Solid-phase substrate and biochip thereof

Abstract

The invention belongs to the technical field of biological detection equipment, and relates to a solid-phase substrate and a biological chip thereof. The surface of the solid-phase substrate provided by the invention is provided with a plurality of groups of regularly arranged marking units, each group of marking units comprises at least three marks arranged according to a preset mode, and each mark comprises a first marking line located in a first direction and a second marking line located in a second direction and intersected with the first marking line. According to the invention, by redesigning the marking unit on the solid-phase substrate, the identifiability of the marking line is obviously improved, so that the accuracy of calibration is improved.

Description

Technical Field

[0001] The present invention relates to the field of gene detection devices, and particularly to a solid-phase substrate and a biochip thereof. Background Art

[0002] As an important consumable for gene sequencing, the gene sequencing chip plays an important role in gene sequencing. Specifically, the surface of the chip can be treated to bind chemical molecules or biomolecules, and nucleic acid molecules can bind to chemical molecules or biomolecules in ways such as covalent bonds and hydrogen bonds, thereby being fixed on the chip surface. During the process of base recognition of the nucleic acid molecules (also called nucleic acid templates) fixed on the chip surface based on fluorescence signals, the fluorescence signals of nucleotides or their analogs introduced into the nucleic acid molecules in each round of base extension reaction can be obtained through optical imaging, and then the base types of the incorporated nucleotides or their analogs can be obtained based on the fluorescence signals.

[0003] In order to be able to simply and accurately identify the nucleotide types introduced in each round of base extension reaction, the sequencing chip needs to be surface-labeled to localize the fluorescence signals in the imaging image. How to reduce the impact on the performance of the sequencing chip on the basis of effectively identifying the label is an issue that the industry has been continuously concerned about. Summary of the Invention

[0004] In a gene sequencing chip, rough positioning of the standard FOV and fine positioning of the signal position can be achieved by designing a trackline on the chip surface. Specifically, the position of the standard FOV is found through a pre-designed trackline (such as the preset shape of the trackline), then the position of the nucleic acid molecules in the sequencing area is determined according to the signal positions shown on the trackline, and finally the base signals of the nucleic acid molecules in the sequencing area are obtained. As an example, as Figure 1 shown, the trackline in a standard FOV consists of 4×6 lines that intersect (such as being perpendicular to each other). These lines are arranged at regular intervals, dividing the FOV into multiple region blocks (blocks) with a certain regular arrangement. Taking two intersecting lines as an example, the enlarged schematic diagram of the trackline is as Figure 2 、 Figure 3 shown. It can be seen from the figure that the area occupied by the trackline in the horizontal direction (subsequently called the X direction) within one FOV is 7 pixel widths; the area occupied in the vertical direction (subsequently called the Y direction) perpendicular to the horizontal direction is 5 pixel lengths, where pixel represents a pixel point. The trackline designed in this way occupies a relatively large area in the FOV, and since no sequencing throughput is generated at the trackline position, the trackline causes a loss in the throughput of the gene sequencing chip.

[0005] To solve the above problems, the present application provides a solid-phase substrate capable of accurately identifying a trackline and facilitating an increase in throughput, and a biochip.

[0006] In a first aspect, the present application provides a solid-phase substrate, on one surface of which there are provided multiple groups of regularly arranged marking units. Each group of the marking units includes at least three marks arranged in a preset manner, and each mark includes a first marking line in a first direction and a second marking line in a second direction and intersecting with the first marking line.

[0007] As a possible implementation manner of the solid-phase substrate of the present application, the solid-phase substrate is provided with at least one detection area, and each detection area is provided with at least one group of the marking units.

[0008] As a possible implementation manner of the solid-phase substrate of the present application, the detection area includes one or more detection units, and each detection unit is provided with one group of the marking units.

[0009] As a possible implementation manner of the solid-phase substrate of the present application, the detection area includes multiple detection rows arranged along the first direction.

[0010] As a possible implementation manner of the solid-phase substrate of the present application, the distance between adjacent detection rows is equal.

[0011] As a possible implementation manner of the solid-phase substrate of the present application, multiple equally spaced detection sites are provided in the same row.

[0012] As a possible implementation manner of the solid-phase substrate of the present application, the detection site is a micro-pore or a nano-pore.

[0013] As a possible implementation manner of the solid-phase substrate of the present application, the detection site is a nano-scale or micro-scale protrusion.

[0014] As a possible implementation manner of the solid-phase substrate of the present application, the sizes of the detection sites in the detection area are the same.

[0015] As a possible implementation manner of the solid-phase substrate of the present application, in two adjacent rows, except for at least one of the two detection sites at both ends of the row, the detection sites on one row are all located on the midline of the connection line between two adjacent detection sites on the other row.

[0016] As a possible implementation manner of the solid-phase substrate of the present application, as a possible implementation manner of the solid-phase substrate of the present application, the detection unit includes a rectangular area block located in the central area of the detection unit.

[0017] As a possible implementation of the solid-phase substrate of the present application, the marking unit is disposed within the rectangular area block.

[0018] As a possible implementation of the solid-phase substrate of the present application, in a group of the marking units, the area of the region enclosed by the marking connection lines accounts for 50% or more of the total area of the rectangular area block.

[0019] As a possible implementation of the solid-phase substrate of the present application, in a group of the marking units, the area of the region enclosed by the marking connection lines accounts for 80% or more of the total area of the rectangular area block.

[0020] As a possible implementation of the solid-phase substrate of the present application, in a group of the marking units, the image enclosed by the marking connection lines is a symmetric image.

[0021] As a possible implementation of the solid-phase substrate of the present application, the center of the symmetric image coincides with the center of the detection unit.

[0022] As a possible implementation of the solid-phase substrate of the present application, each group of the marking units includes three marks arranged in a preset manner.

[0023] As a possible implementation of the solid-phase substrate of the present application, the image enclosed by the connection lines of the three marks is an isosceles triangle.

[0024] As a possible implementation of the solid-phase substrate of the present application, each group of the marking units includes four marks arranged in a preset manner.

[0025] As a possible implementation of the solid-phase substrate of the present application, the image enclosed by the connection lines of the four marks is a rectangle.

[0026] As a possible implementation of the solid-phase substrate of the present application, the detection unit includes a rectangular area block located in the central area of the detection unit, and the sides of the rectangular area block are respectively parallel or perpendicular to the first direction.

[0027] As a possible implementation of the solid-phase substrate of the present application, the four marks are respectively disposed at the four vertices of the rectangular area block.

[0028] As a possible implementation of the solid-phase substrate of the present application, the first direction and the second direction are perpendicular.

[0029] As a possible implementation of the solid-phase substrate of the present application, the total area of the marking unit 11 accounts for 1% or less of the surface area of the detection unit 10A.

[0030] As a possible implementation of the solid-phase substrate of the present application, the aspect ratio of the first marking line is 15 to 30:1.

[0031] As a possible implementation of the solid-phase substrate of the present application, the aspect ratio of the second marking line is 15 to 30:1.

[0032] As a possible implementation of the solid-phase substrate of the present application, the length ratio of the first marking line to the second marking line is 0.8 to 1.2:1.

[0033] As a possible implementation of the solid-phase substrate of the present application, the solid-phase substrate includes a plurality of detection units, and a set of the marking units is arranged in each detection unit; a plurality of detection sites are equidistantly arranged in the first direction in the detection units.

[0034] As a possible implementation of the solid-phase substrate of the present application, taking the distance between the centers of two adjacent detection sites in the first direction as l, the length and width of the first marking line and the second marking line satisfy at least one of the following conditions:

[0035] The length of the first marking line is 80 to 150l,

[0036] The length of the second marking line is 80 to 150l,

[0037] The width of the first marking line is 3 to 8ll,

[0038] The width of the second marking line is 3 to 8ll.

