage tank includes two reaction reagent storage containers and two recovery reagent storage containers.

[0044] Fig. 9 A schematic diagram showing a process flow of a gene sequencing method according to one embodiment of the present invention.

[0045] Fig.10 Schematic diagram showing the fluid flow during the sample addition step (A) and the reagent recovery step (B). DETAILED DESCRIPTION

[0046] The exemplary embodiments of the present invention are described in detail below in conjunction with the accompanying drawings. It should be understood that the scope of the present invention is not limited to the disclosed embodiments, and those skilled in the art may modify and change these exemplary embodiments based on the enlightenment of the present invention after reading the contents disclosed in the present invention without paying creative labor, and these modifications and changes are intended to be included in the scope of the appended claims.

[0047] Figure 1 The sequencing reaction module 100 of the sequencing device according to one embodiment of the present invention is shown, which includes a coaxially arranged carrying and rotating element 10, a sequencing chip 20, a covering element 30 and a liquid storage tank 40. The carrying and rotating element 10 includes a carrying platform 11 for placing the sequencing chip 20, wherein the sequencing chip 20 is detachably connected to the carrying platform 11. For example, a negative pressure air hole (not shown) can be provided on the carrying platform 11 to align the sequencing chip 20 with the center of the carrying platform 11 and fix it. The carrying and rotating element 10 also includes a driver 12 (such as a motor 12) located at the bottom of the carrying platform 11, and the driver 12 can drive the carrying and rotating element 10 and the elements fixedly connected thereto (such as the sequencing chip 20, the covering element 30 and / or the liquid storage tank 40 shown in the figure) to rotate at different angular speeds. The carrying platform 11 can be provided with a groove 112 on its surface facing the sequencing chip 20, and its shape and depth can match the shape and thickness of the sequencing chip 20, so that the sequencing chip 20 is firmly positioned on the carrying platform 11 and does not shift during rotation. Those skilled in the art will appreciate that additional fixing elements may be provided to firmly but detachably combine the sequencing chip 20 and the carrying platform 11 .

[0048] The sequencing chip 20 includes a sequencing surface 21 for sequencing reaction, on which chemical molecules (such as nucleotide primers) for sequencing reaction are covered. Such a sequencing chip 20 can be obtained from the market or can be prepared according to a mature process. For example, the sequencing chip 20 can be a sequencing wafer made of silicon material. Figure 2 A top view of a sequencing chip 20 according to one embodiment of the present invention is shown. In this embodiment, the sequencing chip and its sequencing surface are shown as circular, but the present invention is not limited to circular sequencing chips and is expected to be applicable to sequencing surfaces of any shape. For example, the present invention is expected to be applicable to common rectangular sequencing chips.

[0049] like Figure 2 As shown, in the case where the sequencing chip or sequencing surface is circular, the sequencing chip 20 includes an exhaust sample loading area 211, a sequencing reaction area 212 and a sealing area 213 from the center to the circumference. Preferably, but not necessarily, as shown in the figure, a buffer isolation area 214 can be further provided between the sequencing reaction area 212 and the sealing area 213. The buffer isolation area 214 may destroy the sequencing reaction because of the large flow rate, so it can be left empty and not used for the sequencing reaction. In the present invention, partitioning the sequencing chip 20 does not necessarily mean that each partition of the sequencing chip 20 needs to be specially designed. For example, the exhaust sample loading area 211, the sequencing reaction area 212, the buffer isolation area 214, and the sealing area 213 constitute a flat sequencing wafer, and each area may have no substantial difference in material composition, etching method, surface modification and physical structure. Therefore, the present invention can still use a conventional sequencing chip, but the method of using the sequencing chip 20 in the present invention will be different from the prior art (see below for details). It is understandable that a special sequencing chip can also be used, which does not have a sequencing reaction function (for example, lacks reaction primers) in the areas (exhaust sample addition area 211, buffer isolation area 214, and sealing area 213) other than the sequencing reaction area 212. According to the present invention, the exhaust sample addition area 211, the buffer isolation area 214, and the sealing area 213 do not need to place samples during sequencing, and the data in these areas are not collected for sequencing analysis.

