Sequencing chip and sequencing device

Abstract

The application relates to the field of gene sequencing, aims to solve the problems of long time consumption and high cost of known technology sequencing, and provides a sequencing chip and a sequencing device. The sequencing chip comprises a base layer and a cover layer. The base layer comprises a substrate. The substrate has a first surface and a second surface opposite to each other; the first surface is provided with a sequencing area; the substrate is provided with a liquid inlet hole and a liquid outlet hole. The cover layer is arranged on one side of the first surface of the base layer; a sequencing flow channel is defined between the cover layer and the sequencing area; and the sequencing flow channel is communicated between the liquid inlet hole and the liquid outlet hole. The application has the beneficial effects of high sequencing efficiency and low sequencing cost.

Description

Technical Field

[0001] This application relates to the field of gene sequencing, and more specifically, to sequencing chips and sequencing devices. Background Technology

[0002] With the development of gene sequencing technology, second-generation sequencing technology, which uses array-type reaction cell sequencing chips to perform continuous cycles of chemical reactions such as fluorescent labeling, fluorescent imaging, deblocking, and removal of fluorescent groups, is now widely used in whole genome sequencing, transcriptome sequencing, metagenomic sequencing, and other fields.

[0003] Corresponding sequencing platforms and products are constantly being updated. As an important device in the sequencing process of base sequences of specific DNA fragments, gene sequencing chips are rapidly developing towards high throughput and low cost under the impetus of sequencing technology.

[0004] Some existing sequencers typically have sequencing chips with large integral liquid channels to achieve high-throughput sequencing. This requires large quantities of DNA molecular samples and chemical reaction reagents, which leads to problems such as difficulty in obtaining sequencing samples, high reagent costs, and long testing times, thus limiting their application. Summary of the Invention

[0005] This application provides sequencing chips and sequencing devices to solve the problems of long sequencing time and high cost of known sequencing technologies.

[0006] In a first aspect, embodiments of this application provide a sequencing chip, including a base layer and a capping layer. The base layer includes a substrate. The substrate has a first surface and a second surface facing away from each other; the first surface has a sequencing region; the substrate has an inlet port and an outlet port. The capping layer is stacked on one side of the first surface of the base layer; a sequencing channel is defined between the capping layer and the sequencing region; the sequencing channel communicates between the inlet port and the outlet port.

[0007] In one possible implementation, the outer contour of the sequencing chip projected along its thickness direction is square. The substrate is a strip-shaped hexagon extending diagonally along the outer contour; the inlet and outlet ports are respectively located at both ends of the substrate's extension direction.

[0008] In one possible implementation, the base layer further includes a filler. The filler and the substrate are laterally joined so that the outer contour of the base layer projected along the thickness direction is square, consistent with or substantially consistent with the capping layer.

[0009] In one possible implementation, the surface of the filler on one side along the thickness direction is coplanar with the first surface. The filler also has a surface on the other side along the thickness direction and a second surface.

[0010] In one possible implementation, the sequencing chip has a square outer contour projected along its thickness direction. An inlet and an outlet are located at opposite ends of the square's diagonal. A guide groove is recessed on the first surface; the guide groove includes an inlet guide groove and an outlet guide groove. The inlet guide groove, the sequencing channel, and the outlet guide groove are sequentially connected in series between the inlet and outlet holes along the diagonal of the square.

[0011] In one possible implementation, the first surface is recessed with a guide groove. The guide groove includes an inlet guide groove communicating between the sequencing channel and the inlet port; and / or, the guide groove includes an outlet guide groove communicating between the sequencing channel and the outlet port.

[0012] In one possible implementation, there are multiple sequencing channels. The inlet port is connected to each of the multiple sequencing channels via an inlet guide groove; and / or, the outlet port is connected to each of the multiple sequencing channels via an outlet guide groove.

[0013] In one possible implementation, the outer contour of the sequencing chip projected along its thickness direction is square; the sequencing channels are elongated, and the ratio of channel width to length is no greater than 1 / 7. The extension direction of the sequencing channels is parallel to the diagonal of the outer contour or parallel to one side of the outer contour.

[0014] In one possible implementation, a refrigerant overflow groove is recessed on the first surface of the substrate. The overflow groove is annular and divides the first surface into an inner region and an outer region. The sequencing chip also includes a refrigerant layer; the refrigerant layer is bonded between the outer region of the substrate and the capping layer; the thickness of the sequencing channels is equal to the thickness of the refrigerant layer.

[0015] Secondly, embodiments of this application provide a sequencing device, which includes a sequencing platform, a fixation device, and the aforementioned sequencing chip. The sequencing chip can be mounted on the sequencing platform. The fixation device is connected to the sequencing platform and can fix the sequencing chip on the sequencing platform. The fixation device is either a negative pressure adsorption device or a compression device.

[0016] The implementation of this application enables the convenient design of sequencing chips that are compatible with sequencing platforms and have low throughput, without changing the structure of the sequencing platform, which helps to improve sequencing efficiency and reduce sequencing costs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings in the embodiments will be briefly described below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 This is a schematic diagram of the basic structure of a known sequencing chip.