[0039] As a possible implementation of the solid-phase substrate of the present application, the first marking line and / or the second marking line is a groove formed on the surface, and an identifiable mark is arranged in the groove.

[0040] As a possible implementation of the solid-phase substrate of the present application, the solid-phase substrate includes a plurality of detection units, and a set of the marking units is arranged in each detection unit; a plurality of detection sites are equidistantly arranged in the first direction in the detection units, and the distance between adjacent identifiable marks in the groove is less than the distance between adjacent detection sites in the detection unit.

[0041] As a possible implementation of the solid-phase substrate of the present application, the distance between the identifiable marks in the groove is 1 / 2 or less of the distance between adjacent detection sites in the detection unit.

[0042] As a possible implementation of the solid-phase substrate of the present application, the first marking line and / or the second marking line include holes or protrusions formed on the surface of the solid-phase substrate, and recognizable marks are provided in at least part of the holes or on the surfaces of the protrusions.

[0043] As a possible implementation of the solid-phase substrate of the present application, in the first marking line, the central connection lines of the holes or the protrusions are a straight line, and the straight line is parallel to the first direction.

[0044] As a possible implementation of the solid-phase substrate of the present application, in the second marking line, the connection lines of the centers of the holes or the protrusions are a straight line, and the straight line is perpendicular to the first direction.

[0045] As a possible implementation of the solid-phase substrate of the present application, the distances between adjacent recognizable marks on the first marking line and / or the second marking line are equal respectively.

[0046] As a possible implementation of the solid-phase substrate of the present application, the solid-phase substrate includes a plurality of detection units, and each detection unit is provided with a set of the marking units; the detection units are provided with a plurality of detection sites at equal intervals in the first direction, and the distance between at least part of adjacent recognizable marks is greater than the distance between two adjacent detection sites.

[0047] As a possible implementation of the solid-phase substrate of the present application, taking the distance between the centers of two adjacent detection sites in the first direction as l, the distance between adjacent recognizable marks is n times l, and n is an integer greater than or equal to 1.

[0048] As a possible implementation of the solid-phase substrate of the present application, n is a positive integer from 2 to 4.

[0049] As a possible implementation of the solid-phase substrate of the present application, the recognizable marks are regularly distributed on the first marking line according to a first arrangement rule, and the recognizable marks are regularly distributed on the second marking line according to a second arrangement rule.

[0050] As a possible implementation of the solid-phase substrate of the present application, the recognizable marks are provided in all the holes or on the surfaces of the protrusions of the first marking line and / or the second marking line.

[0051] As a possible implementation of the solid-phase substrate of the present application, the holes are at least one of circular holes, oval holes, polygonal holes and irregular holes.

[0052] As a possible implementation of the solid-phase substrate of the present application, the holes are at least one of circular holes and regular polygonal holes.

[0053] As a possible implementation of the solid-phase substrate of the present application, the holes provided on the surface where the first marking line and / or the second marking line are located have the same shape and size.

[0054] As a possible implementation of the solid-phase substrate of the present application, the protrusion is at least one of a circular protrusion, an elliptical protrusion, a polygonal protrusion, and an irregular protrusion.

[0055] As a possible implementation of the solid-phase substrate of the present application, the protrusion is at least one of a circular protrusion and a regular polygonal protrusion.

[0056] As a possible implementation of the solid-phase substrate of the present application, the protrusions provided on the surface where the first marking line and / or the second marking line are located have the same shape and size.

[0057] As a possible implementation of the solid-phase substrate of the present application, the recognizable mark is a fluorescent mark.

[0058] In a second aspect, the present application provides a biochip, which includes the solid-phase substrate described in the first aspect.

[0059] In the present invention, by designing multiple groups of regularly arranged marking units on the solid-phase substrate, rough positioning of the surface area of the solid-phase substrate and fine positioning of the surface position of the solid-phase substrate can be achieved based on the arrangement rule of the marking units and the relationship between the first marking line and the second marking line. Moreover, by designing the intersecting marking units to achieve the positioning of the solid-phase substrate surface, the area occupied by the marks on the solid-phase substrate surface can be reduced, and the area loss of the solid-phase substrate surface can be reduced, thereby facilitating the improvement of the effective utilization rate of the solid-phase substrate surface. When the solid-phase substrate is used as a biochip, the sequencing throughput can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following will briefly introduce the drawings required for the description of the embodiments. Obviously, the drawings in the following description are only the embodiments of the present application. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.

[0061] Figure 1 It is a schematic structural diagram of a FOV provided with a trackline in the prior art;

[0062] Figure 2 For one Figure 1 An enlarged schematic diagram of the trackline of two intersecting lines in it;

[0063] Figure 3 For one Figure 1Enlarged schematic diagram of the trackline of two intersecting lines;

[0064] Figure 4 Schematic diagram of a solid-phase substrate with a labeling unit provided by an embodiment of the present application;

[0065] Figure 5 Another structural schematic diagram of the solid-phase substrate provided by an embodiment of the present application;

[0066] Figure 6 Partial schematic diagram of the detection unit provided by an embodiment of the present application;

[0067] Figure 7 Schematic diagram of the arrangement of detection sites provided by an embodiment of the present application;

[0068] Figure 8 Schematic diagram of the rectangular area block in the detection unit provided by an embodiment of the present application;

[0069] Figure 9 Effect diagram of setting continuous recognizable signals in the groove provided by an embodiment of the present application;

[0070] Figure 10 Schematic diagram of a labeling unit containing three labels provided by an embodiment of the present application;

[0071] Figure 11 Schematic diagram of a labeling unit containing four labels provided by an embodiment of the present application;

[0072] Figure 12 Schematic diagram of the detection unit containing a labeling unit provided by an embodiment of the present application;

[0073] Figure 13 Partial schematic diagram of the label provided by an embodiment of the present application;

[0074] Figure 14 Another partial schematic diagram of the label provided by an embodiment of the present application;

[0075] Figure 15 Schematic diagram of the first repeating unit provided by an embodiment of the present application;

[0076] Figure 16 Schematic diagram of the second repeating unit provided by an embodiment of the present application;

[0077] Figure 17 Structural schematic diagram provided by this comparative example;

[0078] Figure 18 Q30 data statistical curve graph of Example 1 (V03) and Comparative Example 1 (V02);

[0079] Figure 19Error rate data statistical curves for Example 1 (V03) and Comparative Example 1 (V02);

[0080] Figure 20 Q30 data statistical curve graph for Example 2 (V04) and Comparative Example 1 (V02);

[0081] Figure 21 Error rate data statistical curve graph for Example 2 (V04) and Comparative Example 1 (V02).

[0082] Label description:

[0083] 1. Solid-phase substrate; 10A. Detection unit; 10A1. Rectangular region block; 11. Marking unit; 110. Mark; 1101. First marking line; 1102. Second marking line; A. Detection area; A1 (A11 / A12). Detection row; A10. Detection site; 110, A11 / A12 / A13 / A14 / A15 / A16 / A17 / A19. Detection sites; O1 / O2 / O3 / O4. Intersection points; 101. Detection area; 102. First trackline; 103. Second trackline; 105. Detection site (comparative example). Detailed implementation manners

[0084] The present invention will be further described in detail below through specific implementation manners in conjunction with the accompanying drawings. In the following implementation manners, many detailed descriptions are provided to enable a better understanding of the present application. However, those skilled in the art can easily recognize that some of these features can be omitted in different situations, or can be replaced by other features or methods. In some cases, some operations related to the present application are not shown or described in the specification, which is to avoid the core part of the present application being overwhelmed by excessive descriptions. For those skilled in the art, it is not necessary to describe these related operations in detail, and they can fully understand the related operations based on the descriptions in the specification and general technical knowledge in the art.