[0050] In this embodiment, the exhaust sample addition area 211 is a circular area having a diameter of d1. The sequencing reaction area 212, the buffer isolation area 214, and the sealing area 213 are respectively annular areas having predetermined widths d2, d4, and d3, respectively. Those skilled in the art can reasonably determine the values ​​of d1 to d4 according to actual needs. For example, d1 can be 3 to 30 mm, such as 10 to 30 mm or 15 to 25 mm, such as 18 mm; d2 can be greater than 1 mm and set according to the chip sequencing throughput, such as 5, 10, 15, 20, 25 mm or more; d3 can be 1 to 8 mm, such as 1 to 5 mm, such as 3 mm; d4 can be 0.1 to 10 mm, such as 0.1 to 5 mm, 0.1 to 2 mm, or 0.1 to 1 mm, such as 0.5 mm.

[0051] The covering element 30 covers the sequencing surface 21 of the sequencing chip 20 to form a chamber. Figure 3 and Figure 4 The bottom view and top view of the covering element 30 according to one embodiment of the present invention are respectively shown. As shown in the figure, the covering element 30 is disc-shaped and includes a cover portion 31 corresponding to the sequencing surface 21 of the sequencing chip 20 and a wall portion 32 extending from the circumference of the cover portion 31 to the sequencing surface 21. When assembled together, the lower surface 321 of the wall portion 32 abuts against the sealing area 213 of the sequencing chip 20 and is sealed to the sealing area 213. The covering element 30 can be made of a transparent material (e.g., transparent plastic). Figure 3 and 4 In the illustrated covering element 30 , the cover portion 31 and the wall portion 32 are made of the same transparent plastic.

[0052] In the present invention, the covering element 30 includes a loading channel 33 connected to the chamber fluid, a recovery channel 35 connected to the chamber fluid, and an exhaust channel 34 connecting the chamber to the atmospheric gas. In this embodiment, the loading channel 33 and the recovery channel 35 are both arranged on the cover 31, wherein the loading channel 33 is closer to the center of the disc-shaped covering element 30 than the recovery channel 35, so when the chamber is driven to rotate, the loading channel 33 has a smaller linear velocity than the recovery channel. The recovery channel 35 is shown in the figure as being close to the connection between the covering element cover 31 and the wall 32, and thus has the maximum linear velocity between each point of the covering element cover 31. When the sequencing chip 20 rotates, the reagent will move toward the wall 32 under the action of centrifugal force and finally converge at the wall 32.

[0053] The exhaust channel 34 is shown in the figure as being located in the cover 31 and having its central axis passing through the center of the disc-shaped cover element 30, but those skilled in the art can understand that the exhaust channel 34 can be located in the wall 32 or in other positions of the cover 31 and communicated with the chamber gas. Therefore, the sample loading channel 33 and / or the exhaust channel 34 can correspond to the exhaust loading area 211 of the sequencing chip 20 and be located within the exhaust loading area 211.

[0054] Figure 3 and 4The cover element 30 shown is a schematic diagram of a preferred cover element according to the present invention. In the preferred cover element 30, the cover element 30 also includes a recovery portion 36 extending from the wall portion 32 to the sequencing reaction area 212. The contour 361 of the recovery portion 36 toward the center of the circle is developed in an involute manner relative to the center of the circle, thereby facilitating the reagents to converge from the sample loading channel 33 along the thick arrow 365 to the "potential minimum point", that is, the farthest point from the center of the circle, that is, the position indicated by the arrow 363 in the figure. Placing one end of the recovery channel 35 at the potential minimum point 363 will help to completely recover the reagents in the chamber. Figure 5 A partial schematic bottom view (A) and a partial top view (B) of the location of the recovery channel 35 are respectively shown.