[0019] Figure 2 for Figure 1 A schematic diagram of a modified version of the sequencing chip.

[0020] Figure 3 for Figure 1 A schematic diagram of another variant of the sequencing chip.

[0021] Figure 4 This is a schematic diagram of the sequencing platform in an embodiment of this application.

[0022] Figure 5 This is a schematic diagram of the sequencing chip structure according to an embodiment of this application.

[0023] Figure 6 for Figure 5 A cross-sectional view of the sequencing chip along line AA.

[0024] Figure 7 for Figure 6 Exploded view of the sequencing chip.

[0025] Figure 8 This is a schematic diagram of a modified base layer in this embodiment.

[0026] Figure 9 for Figure 8 A cross-sectional view of the base layer along line BB.

[0027] Figure 10 This is a schematic diagram when the guide channel uses a semi-circular cross-section.

[0028] Figure 11 This is a schematic diagram of another sequencing chip according to an embodiment of this application.

[0029] Figure 12 for Figure 11 A schematic diagram of the basic structure of the sequencing chip.

[0030] Figure 13 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0031] Figure 14 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0032] Figure 15 To adopt Figure 13 A schematic diagram of the mounting method for sequencing chips.

[0033] Figure 16 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0034] Figure 17This is a schematic diagram of another basic structure according to an embodiment of this application.

[0035] Figure 18 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0036] Figure 19 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0037] Figure 20 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0038] Figure 21 This is a schematic diagram of another basic structure according to an embodiment of this application.

[0039] Figure 22 It indicated Figure 20 The guide groove design method in the process.

[0040] Figure 23 This indicates the adoption Figure 20 The liquid state of the sequencing chip at the base layer.

[0041] Figure 24 This is a schematic diagram of the sequencing device according to an embodiment of this application.

[0042] Explanation of key component symbols:

[0043] Figure 1-3 In Chinese: 1-sequencing chip; 1a-sequencing channel.

[0044] Figures 4-24 In the diagram: 100 - Sequencing platform; 110 - Stage; 120 - Compressing device; T1 - Liquid supply channel; T2 - Liquid drainage channel; C1 - Vacuum adsorption tank; 200 - Sequencing chip; 10, 10a, 10b, 10c, 10d, 10e, 10f, 10g, 10h, 10i, 10j - Substrate; 11 - Substrate; 12 - Filler; 20 - Gel layer; 30 - Cap layer; 1000 - Sequencing device; 300 - Fixation device; Q1 - Sequencing channel; S1 - Sequencing region; K1 - Liquid inlet; K2 - Liquid outlet; P1 - First surface; P2 - Second surface; C2 - Guide groove; C21 - Liquid inlet guide groove; C22 - Liquid outlet guide groove; C3 - Glue overflow tank; Z - Thickness direction; P11 - Inner region; P12 - Outer region. Detailed Implementation

[0045] The technical solutions in the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, and not all embodiments.

[0046] It should be noted that when a component is said to be "fixed to" another component, it can be directly on the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component. When a component is said to be "set on" another component, it can be directly set on the other component or there may be an intervening component. The terms "vertical," "horizontal," "left," "right," and similar expressions used in this document are for illustrative purposes only.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0048] Some embodiments of this application are described in detail. Unless otherwise specified, the following embodiments and features can be combined with each other.

[0049] Example

[0050] See Figure 1 One known basic version of sequencing chip 1 has multiple long, wide lanes (also called sequencing channels 1a), thus the sequencing chip 1 has a large throughput.

[0051] If it is necessary to reduce the throughput of sequencing chip 1 in order to improve sequencing efficiency and reduce the amount of sequencing samples and reagents, this can be achieved by narrowing the sequencing channel 1a or reducing the number of sequencing channels 1a.

[0052] For example, the sequencing channel 1a can be narrowed based on the basic sequencing chip 1 described above to obtain... Figure 2 A modified sequencing chip 1 is shown.

[0053] If further throughput reduction is required, the number of sequencing channels 1a can be reduced (e.g., to 2) based on the modified sequencing chip 1 described above, resulting in... Figure 3 Another variant of the sequencing chip 1 is shown.

[0054] The aforementioned methods for reducing throughput may render sequencing chip 1 unsuitable for the sequencing platform, necessitating a change in the sequencing platform and resulting in excessively high improvement costs.

[0055] Figure 4A sequencing platform 100 is shown, which has a stage 110 for carrying a sequencing chip. The stage 110 is generally square and has a liquid supply channel T1 and three liquid drainage channels T2 distributed at its four corners.

[0056] The surface of the stage 110 is also recessed with a vacuum adsorption groove C1 for adsorbing and fixing the sequencing chip placed on the stage 110. When it is necessary to fix the sequencing chip by negative pressure adsorption, the contact surface between the sequencing chip and the sequencing platform 100 needs to cover the area where the vacuum adsorption groove C1 is located, for example, the contact surface is required to be square.