[0085] In addition, the features, operations or characteristics described in the specification can be combined in any appropriate manner to form various implementation manners. At the same time, the steps or actions in the method description can also be reordered or adjusted in a manner obvious to those skilled in the art. Therefore, the various sequences in the specification and drawings are only for clearly describing a certain embodiment and do not mean a necessary sequence, unless it is stated that a certain sequence must be followed.

[0086] The serial numbers assigned to the components herein, such as "first", "second", etc., are only used to distinguish the described objects and do not have any sequential or technical meaning.

[0087] In an embodiment of the present application, the term "sequencing" may also be referred to as "nucleic acid sequencing" or "gene sequencing", and the three can be used interchangeably in expression, all referring to the determination of the types and arrangement orders of bases or nucleotides (including nucleotide analogs) in a nucleic acid molecule. The so-called sequencing includes the process of binding nucleotides to a template and collecting the corresponding signals emitted from the nucleotides (including analogs). The so-called sequencing includes synthesis sequencing (sequencing by synthesis, SBS) and / or ligation sequencing (sequencing by ligation, SBL), including DNA sequencing and / or RNA sequencing, including long-fragment sequencing and / or short-fragment sequencing. The so-called long fragments and short fragments are relative. For example, a nucleic acid molecule longer than 1 Kb, 2 Kb, 5 Kb or 10 Kb can be called a long fragment, and a nucleic acid molecule shorter than 1 Kb or 800 bp can be called a short fragment.

[0088] Sequencing generally includes multiple rounds to achieve the determination of the types and arrangement orders of multiple bases or nucleotides on a nucleic acid template. In an embodiment of the present application, each round of "the process of achieving the determination of the types and arrangement orders of multiple bases or nucleotides on a nucleic acid template" is called "one round of sequencing". "One round of sequencing" (cycle) is also called "sequencing round", and can be defined as a single base extension of four nucleotides / bases. In other words, "one round of sequencing" can be defined as completing the determination of the type of base or nucleotide at any specified position on the template. For a sequencing platform that realizes sequencing based on a polymerization or ligation reaction, one round of sequencing includes the process of enabling four nucleotides (including nucleotide analogs) to bind to the so-called nucleic acid template through base complementarity and collecting the corresponding signals emitted. Among them, for a platform that realizes sequencing based on a polymerization reaction, the reaction system includes reaction substrate nucleotides, polymerase and nucleic acid template. A sequence (sequencing primer) is bound to the nucleic acid template. Based on the base pairing principle and the principle of polymerization reaction, the added reaction substrate nucleotides are connected to the sequencing primer under the catalysis of the polymerase to achieve the binding of the nucleotide to a specific position on the nucleic acid template. Generally, one round of sequencing may include one or more base extensions (repeat). For example, four nucleotides are added to the reaction system in sequence, and base extensions and the collection of corresponding reaction signals are carried out respectively. One round of sequencing includes four base extensions; for another example, four nucleotides are added to the reaction system in any combination, such as in pairs or in a combination of one and three, and the two combinations are respectively subjected to base extension and the collection of corresponding reaction signals. One round of sequencing includes two base extensions; for another example, four nucleotides are added to the reaction system simultaneously for base extension and the collection of reaction signals. One round of sequencing includes one base extension.

[0089] In this article, "trakline" is also called calibration line, identification line or track line. The main function of the trackline is to mark the standard FOV and locate the signal position points, so that the corresponding relationship between the detection position and the detection signal can be confirmed based on the trackline during the detection process.

[0090] In this article, the term "sequencing" can also be referred to as "nucleic acid sequencing" or "gene sequencing", that is, the three can be interchanged in expression, referring to the determination of the base types and arrangement order in a nucleic acid sequence.

[0091] In this article, "FOV" (Field of View) refers to the field of view. During the application process of the solid-phase substrate, it includes the process of performing multiple rounds of optical imaging (taking pictures) on a fixed area of the solid-phase substrate by an optical camera. The area captured by the optical camera each time can be called the FOV (field of view).

[0092] In this article, "Reads" represents "reads", which refers to the sequencing results obtained through a sequencing reaction / or the sequencing data, that is, the read-out fragments. The length of the reads is called the read length.

[0093] When performing optical imaging on the solid-phase substrate, considering the configurations of different optical systems, especially the size of the shooting area of the optical camera, the surface of the solid-phase substrate will be divided into several FOVs according to the size of the shooting area. During the process of performing optical imaging on the solid-phase substrate, in order to determine the standard "FOV" and further locate the detection signals of the detection sites in the standard "FOV" to identify the optical information corresponding to the fixed area of the solid-phase substrate, it is necessary to set marks or marking lines on the surface of the solid-phase substrate. It can be seen that the design of the marks or marking lines is related to the positioning and identification of the signals in the detection area.

[0094] Taking a biochip as an example, when performing optical imaging on the biochip, since only one FOV can be imaged each time, multiple optical images corresponding to multiple FOVs will be obtained when performing optical imaging on the biochip. These different optical images can be located by marks or mark lines on the surface of the biochip. In a current marking method, rough positioning of the standard FOV and fine positioning of the signal position can be achieved by designing tracklines on the chip surface. The trackline usually consists of m×n lines perpendicular to each other, and each line has a certain width. For example, in some biochips, the width of the m lines in the horizontal direction (subsequently referred to as the X direction) is A pixel; the width of the n lines in the horizontal direction (subsequently referred to as the Y direction) is B pixel. If the length of each FOV in the X direction is L1 and the length in the Y direction is L2, then, for each FOV, the area occupied by the trackline is A pixel×L1×m + B pixel×L2×n. Exemplarily, for an FOV with a length of 200 pixel in the X direction and a length of 200 pixel in the Y direction, when the trackline consists of 4×6 lines perpendicular to each other, that is, 4 lines are set in the X direction and 6 lines are set in the Y direction, and the width of the m lines in the X direction is 7 pixel and the width of the n lines in the Y direction is 5 pixel, the area occupied by the trackline is 7 pixel×200 pixel×4 + 5 pixel×200 pixel×6. Thus, the trackline occupies a relatively large area in the FOV, and this part of the area does not generate sequencing data, so it will cause a loss of the throughput of the biochip.

[0095] In view of this, the embodiments of the present application provide a marking method that can replace the trackline for marking and can perform fine positioning through an algorithm. Moreover, the area occupied by the marking provided by the embodiments of the present application is greatly reduced, and it has the advantage of increasing the throughput of the biochip compared with the trackline.

[0096] Taking a biochip for sequencing as an example, the design of the labels on the surface of the solid-phase substrate will be described. In this embodiment, in order to better demonstrate the concept of the present invention, a biochip provided with a microporous structure arranged in an array is used as a representative for description. Specifically, a large number of microporous structures arranged in an array are formed by punching on the surface of the biochip, and biomolecules are bound in the micropores. Taking the biomolecule as a nucleic acid molecule as an example, in a sequencing method using an optical signal as a detection signal, in each round of base extension reaction, at least one nucleic acid template in a micropore binds a nucleotide or nucleotide analog containing an optical label by base complementarity, and the bound nucleotide or nucleotide analog generates an optical signal corresponding to the position where the micropore is located, and this optical signal appears as a bright spot in the optical image. By setting specific labels different from the detection area on the surface of the sequencing chip, the positioning of the micropore array can be achieved. It should be understood that in the embodiments of the present application, the site does not have to exist only in the form of a microporous structure, and each preset position for binding a target sample to be detected and capable of generating a detectable signal in theory after a certain reaction can be regarded as a site. That is: in other embodiments, other methods other than the opening method can also be used to replace the pore structure to form a site, so that the sample to be detected is arranged on the solid-phase substrate in the expected arrangement manner, and a detectable signal (such as an optical signal) is collected at the position where the sample to be detected is located.