[0055] The sequencing reaction module 100 further includes a liquid storage tank 40 which is sealed and connected to the cover element 30 in a detachable and sealed manner (eg, snap-fit, magnetically or vacuum adsorbed). Figure 6, which schematically shows the structure of a reservoir 40 according to one embodiment of the present invention. The reservoir 40 includes a space surrounded by a shell 44, a cover 45 and a base 46, all of which can be made of transparent materials (such as transparent plastics). In the space of the reservoir 40, a reaction reagent storage container 41 (such as 41a and 41b) and a recovery reagent storage container 42 (such as 42a and 42b) that is fluidically connected (not shown) to the reaction reagent storage container 41 are included, wherein each reaction reagent storage container 41 is fluidically connected to a recovery reagent storage container 42. In other embodiments, the recovery reagent storage container 42 may not be fluidically connected to the reaction reagent storage container 41, and for example, the recovered reagent may be added to the reaction reagent storage container by an additional reagent transfer element. According to the size of the reaction reagent storage container and the recovery reagent storage container, the recovered reagent can be immediately transferred or fluidly connected to the reaction reagent storage container after each reaction, or the reagent recovered multiple times can be centrally transferred or fluidly connected to the reaction reagent storage container after multiple (e.g., 5 to 10 times or more) reactions, or the recovered reagent can be transferred together or fluidly connected to the reaction reagent storage container after a reaction cycle (e.g., 300 reactions). Two reaction reagent storage containers 41 and two recovery reagent storage containers 42 are shown in the figure, but the present invention is not limited to the illustrated example. For example, in other embodiments, the reservoir 40 may include a single reaction reagent storage container 41 and a single recovery reagent storage container 42, or include more than two reaction reagent storage containers 41 and recovery reagent storage containers 42. The number of reaction reagent storage containers 41 and recovery reagent storage containers 42 can be reasonably determined according to the gene sequencing process. Only one set of reaction reagent storage container 41 and recovery reagent storage container 42 may be provided. In the gene sequencing process, different reagents are added and recovered through the same set of reaction reagent storage container 41 and recovery reagent storage container 42; or multiple sets of reaction reagent storage containers 41 and recovery reagent storage containers 42 may be provided, each used for different reagents. That is, the number of reaction reagent storage containers 41 and recovery reagent storage containers 42 is between 1 and the number of reagents required for the entire gene sequencing process. In the illustrated embodiment, the liquid storage tank 40 is also provided with an exhaust hole 43 at a position corresponding to the exhaust channel 34. The exhaust hole 43 may also not exist, and the exhaust channel 34 is directly connected to the atmosphere (for example, when the exhaust channel is provided on the wall 32 of the covering element 30).

[0056] When assembled together, the loading channel 33 on the covering element 30 connects the fluid between the chamber formed between the covering element 30 and the sequencing chip 20 and the reaction reagent storage container 41; the recovery channel 35 on the covering element 30 connects the fluid between the chamber and the recovery reagent storage container 42; and the exhaust channel 34 on the covering element 30 connects the gas between the chamber and the atmosphere via the exhaust hole 43.

[0057] Figure 7 The partial cross-sectional views of the recovery channel 35 and the recovery reagent storage container 42 when the carrying and rotating element 10, the sequencing chip 20, the covering element 30 and the liquid storage tank 40 are assembled together are shown from different perspectives. Figure 7 In the A part, the inner diameter of the recovery channel 35 is smaller than the inner diameter of the sample loading channel 33. For example, the inner diameter of the recovery channel 35 is 1 / 5 to 1 / 100 of the inner diameter of the sample loading channel 33, such as 1 / 5 to 1 / 10, or 1 / 10 to 1 / 50, or 1 / 50 to 1 / 100. For example, the recovery channel 35 can be configured as a capillary (with a diameter not exceeding 1 mm). The diameter of the reagent recovery channel 35 is configured to be small enough so that when the chip 20 rotates at the first angular velocity (Ω1), on the one hand, the reagent can fill the cavity through the sample loading channel 33, and on the other hand, it is not enough to break through the surface tension of the tiny pores of the recovery channel 35, so that the reagent cannot reach the recovery reagent storage container 41 above. Figure 7 As shown in part B of , in this embodiment, the recovery reagent storage container 42 includes a first inner diameter 421, a second inner diameter 422, a third inner diameter 423 and a fourth inner diameter 424 in the sample loading direction, wherein the first inner diameter 421 and the third inner diameter 423 are respectively smaller than the second inner diameter 422, thereby forming an "inverted mouth" design to ensure that the reagent will not be thrown out of the storage container during high-speed rotation. The fourth inner diameter 424 is configured to be smaller than the third inner diameter 424 to cooperate with the recovery channel 35, wherein the fourth inner diameter 424 is greater than or equal to the inner diameter of the recovery channel 35. The reaction reagent storage container 41 may also have a similar inverted mouth design in the sample loading direction, wherein the fourth inner diameter of the reaction reagent storage container 41 is greater than or equal to the inner diameter of the sample loading channel 33.