[0057] Of course, when the stage 110 is rectangular, the contact surface between the sequencing chip and the sequencing platform 100 needs to be rectangular.

[0058] Figure 5 This embodiment illustrates a sequencing chip 200, which is related to... Figure 4 The sequencing platforms 100 are mutually compatible. The sequencing chip 200 has a square outer contour and a single square lane. Figure 5 The middle lane (i.e., sequencing channel Q1) is located in the dotted area inside the dashed box. The sequencing chip 200 has one inlet hole K1 and three outlet holes K2 located at the four corners of the sequencing channel Q1.

[0059] See Figure 6 and Figure 7 Along the thickness direction Z of the sequencing chip 200, the sequencing chip 200 includes a base layer 10, a gel layer 20, and a capping layer 30. The capping layer 30 and the base layer 10 are bonded together by the gel layer 20.

[0060] The base layer 10 includes a substrate 11 made of materials such as silicon wafers. The substrate 11 has a first surface P1 and a second surface P2 facing away from each other. The first surface P1 has a sequencing region S1 (i.e., the region on the first surface P1 used to enclose the sequencing flow channel Q1), and the substrate 11 has an inlet port K1 and an outlet port K2. The number of inlet ports K1 and outlet ports K2 can be set as needed, for example, one inlet port K1 and three outlet ports K2.

[0061] The capping layer 30 is bonded and stacked on one side of the first surface P1 of the base layer 10 by the adhesive layer 20. The aforementioned sequencing channel Q1 is defined between the capping layer 30 and the sequencing area S1. The sequencing channel Q1 is connected between the inlet hole K1 and the outlet hole K2.

[0062] The cover layer 30 can be made of a light-transmitting plate such as glass, so that the imaging device can take pictures of the situation inside the sequencing channel Q1 through the cover layer 30.

[0063] The adhesive layer 20 can be a photocurable adhesive. In this embodiment, the sequencing channel Q1 is defined between the sequencing region S1 of the capping layer 30 and the base layer 10, and the thickness of the sequencing channel Q1 is the same as the thickness of the adhesive layer 20.

[0064] To ensure a constant thickness of the sequencing channel Q1, in this embodiment, a certain proportion of microspheres of a fixed size are mixed into the photocurable adhesive to control the thickness of the adhesive layer 20. Specifically, the microspheres are supported between the capping layer 30 and the base layer 10, thereby ensuring a fixed spacing between them. This results in a uniform thickness of the sequencing channel Q1, equal to the diameter of the microspheres. The adhesive material between the microspheres reliably bonds the capping layer 30 and the base layer 10.

[0065] Optionally, the thickness of the adhesive layer 20 is controlled to be around 30-60 μm, for example, 50 μm. Correspondingly, the diameter of the microspheres is approximately 50 μm.

[0066] When bonding the cover layer 30 and the base layer 10 of the sequencing chip 200, a certain amount of photocurable adhesive can be applied as adhesive layer 20 to the area around the outer periphery of the sequencing region S1 on the surface of the base layer 10 using a dispensing machine. The base layer 10, adhesive layer 20 and cover layer 30 are pressed together by external force, and the adhesive layer 20 is irradiated with a lamp source of a certain wavelength for a certain period of time to achieve curing of the adhesive layer 20, ensuring that the adhesive layer 20 reliably bonds the base layer 10 and the cover layer 30.

[0067] The adhesive layer 20 should cover the outer side of the outer contour of the sequencing channel Q1 and maintain a certain adhesive line width so that the adhesion between the capping layer 30 and the substrate 11 is sufficient to withstand the pressure inside the sequencing channel Q1.

[0068] After the sequencing chip 200 is bonded, a certain amount of adhesive can be applied to the inner frame outline and the designated area of ​​the frame using a dispensing machine. The sequencing chip 200 and the protective frame are then aligned and bonded together, completing the assembly of the sequencing chip 200 and the protective frame.

[0069] When the sequencing chip 200 is fixed on the sequencing platform 100, the second surface P2 of the sequencing chip 200 is superimposed on the sequencing platform 100. The inlet port K1 and the supply channel T1 are connected accordingly, and the three outlet ports K2 are connected to the three drainage channels T2 respectively. Thus, when the sequencing chip 200 is fixed on the sequencing platform 100, test liquids (such as samples, reaction reagents, etc.) can enter the sequencing channel Q1 of the sequencing chip 200 from the supply channel T1 through the inlet port K1, and flow out of the sequencing chip 200 from the three outlet ports K2 respectively, and then be discharged through the three drainage channels T2.

[0070] See you again Figure 5The basic sequencing channel Q1 of this sequencing chip 200 is square, with side length a2 approximately equal to the side length a1 of the outer contour of the sequencing chip 200. For example, a2 is 0.8-0.95 times a1. One inlet port K1 and three outlet ports K2 are located at the four corners of the square sequencing channel Q1. This sequencing chip 200 has high throughput and a large imaging area.

[0071] Figure 8 Another layer 10a is shown, which reduces the throughput of the sequencing chip 200 using the layer 10a.