[0097] After the nucleic acid template is bound to the opening site and a nucleotide or nucleotide analog containing an optical label is bound to the nucleic acid template by base complementarity, the optical label in the reaction site generates an optical signal, which appears as a bright spot in the optical image; while in the non-opening area, since there is no nucleic acid template, no optical signal can be detected during the sequencing process, and it appears as a dark spot in the optical image. By forming a band different from the sequencing area, an identification label can be formed, and then each FOV in the sequencing chip can be calibrated, and the sequencing position in the FOV can be located.

[0098] In a first aspect, in an embodiment, a solid-phase substrate is provided. When the image acquisition sensor collects signals from the solid-phase substrate, each FOV is imaged each time, and an optical image of one FOV is obtained. The optical images of multiple FOVs form the total optical image of the solid-phase substrate. By reading the information (such as fluorescence intensity, etc.) in the optical image of the corresponding area and performing algorithm analysis on the optical image, the detection result of this area can be obtained, such as obtaining the type of nucleotide or nucleotide analog bound to the nucleic acid template in the base extension reaction.

[0099] As Figure 4 - 12 shown, a surface of the solid-phase substrate 1 is provided with multiple groups of regularly arranged label units 11, which are used as identifiers for optical signal positioning when detecting a sample to be detected on the surface of the solid-phase substrate 1 based on an optical signal. Figure 4It schematically shows a partial area of the surface of the solid-phase substrate 1 where the labeling unit 11 is provided.

[0100] According to an embodiment of the present application, for the convenience of explaining the setting of the labeling unit 11, the surface of the solid-phase substrate 1 where the labeling unit 11 is provided can be divided into multiple substrate units 10. The substrate unit 10 belongs to a part of the surface, that is, the surface of the solid-phase substrate 1 where the labeling unit 11 is provided is composed of multiple substrate units 10. The substrate unit 10 is not clearly marked on the surface of the solid-phase substrate 1, and the labeling unit 11 is provided in at least some of the substrate units 10.

[0101] According to an embodiment of the present application, the solid-phase substrate 1 is provided with at least one detection area A, and each detection area A is provided with at least one group of labeling units 11. It should be understood that the detection area mentioned in the embodiment of the present application is an area that can be used to carry the sample to be detected. When the solid-phase substrate 1 is used as a detection substrate, the sample to be detected can be carried in all areas of the detection area A, or in some areas of the detection area A. In a possible implementation manner, in the solid-phase substrate 1, the surface where the labeling unit 11 is located is the detection area A, that is, one surface of the entire solid-phase substrate 11 serves as the area for carrying the sample to be detected. In another possible implementation manner, as Figure 5 shown, the solid-phase substrate 1 includes multiple detection areas A and non-detection areas B other than the detection areas A, and the multiple detection areas A are separated by the non-detection areas B. It should be understood that the non-detection area mentioned in the embodiment of the present application, as opposed to the detection area, refers to an area that does not serve as the area for carrying the sample to be detected, and during the signal detection process, this area also does not serve as the signal detection area.

[0102] According to an embodiment of the present application, referring to Figure 6 , the detection area A includes multiple detection rows A1 arranged in the first direction, and each detection row A1 is composed of multiple regularly arranged detection sites A10. In a possible implementation manner, among the multiple detection sites A10 that make up the detection row A1, the distance between adjacent detection sites A10 is equal, that is: multiple equally spaced detection sites A10 are provided in the same detection row A1. In the embodiment of the present application, when the detection site A10 is a figure with a regular shape such as a circle, an ellipse, a rectangle, a square, a triangle, or other polygons, the distance between adjacent detection sites A10 is the length of the line connecting the centers of adjacent detection sites A10; when the detection site A10 is an irregular figure, take the longest line segment connecting any two points on the irregular outer edge, and use the center of this line segment as the center of the irregular figure. At this time, the distance between adjacent detection sites A10 is the length of the line connecting the centers of adjacent detection sites A10. It should be understood that at least the shapes of the detection sites A10 in the same detection area A are the same to facilitate the positioning of each signal site in the detection area A.

[0103] According to an embodiment of the present application, the detection site A10 may have various possible topographies. In one possible implementation, the detection site A10 is set as a pore structure, that is, the detection site 10 is formed by inverting within the surface of the solid-phase substrate 1. Exemplarily, the detection site A10 is a micro-pore or a nano-pore, and during the detection process, the sample to be detected binds within the micro-pore or nano-pore; in another possible implementation, the detection site A10 is set as a convex structure, that is, the detection site 10 is formed by protruding from the surface of the solid-phase substrate 1. Exemplarily, the detection site A10 is a nano-scale convex or a micro-scale convex. In still another possible implementation, the detection site A10 is a pattern provided on the surface of the solid-phase substrate 1, that is, by performing surface treatment on a local part of the solid-phase substrate 1, so that some regions on the surface of the solid-phase substrate 1 can bind the sample, and the other remaining regions cannot bind the sample, thereby realizing the definition of the position of the detection site A10. It should be understood that some regions on the surface of the solid-phase substrate 1 are multiple discrete regions (detection site A10), and these multiple discrete regions (detection site A10) are dispersed among the "other remaining regions" that cannot bind the sample.

[0104] According to an embodiment of the present application, in order to better facilitate the positioning of the signal generated by the sample to be detected bound to the surface of the solid-phase substrate 1, the detection sites A10 within the detection area A have the same size.

[0105] According to an embodiment of the present application, the spacing d between adjacent detection rows A1 (such as Figure 6 A11 and A12 in Figure 7 shown) is equal. In one possible implementation, as Figure 3 shown, in two adjacent detection rows A1 (such as Figure 4 A11 and A12 in Figure 6 shown, Figure 7 A11 and A12 in

[0106] According to an embodiment of the present application, the distribution of the marker units 11 on the surface of the solid-phase carrier 1 can be set according to the size of the FOV when imaging the surface of the solid-phase substrate 1. In one possible implementation, the size of the substrate unit 10 is consistent with the size of the FOV determined in advance when imaging the surface of the solid-phase substrate 1. In some embodiments, the substrate unit 10 is a rectangular unit, a circular unit, or an elliptical unit.

[0107] In a possible implementation, for the detection area A of the solid-phase substrate 1, a set of marking units 11 is provided for each substrate unit 10. For ease of understanding, the substrate unit 10 in the detection area A is defined as the detection unit 10A (the size corresponding to the size of a pre-determined FOV during surface imaging), as Figure 8 shown. That is: the detection area A includes one or more detection units 10A, and a set of marking units 11 is provided for each detection unit 10A. Through this set of marking units 11, the positioning of each optical signal in the surface area corresponding to one FOV, that is, in the detection unit 10A, is achieved.

[0108] According to an embodiment of the present application, the detection unit 10A includes a rectangular area block 10A1 located in the central area of the detection unit 10A, and the rectangular area block 10A1 extends radially from the center of the detection unit 10A to the surroundings. In a possible implementation, the rectangular area block 10A1 is: within the area range formed by shrinking the detection unit 10A by at least 50% on the premise that the center of the detection unit 10A remains unchanged. In a possible implementation, the detection unit 10A is rectangular, and the rectangular area block 10A1 is: a rectangular area formed by shrinking each of the two pairs of opposite sides of the rectangular detection unit 10A along the central symmetry axis by at least 50%. It should be understood that the percentage of shrinkage of the two pairs of opposite sides of the rectangular detection unit 10A can be the same, such as both being 50%; or different, such as one pair of opposite sides of the rectangular detection unit 10A shrinking by 50% along the central symmetry axis, and the other pair of opposite sides of the rectangular detection unit 10A shrinking by 60% along the central symmetry axis. When the two pairs of opposite sides of the rectangular detection unit 10A do not shrink in equal proportion, the rectangular area block 10A1 is the area enclosed by the extension lines of the two pairs of opposite sides after shrinkage.