[0058] In the present invention, the covering element 30 can be separately provided with the sequencing chip 20 or can be provided integrally. When provided separately, the sequencing chip 20 is a disposable consumable, and the covering element 30 and the liquid reservoir 40 are reusable; when provided integrally, the sequencing chip 20 and the covering element 30 are used together as disposable consumables, and the liquid reservoir 40 is reusable. Therefore, another aspect of the present invention provides a sequencing consumable, which includes a sequencing chip 20 and a covering element 30. All the above descriptions of the present invention regarding the sequencing chip 20 and the covering element 30 are applicable to the sequencing consumable.

[0059] In the embodiment where the liquid storage tank 40 includes two reaction reagent storage containers 41 and two recovery reagent storage containers 42, an additional rotary drive (not shown) is required to drive the liquid storage tank to rotate to achieve reagent switching. Figure 8 As shown, the liquid storage tank 40 is rotated clockwise so that the reagent recovery channel 35 and the reagent injection channel 33 are connected to different recovery reagent storage containers 42 and different reaction reagent storage containers 41 respectively.

[0060] exist Figure 1 and Figure 8 In the illustrated embodiment, there is no valve between the recycling channel 35 and the recycling reagent storage container 42, and there is no valve between the sample addition channel 33 and the reaction reagent storage container 41. The present invention reasonably configures the positions of the recycling channel 35 and the sample addition channel 33, and utilizes the difference in rotation angular velocity to realize the sample addition and recycling actions, so that the sample addition and recycling processes can be controlled without the aid of an additional valve structure. This will be described in more detail below.

[0061] Fig. 9 A schematic flow chart of a gene sequencing method according to one embodiment of the present invention is shown. The method starts at step 91, which provides a gene sequencing device of the present invention or a sequencing device comprising a gene sequencing consumable provided by the present invention. As described above, the gene sequencing device includes a coaxially arranged bearing and rotating element, including a bearing platform and a driver; a sequencing chip, including a sequencing surface for sequencing reaction, and the sequencing chip is detachably carried on the bearing platform; a covering element, which covers the sequencing surface to form a chamber, and a liquid storage tank, which includes a reaction reagent storage container and a recovery reagent storage container; wherein the covering element includes (i) a sample loading channel for fluid communication between the chamber and the reaction reagent storage container; (ii) a recovery channel for fluid communication between the chamber and the recovery reagent storage container; and (iii) an exhaust channel for gas communication between the chamber and the atmosphere, wherein, when the chamber is driven to rotate, the sample loading channel has a linear velocity smaller than that of the recovery channel.

[0062] The method then performs a sample loading step (step 92), driving the bearing and rotating element to rotate the entire gene sequencing device at a first angular velocity Ω1, at which the reaction reagent enters the chamber from the reaction reagent storage container via the sample loading channel and fills the chamber. The amount of reaction reagent in the reaction reagent storage container can be set to be equal to the volume of the chamber or 10% to 100% (e.g., 10%, 20%, 50%, 80% or 100%) greater than the volume of the chamber. In other embodiments, the amount of reaction reagent in the reaction reagent storage container can be set to be more or less.