[0072] The sequencing region S1 of the base layer 10a is a square with a smaller side length a3. For example, a3 is 0.4-0.6 times that of a2. This reduces the area of ​​the sequencing channel Q1, thereby reducing the amount of sample and reaction reagents used and shortening the testing time. For example, when a3 is 0.5 times that of a2, the area of ​​the sequencing channel Q1 is reduced to 1 / 4 of its original size, and the corresponding sample and reaction reagent usage can be reduced to approximately 1 / 4 of its original size. It should be noted that the size (or throughput) of the sequencing channel Q1 can be designed based on the mainstream sample length.

[0073] To ensure that the sequencing chip 200 with reduced throughput is still compatible with the sequencing platform 100, the positions of the liquid inlet K1 and liquid outlet K2 of the base layer 10a are as follows: Figure 5 The grassroots level 10 remains consistent.

[0074] See Figure 8 The first surface of the base layer 10a is also recessed with a guide groove C2, which connects the sequencing area S1 and the liquid outlet K2. Thus, in the sequencing chip 200 using the base layer 10a, the guide groove C2 can connect the sequencing channel Q1 and the liquid outlet K2.

[0075] The number of guide grooves C2 can be set as needed. For example, there are two guide grooves C2, which connect the sequencing region S1 and the two outlet holes K2 respectively. One guide groove C2 forms a 45° angle with the two sides of the square to connect to the outlet hole K2 located diagonally opposite the inlet hole K1; the other guide groove C2 is close to the bottom edge of the square and parallel to the bottom edge to connect to the inlet hole K1 at the bottom edge. The other outlet hole K2 is not used and does not need to be connected to the sequencing region S1.

[0076] Alternatively, the unused liquid outlet K2 can be omitted and not machined, or it can be machined and then unused.

[0077] In this embodiment, the outer sides of sequencing channel Q1 and guide groove C2 are sealed with adhesive layer 20 to ensure the sealing of sequencing channel Q1 and guide groove C2.

[0078] Figure 8The substrate 10a shown directly selects a small square region on the substrate 11 as the sequencing region S1 that encloses the sequencing flow channel Q1. As a mature online product, modifying the sequencing platform 100 has a long development cycle and high R&D costs. Compared to the reagent cost per sequencing run, the cost of changing the substrate is relatively small.

[0079] Therefore, relative Figure 5 200 sequencing chips Figure 8 The outer contour of the sequencing chip 200 remains unchanged, and the positions of the inlet hole K1 and the outlet hole K2 remain unchanged. By reducing the area of ​​the sequencing region S1 and adding a guide groove C2 to connect the outlet hole K2, a sequencing chip 200 with a smaller throughput can be obtained while maintaining compatibility with the sequencing platform 100.

[0080] Compared to Figure 5 The sequencing chip 200, using the adjusted... Figure 8 When using the sequencing chip 200 with the base layer 10a shown, the reagent substitution ratio did not change significantly, the sequencing time was accelerated, and the total reagent cost was reduced.

[0081] The function of the guide groove C2 in this embodiment is: 1) to allow the reagent to better cover the entire square sequencing chip 200, so that the amount of reagent replacement in the sequencing channel Q1 (referring to the amount of reagent that the next reagent completely replaces the previous reagent) is within a reasonable range; 2) to connect the sequencing channel Q1 to the liquid outlet K2, and then to the liquid drainage channel T2 of the sequencing platform 100.

[0082] Optionally, the cross-section of the sequencing channel Q1 and the connection point should be kept consistent to ensure smooth connection and stable liquid flow.

[0083] In this example, although guide channel C2 is not involved in image sequencing, it still serves the function of reagent transport. Therefore, in the design, the flow resistance at guide channel C2 should not be too large. It needs to be a cross-section with a certain depth and width, and its size is generally an order of magnitude larger than the thickness of sequencing channel Q1 (e.g., 50 μm) (e.g., about 0.3 mm, approximately half the thickness of the 10a base layer), to ensure that the main pressure loss is still at sequencing channel Q1.

[0084] The cross-sectional shape of the guide groove C2 in this embodiment can be selected as needed.

[0085] For example Figure 9 In the middle, the cross-sectional shape of the guide groove C2 is rectangular.

[0086] See Figure 9 Optionally, the guide groove C2 has a rectangular cross-section with a depth of 0.3 mm and a width of 0.6 mm. The shallow depth of the guide groove C2 facilitates laser processing.

[0087] The advantage of using a rectangular cross-section guide groove C2 is that it is easier to process and the cross-sectional shape is easier to control, but it is not advisable to use a rectangle with a small width-to-depth ratio. The disadvantage of using a rectangular cross-section guide groove C2 is that reagent substitution at the two right angles at the bottom of the guide groove C2 is relatively difficult.

[0088] Figure 10 The cross-sectional shape of the guide groove C2 shown is semi-circular.

[0089] See Figure 10 The guide groove C2 has a semi-circular cross-section with a radius of 0.3 mm. The guide groove C2 can be machined using sandblasting.