[0109] According to an embodiment of the present application, the marking unit 11 is arranged within the rectangular area block 10A1. Thus, through the marking unit 11 arranged within 50% of the center of the detection unit 10A area and the design features of the marking unit 11, each signal generated within the detection unit 10A can be achieved. In some embodiments, in a set of marking units 11, the area of the region enclosed by the connection lines of the respective marks 110 accounts for 50% or more of the total area of the rectangular area block 10A1. Exemplarily, the area of the region enclosed by the connection lines of the respective marks 110 is 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, etc. of the total area of the rectangular area block 10A1. In a specific embodiment, in a set of marking units 11, the area of the region enclosed by the connection lines of the respective marks 110 accounts for 80% or more of the total area of the rectangular area block 10A1.

[0110] According to an embodiment of the present application, in each group of marking units 11, at least three markings 110 arranged in a preset manner are included. Thus, the positioning and attribution of each signal within one FOV can be achieved through the setting manner and distribution of three or more markings 110 in the detection unit 10A. In the embodiment of the present application, for a group of marking units 11, the number of markings 110 included is at least three, and can be 3, 4, 5, 6, 7, 8 or even more. However, it should be understood that for markings 110 of the same size, the more the number, the more beneficial it is for positioning the signals within the detection unit 10A. At the same time, the more area may be occupied, reducing the actual effective area. Therefore, in a possible implementation manner, each group of marking units 11 includes 3 - 6 markings 110 arranged in a preset manner.

[0111] In some possible implementation manners, in a group of marking units 11, the image formed by connecting the lines of each marking 110 is a symmetric image. Exemplarily, an isosceles triangle, especially an equilateral triangle, a rectangle such as a rectangle or a square, and other symmetric polygons such as a rhombus, a regular pentagon, a regular hexagon, etc. Theoretically, it can also be set to shapes such as a circle or an ellipse. Thus, by making the image formed by connecting the lines of each marking 110 a symmetric image, the positioning of each signal inside and outside the region of this symmetric image can be achieved with the help of an algorithm. It should be understood that the image formed by connecting the lines of each marking 110 can be the image formed by sequentially connecting the centers of each marking 110. In some embodiments, the center of the symmetric image coincides with the center of the detection unit 10A.

[0112] In a specific embodiment, each group of marking units 11 includes three markings 110 arranged in a preset manner. In some embodiments, the image formed by connecting the lines of the three markings 110 is an isosceles triangle. In some embodiments, the area of the isosceles triangle accounts for 20% or more of the total area of the detection unit 10A. Exemplarily, the area of the isosceles triangle accounts for 20%, 25%, 30%, 35%, 40%, 45%, 50% or other such situations of the total area of the detection unit 10A.

[0113] In another specific embodiment, each group of marking units 11 includes 4 markings 110 arranged in a preset manner. In some embodiments, the image formed by connecting the lines of the four markings 110 is a rectangle. In some embodiments, the area of the rectangle accounts for 20% or more of the total area of the detection unit 10A. Exemplarily, the area of the rectangle accounts for 20%, 25%, 30%, 35%, 40%, 45%, 50% or other such situations of the total area of the detection unit 10A.

[0114] According to an embodiment of the present application, as Figure 8As shown, each marker 110 includes a first marker line 1101 in the first direction and a second marker line 1102 in the second direction and intersecting with the first marker line. In the embodiments of the present application, the intersection point of the first marker line 1101 and the second marker line 1102 is denoted as the center of the marker 110. In each group of marker units 11, there are 4 markers 110 arranged in a preset manner, and in an embodiment where the image formed by connecting the four markers 110 is a rectangle, the sides of the rectangle are respectively parallel or perpendicular to the first direction.

[0115] In a possible implementation manner, in the marker unit 11, at least one of the first marker line 1101 and the second marker line 1102 of each marker 110 respectively coincides with the four sides of the rectangular region block 10A1 or their extensions. In some embodiments, the detection unit 10A includes a rectangular region block 10A1 in the central region of the detection unit 10A, and the sides of the rectangular region block 10A are respectively parallel or perpendicular to the first direction. Exemplarily, when the marker unit 11 is composed of three markers 110, the image formed by connecting the three markers 110 is an isosceles triangle, and the first marker lines 1101 of the three markers 110 are respectively arranged on a set of opposite sides of the rectangular region block 10A. When the marker unit 11 is composed of four markers 110, the image formed by connecting the four markers 110 is a rectangle, and the first marker lines 1101 of the four markers 110 are arranged on a set of opposite sides of the rectangular region block 10A.

[0116] According to the embodiments of the present application, the first marker line 1101 and the second marker line 1102 intersect at a preset angle, and through the preset angle and the positions of the first marker line 1101 and the second marker line 1102 in the detection unit 10A, the attribution of each signal in the detection unit 10A can be calculated and located by means of an algorithm.

[0117] According to the embodiments of the present application, the included angle between the first marker line 1101 and the second marker line 1102 can be set according to the number of markers 11 in the detection unit 10A and the included angles formed by the first marker line 1101 and the second marker line 1102 with the detection row A10, so that through these parameters, the attribution of each signal in the detection unit 10A can be realized.

[0118] According to the embodiments of the present application, the included angle between the first marker line 1101 and the detection row A10 can be 0 - 180°. In some embodiments, the first marker line 1101 is parallel to the detection row A10. In one embodiment, the first direction and the second direction are perpendicular. That is: the first marker line 1101 and the second marker line 1102 are perpendicularly arranged.

[0119] In a possible implementation manner, refer to Figure 8, the detection unit 10A includes a group of marking units 11, and the marking unit 11 includes four markings 110. The marking 110 includes a first marking line 1101 and a second marking line 1102 that are perpendicular to each other, wherein the first marking line 1101 is parallel or perpendicular to the detection row A10 (not shown in the figure).

[0120] According to an embodiment of the present application, the setting manner of the marking 110 in the detection unit 10A can be flexibly set on the basis of being able to achieve marking. As a possible implementation manner of the present application, the detection site A10 in the detection area A is a groove, a protrusion formed on the surface of the solid-phase substrate 1, or a pattern formed on the surface of the solid-phase substrate 1 through physical and / or chemical treatment.

[0121] As a possible implementation manner, the first marking line 1101 and / or the second marking line 1102 are grooves formed on the surface of the solid-phase substrate 1, specifically on the detection unit 10A. Thus, a detection signal different from the surrounding detection sites A10 can be formed in the groove to realize the identification and positioning of the signals in the detection unit 10A.

[0122] In a possible embodiment, an identifiable marker is provided in the groove. The detection site A10 itself can bind to the identifiable marker or can bind to the identifiable marker through the bound sample, and the setting manner of the identifiable marker in the groove is different from the setting manner of the detection site A10 outside the groove. Thus, through the difference in the identifiable signals generated by the identifiable marker, the identifiable marker unit 11 is identified, and then, by means of the positions and setting manners of the respective markings 110 in the marking unit 11 in the detection unit 10A, the positioning result of the detectable signals in all the detection sites A10 in the detection unit 10A is obtained. In the embodiments of the present application, the identifiable marker is a marker that can be recognized when detecting the sample on the solid-phase substrate 1. Exemplarily, a fluorescent marker, but not limited thereto.

[0123] In a specific embodiment, each detection area A includes a plurality of detection units 10A, and a set of marking units 11 is arranged in each detection unit 10A; the detection unit 10A is provided with a plurality of detection rows A10 in the first direction, and a plurality of detection sites A10 are arranged at equal intervals in each detection row A10. The marking unit 11 includes at least three marks 110, and each mark 110 includes a first marking line 1101 located in the first direction and a second marking line 1102 located in the second direction and intersecting with the first marking line 1101. The first marking line 1101 and the second marking line 1102 are grooves formed on the detection unit 10A, and recognizable marks are arranged in the grooves. The distance between adjacent recognizable marks in the groove is smaller than the distance between adjacent detection sites in the detection unit. Thus, the first marking line 1101 and the second marking line 1102 form a recognizable signal stronger than the surrounding area, so as to realize the positioning of the marking unit 11. Then, according to the positions of the marks 110 in the marking unit 11 and the setting characteristics of the first marking line 1101 and the second marking line 1102 in each mark 110, the positioning of the signals generated in each detection site 10A in the detection unit 10A is realized.