[0063] As mentioned above, when the liquid storage tank includes two or more reaction reagent storage containers and two or more recovery reagent storage containers, before each sample addition, the liquid storage tank can be driven to rotate separately so that one of the reaction reagent storage containers and one of the recovery reagent storage containers are connected to the sample addition channel and the recovery channel fluid respectively.

[0064] Fig.10Part A of the figure shows a schematic diagram of fluid flow during the loading step. The driver drives the device to rotate at a first angular velocity Ω1, and the reagent stored in the reaction reagent storage container 41 enters the chamber 25 defined by the covering element 30 and the sequencing chip 20 through the loading channel 33 under the action of centrifugal force, and fills the chamber 25. As described above, since the diameter of the recovery pipe 35 is smaller than the loading channel 33 and is set small enough, the reagent fills the chamber 25 at a rotation speed of Ω1, but is not enough to break through the surface tension of the tiny pore size of the recovery pipe 35, so the reagent cannot reach the recovery reagent storage container 42. In the process of the reagent filling the chamber 25, the gas retained in the chamber can be discharged from the chamber 25 through the gas channel 34 (as shown by the dotted arrow).

[0065] Subsequently, the method proceeds to step 93, wherein the reaction reagent is contacted with the sequencing chip under predetermined conditions for a predetermined time. The predetermined conditions may include placing the sequencing chip at a predetermined temperature or temperature range (e.g., 60° C. or 58° C.) to achieve a base pairing reaction. The predetermined time may be, for example, 1 to 5 seconds or longer. Those skilled in the art may determine the predetermined conditions and predetermined time as needed, and generally the conditions and time may be pre-set and executed by a computer.

[0066] After the single sequencing reaction in step 93 is completed, step 94 is performed to drive the carrying and rotating elements to rotate the entire gene sequencing device at a second angular velocity Ω2 that is greater than the first angular velocity Ω1. At the second angular velocity Ω2, the reaction reagent enters the recovery reagent storage container through the recovery channel.

[0067] Fig.10 Part B of FIG. 9 shows a schematic diagram of fluid flow during the reagent recovery step 94. At the second angular velocity Ω2, the reagent in the chamber 25 breaks through the surface tension of the recovery channel 35 and enters the recovery reagent storage container 42, and the gas enters the chamber 25 again via the gas channel 34 (as shown by the dotted arrow).

[0068] It is particularly advantageous in the present invention to set the recovery channel 35 as a capillary tube, because due to the capillary action, under the action of gravity, the reagent in the capillary tube cannot flow into the cavity. This design can eliminate the need to set a valve in the recovery channel, simplify the design of consumables, reduce the complexity of the production process, and reduce production costs. In addition, the first angular velocity Ω1 and the second angular velocity Ω2 can be defined by the critical rotation speed Ω0. For example, when adding a sample, Ω1 < Ω0, and when the reagent is recovered, the rotation speed Ω2 > Ω0. Ω0 can be determined experimentally. Generally, Ω0 is related to factors such as the diameter of the sequencing chip, the diameter of the reagent recovery channel (i.e., the capillary channel), and the viscosity of the reagent. The overall rotation speed can be gradually increased, and the overall rotation speed at the moment when the reagent breaks through the surface tension of the reagent recovery channel (i.e., the capillary channel) is defined as the critical speed Ω0.

[0069] Subsequently, in step 95, the recovery reagent in the recovery reagent storage container is transferred to the reaction reagent storage container for the next reaction cycle. Step 96 repeats steps 92 to 95, for example, 2 to 300 cycles, wherein 300 times is generally the upper limit of the cycle required for sequencing reaction), until the active ingredient in the reagent cannot effectively complete the reaction. It is worth noting that in multiple cycles, what is reduced is the active ingredient participating in the reaction in the reagent, and the total volume of the reagent changes very little. At this time, the reagent recovered in the recovery reagent storage container is discharged as waste liquid, and then new reagents are added to the reaction reagent storage container or a new liquid storage tank is replaced, and the aforementioned cycle is carried out until the sequencing reaction is completed. By this, there is no need to consume a large amount of reagents for each reaction, and the active ingredient in the reagent can be fully utilized, which greatly reduces the reagent dosage required for sequencing reaction, and greatly reduces the sequencing cost.