[0090] With a fixed thickness of the silicon wafer base layer 10a, the advantage of the semi-circular cross-section guide groove C2 is that it has a smaller cross-sectional area and no right-angled areas that are difficult to replace, resulting in more complete overall reagent replacement. Furthermore, if this sequencing chip 200 subsequently requires reverse pumping of reagents from the outlet K2, the semi-circular cross-section guide groove C2 can recover a higher proportion of high-purity reagents.

[0091] Compared to rectangular cross-sections, the semi-circular cross-section guide groove C2 is relatively difficult to process, requiring high-precision laser grooving or sandblasting processes. If the surface of the semi-circular guide groove C2 is not smooth, other problems may arise.

[0092] Figure 11 Another sequencing chip 200 is shown, which can also be used with the sequencing platform 100 described above.

[0093] See Figure 11 The sequencing chip 200 has a square outer contour. In the thickness direction Z, the sequencing chip 200 is formed by stacking a base layer 10b, a gel layer 20, and a capping layer 30. For the specific stacking method, please refer to [reference needed]. Figure 6 and Figure 7 I will not go into details here.

[0094] Figure 12 for Figure 11 A schematic diagram of the structure of the base layer 10b of the sequencing chip 200.

[0095] See Figure 12 The base layer 10b is composed of a square substrate 11 (such as a silicon wafer). The first surface P1 of the substrate 11 has a recessed adhesive overflow groove C3. The adhesive overflow groove C3 is annular and divides the first surface P1 into an inner surface region P11 and an outer surface region P12. The inner surface region P11 is located inside the inner periphery of the adhesive overflow groove C3, i.e. Figure 12 The dot-filled region in the middle serves as the sequencing region S1 that encloses the sequencing flow channel Q1. The outer region P12 is located outside the outer periphery of the overflow groove C3. The adhesive layer 20 is bonded between the outer region P12 of the substrate 11 and the capping layer 30.

[0096] Adhesive layer 20 (such as photocurable adhesive) can be applied to the entire outer area P12, or adhesive layer 20 can be applied to the area surrounding the overflow groove C3 on the outer area P12. The width of adhesive layer 20 (i.e., the distance between the inner and outer circumferential contours of adhesive layer 20) should be set to be relatively large (e.g., greater than 1 mm) to ensure reliable adhesion between the base layer 10b and the capping layer 30 and to ensure reliable sealing of the sequencing channel Q1.

[0097] In this embodiment, an overflow groove C3 is recessed on the base layer 10b. Thus, during the glue application and stacking process, when the glue flows to the overflow groove side, it will be collected by the overflow groove C3 and will not easily enter the inner surface area P11. This makes it less likely for protruding glue to appear on the sidewall of the sequencing channel Q1, ensuring that the sidewall of the sequencing channel Q1 is uniform and flat. This reduces the obstruction of the sidewall of the sequencing channel Q1 to reagents, ensuring that reagents flow smoothly into and out of the sequencing channel Q1 and are less likely to remain in the sequencing channel Q1.

[0098] In this embodiment, the overflow groove C3 can be processed on the first surface P1 of the substrate 11 by sandblasting / laser processing / tool ​​wheel processing.

[0099] In this embodiment, the gel layer 20 can also be controlled by adding microspheres of a fixed size, for example, around 40 μm. The thickness of the sequencing channel Q1 is determined by the thickness of the gel layer 20.

[0100] It is understood that in this embodiment, the overflow groove can also be set on the lower surface of the cover layer 30.

[0101] See you again Figure 12 In this embodiment, sequencing channel Q1 is composed of... Figure 5 The single square shape is transformed into a long strip. In this way, a low-throughput chip can still be obtained. The inlet port K1 and outlet port K2 are respectively opened at both ends of the long strip, which are directly connected to the sequencing channel Q1. There is no need to open an additional guide groove C2 to connect the sequencing channel Q1 and the inlet port K1 or outlet port K2, thus saving the processing cost of the guide groove C2.

[0102] Optionally, the sequencing channel Q1 can be designed as an elongated strip extending diagonally along the base layer 10b, including a rectangular segment in the middle and triangular segments at both ends. The inlet port K1 and outlet port K2 are respectively located at the apex of the two triangular segments. The flow sequence of the sequencing channel Q1 is as follows: liquid flows from the inlet port K1 through the sequencing channel Q1 and then out through the outlet port K2. Relative to... Figure 5 In this sequencing chip 200, only one liquid outlet hole K2 of the base layer 10b is retained, while the other two liquid outlet holes K are either discontinued or not processed.

[0103] Figure 11 and Figure 12In this embodiment, the sequencing channel Q1 extends at a 45° angle to the square outer frame, connecting the inlet port K1 and the outlet port K2 located diagonally. In other embodiments, the sequencing channel Q1 may also extend along the direction connecting the inlet port K1 and the outlet port K2 located on the same horizontal or vertical side; this is not limited here.

[0104] Referring to equation (1) below, Figure 11 and Figure 12 In the illustrated scheme, because the sequencing channel Q1 is long and narrow, the ratio of channel length L to width W is relatively large, resulting in relatively high flow resistance, the liquid flow rate cannot be too fast, and the sequencing time is relatively long.