[0124] In some embodiments, the distance between the recognizable marks in the groove is 1 / 2 or less of the distance between adjacent detection sites 10A in the detection unit 10A. Thus, the first marking line 1101 and the second marking line 1102 form a signal with an intensity significantly higher than that of the detection site 10A, so as to realize the recognition of information such as the number, position, setting direction, size, etc. of the marks 110 in the detection unit 10A through the intensity difference. Furthermore, with the help of this information, the positioning of the signals at the positions of the detection sites 10A in the detection unit 10A can be realized. In some embodiments, the density of the recognizable marks in the groove is 4 times or more of the density of the detection sites 10A in the detection unit 10A, so that a dense strong signal band can be generated in the whole groove to distinguish the signals generated by the detection sites 10A in the detection unit 10A and accelerate the recognition of the first marking line 1101 and the second marking line 1102. Exemplarily, as Figure 9 shown, the marks 110 form a continuous recognizable signal.

[0125] In another possible embodiment, no recognizable marks are arranged in the groove, and the detection site A10 itself can bind to recognizable marks or can bind to recognizable marks through the bound sample. Thus, when signal acquisition is performed on the detection unit 10A, since there are no recognizable marks in the groove, no recognizable signal is generated. During image acquisition, the marking unit 11 can be recognized by means of signal differences, and then, with the help of the positions and setting modes of the marks 110 in the marking unit 11, the detectable signals in the detection site A10 in the detection unit 10A can be obtained, and the positioning of these detectable signals can be realized.

[0126] As a possible implementation, the first marking line 1101 and / or the second marking line 1102 include holes or protrusions formed on the surface of the solid-phase substrate 1, and recognizable marks are provided in at least part of the holes or on the surfaces of the protrusions. Thus, by forming recognizable marks in the first marking line 1101 and / or the second marking line 1102 that are different from the setting density and arrangement pattern of the surrounding detection sites A10, the recognition of each mark 110 in the detection unit 10A can be achieved. Furthermore, based on the positions of the marks 110 in the marking unit 11 and the setting characteristics of the first marking line 1101 and the second marking line 1102 in each mark 110, the recognition and positioning of the signals in the detection unit 10A can be realized. In one embodiment, the recognizable marks are provided in all the holes or on the surfaces of the protrusions of the first marking line 1101 and / or the second marking line 1102.

[0127] In one possible embodiment, in the first marking line 1101, the connecting lines of the centers of the holes or protrusions are a straight line, that is: the centers of the holes or protrusions on the first marking line 1101 are located on the same straight line, and this straight line is parallel to the first direction.

[0128] In one possible embodiment, in the second marking line 1102, the connecting lines of the centers of the holes or protrusions are a straight line, that is: the centers of the holes or protrusions on the second marking line 1102 are located on the same straight line, and this straight line is perpendicular to the first direction.

[0129] In some embodiments, the distances between adjacent recognizable marks on the first marking line 1101 are equal, that is: several recognizable marks are arranged at equal intervals on the first marking line 1101, and the distance between adjacent recognizable marks is different from the distance between adjacent detection sites 10A. In some embodiments, the distances between adjacent recognizable marks on the second marking line 1102 are equal, that is: several recognizable marks are arranged at equal intervals on the second marking line 1102, and the distance between adjacent recognizable marks is different from the distance between adjacent detection sites 10A. In some embodiments, the distances between adjacent recognizable marks on the first marking line 1101 and the second marking line 1102 are respectively equal.

[0130] In some other embodiments, the distances between adjacent recognizable marks on the first marking line 1101 are not completely equal. In some embodiments, the distances between adjacent recognizable marks on the second marking line 1102 are not completely equal.

[0131] In one implementation scenario, the solid-phase substrate 1 includes a plurality of detection units 10A, and each detection unit 10A is provided with a set of marking units 11. The marking unit 11 includes at least three marks 110, and each mark 110 includes a first marking line 1101 in a first direction and a second marking line 1102 in a second direction and intersecting with the first marking line 1101. The first marking line 1101 and the second marking line 1102 include holes or protrusions formed on the surface of the solid-phase substrate 1, and recognizable marks are provided in the holes or on the surfaces of the protrusions. The detection unit 10A is provided with a plurality of detection sites A10 at equal intervals in the first direction, and the distance between at least some adjacent recognizable marks on the first marking line 1101 and the second marking line 1102 is greater than the distance between two adjacent detection sites A10. Exemplarily, the distance between all adjacent recognizable marks on the first marking line 1101 and the second marking line 1102 is greater than the distance between two adjacent detection sites A10. It should be understood that in the embodiments of the present application, the distance between adjacent detection sites A10 refers to the length of the line connecting the centers of two adjacent detection sites A10, and the definition of the center is as described above and will not be elaborated here.

[0132] In some embodiments, taking the spacing between the centers of two adjacent detection sites A10 in the first direction as l, the distance between adjacent recognizable marks on the first marking line 1101 and the second marking line 1102 is n times l, where n is an integer greater than or equal to 1. Thus, the signals generated by the recognizable marks in the first marking line 1101 and the second marking line 1102 are significantly different from the signal distribution in the region where the detection site A10 is located, so as to realize the positioning of the marking unit 11. Then, according to the positions of the marks 110 in the marking unit 11 and the setting characteristics of the first marking line 1101 and the second marking line 1102 in each mark 110, the positioning of the signals generated in each detection site 10A in the detection unit 10A is realized. In some embodiments, n is a positive integer from 2 to 4. Exemplarily, n is 2, 3 or 4.

[0133] In the implementation scenario where the first marking line 1101 and the second marking line 1102 include holes formed on the surface of the solid-phase substrate 1, the holes are at least one of circular holes, oval holes, polygonal holes and irregular holes. In some embodiments, the holes are at least one of circular holes and regular polygonal holes. In order to facilitate the recognition of the signals of the marks 110, the shapes and sizes of the holes provided on the surfaces where the first marking line 1101 and the second marking line 1102 are located are preferably the same.

[0134] In an implementation where the first marking line 1101 and the second marking line 1102 include protrusions formed on the surface of the solid-phase substrate 1, the protrusions are at least one of circular protrusions, elliptical protrusions, polygonal protrusions, and irregular protrusions. In some embodiments, the protrusions are at least one of circular protrusions and regular polygonal protrusions. To facilitate the identification of the signals of the markings 110, the shapes and sizes of the protrusions provided on the surfaces where the first marking line 1101 and the second marking line 1102 are located are the same.

[0135] In some embodiments, the area where the marking lines (including the first marking line 1101 and the second marking line 1102) are located can also be set as a groove, and then holes or protrusions are provided in the groove, and recognizable markings are provided in the holes or protrusions. The setting method of the holes or protrusions and the situation of providing recognizable markings in the holes or protrusions can refer to the situation of "the first marking line 1101 and / or the second marking line 1102 include holes or protrusions formed on the surface of the solid-phase substrate 1, and recognizable markings are provided in at least part of the holes or on the surfaces of the protrusions" above, and will not be elaborated here.

[0136] According to the embodiments of the present application, the position of the marking unit 11 in the detection unit 10A can be determined by the signal distribution characteristics and positions of the markings 110 in the marking unit 11 in the detection unit 10A. Further, by combining the position of the marking unit 11 in the detection unit 10A and the distribution characteristics of the marking unit 11, the signals of the detection sites A10 in the detection unit 10A can be identified.