[0070] The above are all representative examples of embodiments of the present invention and are provided for illustrative purposes only. The present invention contemplates that one or more technical features used in one embodiment may be added to another embodiment to form an improved or alternative embodiment without violating the purpose of the embodiment. Similarly, one or more technical features used in one embodiment may be omitted or replaced to form an alternative or simplified embodiment without violating the purpose of the embodiment. In addition, one or more technical features used in one embodiment may be combined with one or more technical features in another embodiment to form an improved or alternative embodiment without violating the purpose of the embodiment. The present invention is intended to include all of the above improved, alternative, and simplified technical solutions.

Claims

1. A gene sequencing device, characterized in that: Including coaxial setup: (a) Load-bearing and rotating components, including load-bearing platforms and drives; (b) a sequencing chip, comprising a sequencing surface for sequencing reaction, wherein the sequencing chip is detachably carried on the carrying platform; (c) a covering element covering the sequencing surface to form a chamber, and (d) a liquid storage tank, which includes a reaction reagent storage container and a recovery reagent storage container; in The covering element includes (i) a sample loading channel for fluid communication between the chamber and the reaction reagent storage container; (ii) a recovery channel for fluid communication between the chamber and the recovery reagent storage container; and (iii) an exhaust channel for gas communication between the chamber and the atmosphere, wherein, when the chamber is driven to rotate, the sample loading channel has a smaller linear velocity than the recovery channel, and the inner diameter of the recovery channel is smaller than the inner diameter of the sample loading channel.

2. The gene sequencing device according to claim 1, characterized in that: The sequencing surface of the sequencing chip includes, from the center to the periphery, an exhaust and sample loading area, a sequencing reaction area, and a sealing area; preferably, a buffer isolation area is also provided between the sequencing reaction area and the sealing area.

3. The gene sequencing device according to claim 2, characterized in that: The covering element includes a cover portion corresponding to a sequencing surface of the sequencing chip and a wall portion extending from the periphery of the cover portion toward the sequencing surface, wherein the wall portion abuts against a sealing area of ​​the sequencing chip and is sealingly connected to the sealing area.

4. The gene sequencing device according to claim 3, characterized in that: The covering element further comprises a recovery portion extending from the wall portion toward the sequencing reaction area, wherein a contour of the recovery portion toward the center is unfolded in an involute manner relative to the center; preferably, one end of the recovery channel is located at the farthest point from the center of the contour of the recovery portion.

5. The gene sequencing device according to claim 3, characterized in that: in: (i) the sample loading channel and / or the exhaust channel corresponds to the exhaust loading area; preferably, the central axis of the exhaust channel passes through the center; (ii) the inner diameter of the recovery channel is 1 / 5 to 1 / 100 of the inner diameter of the sample addition channel; preferably, the recovery channel is a capillary; (iii) there is no valve between the recovery channel and the recovery reagent storage container; and / or there is no valve between the sample addition channel and the reaction reagent storage container; (iv) the sequencing surface is circular; and / or (v) The covering element is disc-shaped.

6. The gene sequencing device according to claim 1, characterized in that: The liquid storage tank also has one or more of the following features: (i) comprising an exhaust hole, wherein the exhaust hole is in gas communication with the exhaust channel; (ii) comprising at least two reaction reagent storage containers and at least two recovery reagent storage containers, preferably each reaction reagent storage container is in fluid communication with one recovery reagent storage container; (iii) comprising a second rotary drive for driving the liquid storage tank to rotate; (iv) the reaction reagent storage container and / or the recovery reagent storage container sequentially include a first inner diameter, a second inner diameter, a third inner diameter and a fourth inner diameter in the sample loading direction, the first inner diameter and the third inner diameter are respectively smaller than the second inner diameter, and the fourth inner diameter is smaller than the third inner diameter; preferably, the fourth inner diameter of the reaction reagent storage container is greater than or equal to the inner diameter of the sample loading channel; preferably, the fourth inner diameter of the recovery reagent storage container is greater than or equal to the inner diameter of the recovery channel; (v) the recovery reagent storage container is in fluid communication with the reaction reagent storage container; and (vi) The liquid storage tank and the covering element are detachably sealed and connected.