[0105]

[0106] In equation (1), ΔP is the pressure drop, L is the channel length, W is the channel width, H is the channel depth, Q is the flow rate, and μ is the dynamic viscosity.

[0107] Optionally, in this embodiment, the ratio of the channel width W to the length L is not greater than 1 / 7, so as to avoid excessive reagent loss at the edges.

[0108] See Figure 13 This embodiment also provides another base layer 10c, which is for use in... Figure 12 Based on the base layer 10b, material in the triangular areas at the lower left and upper right corners is cut away, making the base layer 10c a strip-shaped hexagon extending diagonally along the outer contour of the sequencing chip 200. This reduces the amount of material used in the base layer 10c (such as a silicon wafer), thus lowering costs.

[0109] Figure 14 Another type of substrate 10d is shown, which includes a substrate 11 made of silicon wafer and a filler 12 made of other materials (such as plastic, glass, etc.). For example Figure 14 The 10d base level Figure 13 A triangular filler 12 is attached to each side of the base 11, so that the outer contour of the projection of the base layer 10d along the thickness direction Z is square, which can better adapt to the aforementioned sequencing platform 100.

[0110] By replacing silicon wafers with other materials, the amount of silicon wafers used can be reduced, thus saving costs.

[0111] To ensure the overall flatness of the base layer 10d, the surface of the filler 12 along the thickness direction on one side is coplanar with the first surface P1, and the surface of the filler 12 along the other side along the thickness direction is the second surface P2.

[0112] In this embodiment, optionally, the sharp angles of the triangular segments at both ends of the sequencing region S1 are set to no more than 90 degrees, for example, 45 degrees.

[0113] For adoption Figure 14 The sequencing chip 200 of the base layer 10d shown is suitable for fixation onto the sequencing platform 100 of this embodiment by negative pressure adsorption.

[0114] For adoption Figure 13 The sequencing chip 200 of the base layer 10c shown may not be suitable for fixation to the sequencing platform 100 of this embodiment by negative pressure adsorption. In this case, the sequencing chip 200 can be pressed against the sequencing platform 100 by auxiliary means such as the pressing device 120, as can be seen in [reference needed]. Figure 15 .

[0115] Figure 16 Another base layer 10e is shown, with a square outer contour, which can also be adapted to the aforementioned sequencing platform 100.

[0116] See Figure 16 The 10e level at the grassroots level is in Figure 12 Based on the base layer 10b, the size of sequencing channel Q1 and sequencing region S1 is reduced, and the two ends of sequencing region S1 in the longitudinal direction are connected to the inlet hole K1 or the outlet hole K2 through guide groove C2 respectively.

[0117] Specifically, in addition to the overflow groove C3, the first surface P1 of the base layer 10e also has two guide grooves C2 (namely, the liquid inlet guide groove C21 and the liquid outlet guide groove C22). The liquid inlet hole K1 and the liquid outlet hole K2 are respectively located at the two ends of the diagonal of the square base layer 10e. The liquid inlet guide groove C21, the sequencing flow channel Q1, and the liquid outlet guide groove C22 are connected in series along the diagonal of the square between the liquid inlet hole K1 and the liquid outlet hole K2.

[0118] By further reducing the size of the sequencing region S1 and the sequencing channel Q1, the sequencing chip 200 using this 10e base layer has a lower throughput, which can further reduce the reagents required for sequencing. It should be noted that this approach may make the sequencing region S1 and the sequencing channel Q1 very thin and long, further increasing the ratio of channel length L to width W, and further increasing flow resistance.

[0119] The shape and arrangement of the guide groove C2 can be found in the foregoing. Figures 8-10 And related information.

[0120] Figure 8 In the process, the sequencing region S1 and the sequencing channel Q1 are square, resulting in low overall flow resistance. However, due to the large cross-sectional width of the liquid flow channel, a guide groove C2 needs to be opened on the first surface P1 to ensure uniform liquid spreading, which leads to higher process costs. Figures 11-15 In this method, the sequencing region S1 and sequencing channel Q1 are elongated strips, eliminating the need for a guide channel C2 to ensure uniform liquid flow. However, due to the narrowness and high flow resistance of sequencing channel Q1, reagents cannot pass through sequencing channel Q1 quickly, resulting in a certain disadvantage in sequencing time.

[0121] In view of this, Figure 17 Another base layer 10f was proposed. Its outer contour is square, and it can also be adapted to the aforementioned sequencing platform 100. Figure 17 The 10f level of this grassroots unit is in Figure 16 Based on this, the sequencing region S1 is changed to multiple (e.g., two). Accordingly, one end of the liquid inlet guide channel C21 is connected to the liquid inlet hole K1, and multiple branches at the other end are connected to multiple sequencing regions S1 respectively; one end of the liquid outlet guide channel C22 is connected to the liquid outlet hole K2, and multiple branches at the other end are connected to multiple sequencing regions S1 respectively.