[0137] According to the embodiments of the present application, the distribution characteristics of the marking unit 11 at least include the number and arrangement of the markings 110 that make up the marking unit 11, and the position of each marking 110, and the intersection manner of the first marking line 1101 and the second marking line 1102 in the marking 110, such as the included angle, the lengths and widths of the first marking line 1101 and the second marking line 1102 relative to the detection unit 10A.

[0138] In a possible implementation manner, the markings 110 in the marking unit 11 have a definite number and preset positions. The number of the markings in the marking unit 11 is not described here again for the sake of saving space as described above.

[0139] The marker 110 in the marker unit 11 has a definite quantity and a preset position, which can be determined based on the shape and size of the detection unit 10A. In a rectangular detection unit 10A corresponding to the shape and size of the FOV, more than three markers 110 are arranged in a rectangular region block 10A1 formed by inwardly contracting the two pairs of opposite sides of the rectangular detection unit 10A by 2-4 times respectively. It should be understood that the inward contraction of the two pairs of opposite sides of the rectangular detection unit 10A by 2-4 times respectively does not mean that the two pairs of opposite sides of the rectangular detection unit 10A are inwardly contracted by the same multiple at the same time. Exemplarily, one pair of opposite sides of the rectangular detection unit 10A is inwardly contracted by 2 times, and the other pair of opposite sides is inwardly contracted by 3 times. At this time, the rectangle enclosed by the extension lines of the two pairs of opposite sides is the rectangular region block 10A1.

[0140] Exemplarily, the marker unit 11 includes four markers 110. In the four markers 110, the first marker line 1101 and the second marker line 1102 intersect perpendicularly, and the figure formed by connecting the four intersection points is a rectangle. At this time, the positions of the four markers 110 are used to roughly locate the detection unit 10A, and the detection unit 10A is determined as multiple regions; furthermore, through the relative positions of the four intersection points in the detection unit 10A, as well as the lengths and intersection manners of the first marker line 1101 and the second marker line 1102, the precise positions of the signals generated at the detection sites A10 in each region are determined.

[0141] In a possible implementation manner, the first marker line 1101 and the second marker line 1102 may intersect to form a symmetric figure, or may intersect in an asymmetric form. In the embodiments of the present application, the rough positioning of the detection unit 10A can be realized by setting the intersection manner of the first marker line 1101 and the second marker line 1102, and the detection unit 10A is determined as multiple regions.

[0142] Exemplarily, such as Figure 10As shown, the marking unit 11 includes three markings 110. Among the three markings 110, the first marking line 1101 and the second marking line 1102 are respectively perpendicular to each other and intersect, and the three intersections formed are O1, O2, and O3 respectively. The connection lines of the three intersections form an isosceles triangle. Among them, in the marking 110 where the vertex is located, the first marking line 1101 and the second marking line 1102 intersect at their respective center points; among the two markings 110 located at the base angles, the intersection point O2 is close to the second ends of the first marking line 1101 and the second marking line 1102 in the marking 110 where it is located, and the intersection point O2 is close to the second ends of the first marking line 1101 and the second marking line 1102 in the marking 110 where it is located. Thus, the positions of the three markings 110 can be determined through the signals of the markings 110 included in the marking unit 11, realizing the regional division of the detection unit 10A, that is, realizing the rough positioning of the signals in the detection unit 10A; furthermore, based on the intersection manner of the first marking line 1101 and the second marking line 1102 in each marking 110, the signals for each marking 110 to distinguish its surrounding area are obtained, realizing the positioning of the signals generated by each detection site A10 in the detection unit 10A.

[0143] In another example, as Figure 11 , Figure 12 shown, the marking unit 11 includes four markings 110. Among the four markings 110, the first marking line 1101 and the second marking line 1102 are respectively perpendicular to each other and intersect, and the three intersections formed are O1, O2, O3, and O4 respectively. The connection lines of the four intersections form a rectangle. Through the signals of the markings 110 included in the marking unit 11, the positions of the four markings 110 are determined, realizing the regional division of the detection unit 10A. For example, the extension lines formed by the four sides of the rectangle divide the detection unit 10A into 3×3 regional blocks, that is, realizing the rough positioning of the signals in the detection unit 10A. Among them, the intersection manners of the four markings 110 are different. For the marking 110 where the intersection point O1 is located, the intersection point O1 is close to the first end of the first marking line 1101 and the first end of the second marking line 1102; for the marking 110 where the intersection point O2 is located, the intersection point O2 is close to the first end of the first marking line 1101 and the second end of the second marking line 1102; for the marking 110 where the intersection point O3 is located, the intersection point O3 is close to the second end of the first marking line 1101 and the second end of the second marking line 1102; for the marking 110 where the intersection point O4 is located, the intersection point O4 is close to the second end of the first marking line 1101 and the first end of the second marking line 1102. Thus, on the basis of the 9 regional blocks, by means of the intersection manner of the first marking line 1101 and the second marking line 1102 in each marking 110, the signals in each regional block are obtained, realizing the fine positioning of the signals generated by each detection site A10 in the detection unit 10A.

[0144] It should be understood that each marking line respectively includes a first end and a second end that are away from each other. When one end where an end point of the marking line is located is taken as the first end, the other end where the other end point is located is the second end. In the above illustrated example, for the first marking line 1101, the leftward end is named the first end, and the rightward end is named the second end; for the second marking line 1102, the upward end is named the first end, and the downward rightward end is named the second end.

[0145] According to an embodiment of the present application, the first marking line 1101 and the second marking line 1102 are identification lines with a certain aspect ratio. As a possible implementation manner, the aspect ratio of the first marking line 1101 is 15 to 30:1. Exemplarily, the aspect ratio of the first marking line 1101 is 15:1, 18:1, 20:1, 22:1, 25:1, 28:1, 30:1 and other specific cases. As a possible implementation manner, the aspect ratio of the second marking line 1102 is 15 to 30:1. Exemplarily, the aspect ratio of the second marking line 1102 is 15:1, 18:1, 20:1, 22:1, 25:1, 28:1, 30:1 and other specific cases. By setting the aspect ratios of the first marking line 1101 and the second marking line 1102 within the above range, on the one hand, the difference between the area where the marking 110 is located and the surrounding detection area can be enhanced, and the recognition intensity of the marking 110 can be improved; on the other hand, by controlling the aspect ratios of the first marking line 1101 and the second marking line 1102 within the above range, the recognition intensity of the marking 110 can be improved within a limited area, and the area ratio of the marking unit 11 in the detection unit 10A can be reduced. In some embodiments, in a detection unit 10A, the area of the marking unit 11 accounts for less than 1% of the area of the detection unit 10A, and the recognition of the marking unit 11 can still be achieved. Exemplarily, the area of the marking unit 11 can account for 1%, 0.9%, 0.8%, 0.7% and other cases of the area of the detection unit 10A.

[0146] According to an embodiment of the present application, by setting the intensities of the first marking line 1101 and the second marking line 1102 in the detection unit 10A, it can contribute to the recognition of the marking 110, and on this basis, reduce the area ratio of the marking unit 11 in the detection unit 10A. In a possible implementation manner, taking the distance between the centers of two adjacent detection sites A10 in the first direction as l, at least one of the following conditions is satisfied by the lengths and widths of the first marking line 1101 and the second marking line 1102:

[0147] The length of the first marking line 1101 is 80 to 150l. Exemplarily, the length of the first marking line 1101 is 80l, 85l, 90l, 95l, 100l, 105l, 110l, 115l, 120l, 125l, 130l, 135l, 140l, 145l, 150l, etc.;

[0148] The length of the second marking line 1102 is 80 to 150l. Exemplarily, the length of the second marking line 1101 is 80l, 85l, 90l, 95l, 100l, 105l, 110l, 115l, 120l, 125l, 130l, 135l, 140l, 145l, 150l, etc.;

[0149] The width of the first marking line 1101 is 3 to 8l. Exemplarily, the width of the first marking line 1101 is 3l, 4l, 5l, 6l, 7l, 8l, etc.;

[0150] The width of the second marking line 1102 is 3 to 8l. Exemplarily, the width of the second marking line 1102 is 3l, 4l, 5l, 6l, 7l, 8l, etc.