7. A gene sequencing consumables assembly, characterized in that: Including coaxial setup: (a) a sequencing chip, comprising a sequencing surface for sequencing reaction; and (b) a covering element covering the sequencing surface to form a chamber; wherein The covering element includes (i) a sample loading channel connected to the chamber fluid; (ii) a recovery channel connected to the chamber fluid; and (iii) an exhaust channel connected to atmospheric gas, wherein, when the chamber is driven to rotate, the sample loading channel has a smaller linear velocity than that of the recovery channel, and the inner diameter of the recovery channel is smaller than the inner diameter of the sample loading channel.

8. The gene sequencing consumables assembly according to claim 7, characterized in that: The sequencing surface of the sequencing chip includes, from the center to the periphery, an exhaust and sample loading area, a sequencing reaction area, and a sealing area; preferably, a buffer isolation area is also provided between the sequencing reaction area and the sealing area.

9. The gene sequencing consumables assembly according to claim 8, characterized in that: The covering element includes a cover portion corresponding to the sequencing surface of the sequencing chip and a wall portion extending from the periphery of the cover portion toward the sequencing surface, wherein the wall portion abuts against the sealing area of ​​the sequencing chip and is sealed and connected to the sealing area.

10. The gene sequencing consumables assembly according to claim 9, characterized in that: The covering element further comprises a recovery portion extending from the wall portion toward the sequencing reaction area, wherein a contour of the recovery portion toward the center is unfolded in an involute manner relative to the center; preferably, one end of the recovery channel is located at the farthest point from the center of the contour of the recovery portion.

11. The gene sequencing consumables assembly according to claim 8, characterized in that: in: (i) the sample loading channel and / or the exhaust channel corresponds to the exhaust loading area; preferably, the central axis of the exhaust channel passes through the center; (ii) the inner diameter of the recovery channel is 1 / 5 to 1 / 100 of the inner diameter of the sample addition channel; preferably, the recovery channel is a capillary; (iii) the sample loading channel and / or the recovery channel do not include a valve; (iv) the sequencing surface is circular; and / or (v) The covering element is disc-shaped.

12. A gene sequencing method, comprising: (a) providing a gene sequencing device according to any one of claims 1 to 6; (b) driving the bearing and rotating element to rotate the gene sequencing device as a whole at a first angular velocity, wherein at the first angular velocity, the reaction reagent enters the chamber from the reaction reagent storage container through the sample loading channel and fills the chamber; (c) contacting the reaction reagent with the sequencing chip under predetermined conditions and for a predetermined time; (d) driving the bearing and rotating element to rotate the entire gene sequencing device at a second angular velocity greater than the first angular velocity, wherein at the second angular velocity, the reaction reagent enters the recovery reagent storage container through the recovery channel; (e) transferring the recovery reagent in the recovery reagent storage container to the reaction reagent storage container; and (f) Repeat steps (b) to (e) 2 to 300 times until the sequencing reaction is completed.

13. The method according to claim 12, wherein: (i) when the liquid storage tank comprises at least two reaction reagent storage containers and at least two recovery reagent storage containers, before step (b), the liquid storage tank is driven to rotate separately so that one of the reaction reagent storage containers and one of the recovery reagent storage containers are connected to the sample loading channel and the recovery channel fluid respectively; (ii) in step (a), the sequencing chip and the covering element are set to be disposable; and / or (iii) In step (a), the amount of the reaction reagent in the reaction reagent storage container is set to be equal to the volume of the chamber.

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