[0122] Compared to Figure 8 and Figures 11-15 The plan, Figure 17 The proposed scheme ensures that sequencing time is not significantly affected, and that processing costs and flow resistance are moderate.

[0123] In this embodiment, the sequencing region S1 requires a special surface treatment process, and the guide groove C2 requires a special size design and processing (refer to the previous description of the guide groove C2). Other areas do not require special treatment. On the first surface P1, the sequencing region S1 needs to be separated from other areas (e.g., separated by the adhesive layer 20), and the area where the guide groove C2 is located also needs to be separated from other areas (e.g., separated by the adhesive layer 20).

[0124] Figure 17 In the middle, the total width of the two sequencing regions S1 is Figure 16 The width of a single sequencing region S1 is twice that of the sequencing region S1. According to formula (1), the flow resistance of the sequencing channel Q1 corresponding to the sequencing region S1 is reduced.

[0125] Figure 17 In sequencing region S1, the width-to-length ratio should ideally not exceed 1:7 to avoid excessive reagent loss at the edges. Optionally, the sharp angles of the triangular segments at both ends of sequencing region S1 should be set to no more than 90 degrees, for example, 60 degrees.

[0126] The guide channel C2 is used to transport reagents to different zones. The width and depth of the flow channel at different positions of the guide channel C2 can vary to achieve uniform liquid flow. The cross-sectional shape of the guide channel C2 can be rectangular, semi-circular, or other shapes. To avoid excessive resistance, the guide channel C2 should not be too shallow or too narrow. To avoid problems such as excessive reagent loss, excessively thin walls, and decreased strength, the guide channel C2 should not be too deep.

[0127] Figure 17In the sequencer, the sequencing region S1 is relatively long and thin, and pressure-sensitive adhesive (PSA) can be used as the adhesive layer 20 for encapsulation. During encapsulation, the PSA material is cut into the shape of the flow channel, adhered to the first surface P1 of the base layer 10f, and then the capping layer 30 is attached to it using a tooling alignment. Finally, a thermoforming press is used to cure the PSA, bonding the base layer 10f and the capping layer 30 together. The shape obtained by cutting the PSA material forms the sequencing flow channel Q1. One advantage of PSA is that the encapsulation shape can be arbitrarily changed, and the resulting sequencing flow channel Q1 is relatively uniform. Another advantage is that the gap tolerance of the encapsulated sequencing flow channel Q1 is small. A third advantage is that there is no need to open the overflow groove C3 to assist in the formation of the sequencing flow channel Q1.

[0128] Figure 18 Another substrate 10g is shown. This substrate 10g has a square outer contour and can also be adapted to the aforementioned sequencing platform 100.

[0129] Unlike the aforementioned embodiments, Figure 18 In the process, the inlet hole K1 and the outlet hole K2 are not located on a diagonal line of the outer contour of the base layer 10g, but on a straight line parallel to one side of the base layer 10g. Other outlet holes K2 are not processed, or are processed but not used.

[0130] Figure 18 In the process, ≥2 elongated sequencing regions S1 are divided on the base layer 10g. Then, guide grooves C2 are processed on the first surface P1 of the base layer 10g to connect the liquid inlet hole K1 and each sequencing channel Q1, as well as to connect the liquid outlet hole K2 and each sequencing channel Q1.

[0131] Figure 18 In this study, the number of sequencing regions S1 is 2.

[0132] See Figure 19 If the sequencing chip 200 itself has a method for fixation (such as using the aforementioned clamping device 120) without the need for vacuum adsorption, a base layer 10h larger than the sequencing platform 100 can be used to increase throughput by increasing the number of sequencing regions S1 and sequencing channels Q1. For example... Figure 19 In this configuration, the area of ​​the base layer 10h is larger than that of the sequencing platform 100, and the sequencing chip 200 has a portion located outside the sequencing platform 100, and has more sequencing regions S1 and sequencing channels Q1. At this time, the sequencing platform 100 can still be fixed to the sequencing platform 100 by means of a clamping device 120 or similar means.

[0133] See Figure 20 When there is no need to consider the vacuum adsorption of the sequencing chip 200, it is possible to... Figure 18The base layer 10g is cut, retaining only the lower half to obtain the base layer 10i, thus saving silicon wafer usage for the base layer 10i. Of course, the upper half can also be replaced with filler 12 of other materials.

[0134] Figure 21 Another base layer 10j is shown. See also Figure 21 The outer contour of the base layer 10j is square. There are four sequencing regions S1. The base layer 10j has one inlet port K1 and two outlet ports K2. One outlet port K2 is located diagonally opposite the inlet port K1, and the other outlet port K2 is located at a corner adjacent to the inlet port K1. The inlet port K1 is connected to each of the four sequencing channels Q1 via an inlet guide channel C21. There are two outlet guide channels C22. One outlet guide channel C22 connects two sequencing channels Q1 to one outlet port K2, and the other outlet guide channel C22 connects the other two sequencing channels Q1 to the other outlet port K2. By utilizing multiple outlet ports K2, the flow rate limitation of a single outlet port K2 on the sequencing channels Q1 can be avoided, thus accelerating the sequencing process.

[0135] For example, if the number of sequencing regions S1 and sequencing channels Q1 is large (e.g., ≥3), in a single-inlet, single-outlet configuration, the diameter of the outlet well K2 may become a bottleneck for speed improvement. In this case, if... Figure 21 As shown, by using multiple outlet holes K2 to achieve liquid discharge from different sequencing channels Q1, the flow diameter can be increased and the sequencing speed can be improved.

[0136] Figure 22 This indicates the adoption Figure 20 The design method of the guide groove C2 of the sequencing chip 200 of the base layer 10i is shown.

[0137] See Figure 22 By adjusting the length (L1, L2, L3, and L4 in the figure) and cross-sectional area of ​​the groove segment C2 used to connect each sequencing channel Q1, the flow resistance and internal volume of different sequencing channels Q1 can be adjusted, thereby achieving uniform liquid flow through different sequencing channels Q1 (see Figure 1). Figure 23 Specifically, the inlet guide channel C21 and outlet guide channel C22 of the sequencing chip 200 adopt an "F"-shaped flow channel design, which is an asymmetric flow channel. The flow channel design method can be found in the Chinese patent application document with application number 202510063923.X and application date of January 15, 2025.

[0138] See Figure 24 This embodiment also provides a sequencing device 1000, including a sequencing platform 100, a sequencing chip 200, and a fixing device 300. The sequencing chip 200 can be carried on the sequencing platform 100. The fixing device 300 can fix the sequencing chip 200 to the sequencing platform 100. The fixing device 300 may include a clamping device 120.

[0139] In other embodiments, the fixing device 300 may also include a negative pressure adsorption device.

[0140] The above embodiments are only used to illustrate the technical solutions of this application and are not intended to limit it. Although this application has been described in detail with reference to the above preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions to the technical solutions of this application should not depart from the spirit and scope of the technical solutions of this application.

Claims

1. A sequencing chip, characterized in that, include: The base layer includes a substrate; the substrate has a first surface and a second surface facing away from each other; the first surface has a sequencing region; the substrate has a liquid inlet and a liquid outlet. as well as, A capping layer is stacked on one side of the first surface of the base layer; a sequencing channel is defined between the capping layer and the sequencing region; the sequencing channel is connected between the inlet and the outlet.

2. The sequencing chip according to claim 1, characterized in that: The outer contour of the sequencing chip projected along its thickness direction is square. The substrate is a strip-shaped hexagon extending diagonally along the outer contour; the liquid inlet and the liquid outlet are respectively located at both ends of the extending direction of the substrate.

3. The sequencing chip according to claim 2, characterized in that: The base layer also includes a filler; The filler and the substrate are joined laterally so that the outer contour of the base layer projected along the thickness direction is square, consistent with or substantially consistent with the capping layer.

4. The sequencing chip according to claim 3, characterized in that: The surface of the filler along the thickness direction is coplanar with the first surface; The filler has a surface on the other side of the thickness direction and a second surface.

5. The sequencing chip according to claim 1, characterized in that: The outer contour of the sequencing chip projected along its thickness direction is square. The inlet and outlet are respectively located at the two ends of the diagonal of the square. The first surface is recessed with a guide groove; the guide groove includes an inlet guide groove and an outlet guide groove; The inlet guide channel, the sequencing channel, and the outlet guide channel are connected in series along the diagonal of the square between the inlet hole and the outlet hole.

6. The sequencing chip according to any one of claims 1-4, characterized in that: The first surface is recessed with a guide groove; The guide channel includes an inlet guide channel, which is connected between the sequencing channel and the inlet port; and / or, the guide channel includes an outlet guide channel, which is connected between the sequencing channel and the outlet port.

7. The sequencing chip according to claim 6, characterized in that: The sequencing channels have multiple components; The inlet port is connected to multiple sequencing channels through the inlet guide groove; and / or, the outlet port is connected to multiple sequencing channels through the outlet guide groove.

8. The sequencing chip according to claim 1, characterized in that: The outer contour of the sequencing chip projected along its thickness direction is square; the sequencing channel is elongated, and the ratio of the channel width to its length is no greater than 1 / 7. The sequencing channel extends parallel to the diagonal of the outer contour or parallel to one side of the outer contour.

9. The sequencing chip according to claim 1, characterized in that: The first surface of the substrate is recessed with an overflow groove, which is annular and divides the first surface into an inner area and an outer area. The sequencing chip further includes a gel layer; the gel layer is bonded between the outer region of the substrate and the capping layer; the thickness of the sequencing channel is equal to the thickness of the gel layer.

10. A sequencing device, characterized in that, include: Sequencing platform; The sequencing chip according to any one of claims 1-9, wherein the sequencing chip can be carried on the sequencing platform; as well as, A fixing device is connected to the sequencing platform and is capable of fixing the sequencing chip to the sequencing platform; Wherein, the fixing device is a negative pressure adsorption device, or the fixing device is a pressing device.

Images