[0151] In some embodiments, the length of the first marking line 1101 is 100 to 120l; the length of the second marking line 1102 is 100 to 120l; the width of the first marking line 1101 is 4 to 6l; the width of the second marking line 1102 is 4 to 6l.

[0152] According to the embodiments of the present application, the distinction of the marking units can be enhanced by setting the length ratio of the first marking line 1101 and the second marking line 1102. In some embodiments, the length ratio of the first marking line 1101 and the second marking line 1102 is 0.8 to 1.2:1. Exemplarily, the length ratios of the first marking line 1101 and the second marking line 1102 are 0.8:1, 0.9:1, 1:1, 1.1:1, 1.2:1, etc.

[0153] Thus, in the embodiments of the present application, by designing multiple groups of regularly arranged marking units 11 on the solid-phase substrate, rough positioning of the surface area of the solid-phase substrate 1 and fine positioning of the surface position of the solid-phase substrate 1 can be achieved based on the arrangement rule of the marking units 11, the number and position of the markings 110, and the relationship and characteristics between the first marking line 1101 and the second marking line 1102. Moreover, by designing intersecting marking units to achieve positioning of the surface of the solid-phase substrate 1, the area occupied by the markings on the surface of the solid-phase substrate 1 can be reduced, and the area loss of the surface of the solid-phase substrate 1 can be decreased, thereby facilitating improvement of the effective utilization rate of the surface of the solid-phase substrate 1. When the solid-phase substrate 1 is used as a biochip, the sequencing throughput can be increased.

[0154] According to an embodiment of the present application, the recognizable marks are regularly distributed on the first mark line 1101 according to a first arrangement rule, and the recognizable marks are regularly distributed on the second mark line 1102 according to a second arrangement rule. It should be understood that the recognizable marks in the embodiments of the present application, whether they are the recognizable marks arranged in the grooves, or the recognizable marks arranged in the holes or protrusions or even on the processed surface patterns, can be formed by direct bonding, or by forming a detection site A10 at the corresponding position, and placing the same sample as that in the detection site A10 in the area other than the marking unit 11 in the detection unit 10A, and performing the same operations to introduce the recognizable marks. In actual operation, for the sake of simplicity of operation, a detection site A10 identical to the area other than the marking unit 11 is formed in the detection unit 10A, and then the recognizable marks are introduced through the same operations. In the following text, for the sake of easy understanding, the recognizable marks are described by introducing the detection site A10 into the mark 110. It should be understood that for each detection site A10 in the mark 110, there corresponds a recognizable mark.

[0155] For a clearer description of the setting of the mark 110, the following takes the solid-phase substrate 1 as a biochip for sequencing and the recognizable mark as an optical mark such as a fluorescent mark as an example for illustration.

[0156] In a possible implementation, with reference to Figure 13 - 16 , the first mark line 1101 has first repeating units arranged periodically in a first direction. The first repeating unit includes a plurality of detection sites A10 arranged in the first direction. The plurality of detection sites A10 on the first repeating unit are located on the same straight line, as shown in Figure 13 , Figure 14 . That is, all the detection sites A10 on the first mark line 1101 are located on the same straight line. Thereby, the recognition degree of the detection sites A10 on the first mark line 1101 can be improved, which is beneficial to the recognition of the first mark line 1101. In some embodiments, the centers of all the detection sites A10 on each first mark line 1101 are located on the same straight line in the same direction as the first direction. In this case, the centers of the optical signals generated by all the detection sites A10 on the first mark line 1101 are usually also on the same straight line, which is more beneficial to the recognition of the first mark line 1101.

[0157] The second mark line 1102 has second repeating units arranged periodically in a second direction. The second repeating unit includes a plurality of detection sites A10 arranged in the second direction. The plurality of detection sites A10 on the second repeating unit are located on the same straight line. That is, all the detection sites A10 on the second mark line 1102 are located on the same straight line, as shown in Figure 13 , Figure 14As shown, the recognition rate of the detection site A10 of the second marking line 1102 can be improved, which is conducive to identifying the second marking line 1102. In some embodiments, the centers of all the detection sites A10 on each second marking line 1102 are on the same straight line. In this case, the centers of the optical signals generated by all the detection sites A10 on the second marking line 1102 are usually also on the same straight line, which is more conducive to identifying the second marking line 1102.

[0158] By arranging the repeating units in a periodic pattern, the marking lines in two directions (including the first marking line 1101 and the second marking line 1102) present regular optical signals with dark and bright intervals, so as to facilitate the accurate identification of the marking lines in two directions (including the first marking line 1101 and the second marking line 1102) during optical detection, realize the calibration of the detection unit 10A, and improve the detection accuracy.

[0159] In an embodiment of the present application, the first repeating unit includes at least one set of consecutive sites, the consecutive sites include at least two first detection sites, and the distance between the two first detection sites is the first distance. Exemplarily, the consecutive sites include two first detection sites. In some embodiments, the first repeating unit further includes a second detection site provided on the first side of the consecutive sites, and the second distance between the first detection site adjacent to the second detection site in the consecutive sites and the second detection site is greater than the first distance. Thus, a blank interval without detection sites is formed between the consecutive sites and the second detection site, and this region appears as a signal-free area, such as a fluorescent dark area, in the optical image obtained by optical detection.

[0160] In some embodiments, the second distance ≥ the first distance × 2. This can enhance the brightness and darkness difference between the consecutive sites and the second detection site, which helps to identify the first marking line 1101. Exemplarily, the second distance = the first distance × 2.

[0161] In some embodiments, the first repeating unit includes a second detection site disposed on a first side of the continuous site and a third detection site disposed on a second side. The second distance between the first detection site adjacent to the second detection site in the continuous site and the second detection site is greater than the first distance, and the third distance between the first detection site adjacent to the third detection site in the continuous site and the third detection site is greater than the first distance. It should be understood that the first side and the second side are opposite sides based on a reference object. Taking the first repeating unit located on the horizontal axis as an example, the first side of the continuous site refers to the side on the left (or right) of the continuous site. Correspondingly, the second side of the continuous site refers to the side on the right (or left) of the continuous site. Thus, an interval without detection sites is formed between the continuous site and the second detection site, and between the continuous site and the third detection site. This region appears as a dark area in the optical image obtained by optical detection, thereby forming a strip of bright (second detection site)-dark-bright (continuous site)-dark-bright (third detection site), making the dark and bright intervals arranged in the first direction show higher regularity, which helps to improve the recognizability of the first marking line 1101. In one embodiment, when the third distance ≥ 2× the first distance, the light and dark difference between the continuous site and the third detection site can be enhanced, which helps to improve the recognition accuracy of the marking unit 11. Exemplarily, the third distance = 3× the first distance.

[0162] In the embodiments of the present invention, the second distance and the third distance may be the same or different. In one embodiment, the second distance is not equal to the third distance. Thus, the second detection site - continuous site (first detection site) - third detection site is arranged asymmetrically, which is beneficial to distinguish the direction of the mark 110 and realize the regional recognition of the detection unit 10A through this difference.

[0163] In one embodiment, the first repeating unit includes a set of consecutive sites, a second detection site disposed on the first side of the consecutive sites, and the distance between the next detection site in the extending direction of the second detection site and the second detection site is greater than the first distance. At this time, an interval without detection sites is formed on each side of the second detection site, so that a strip of dark-(second detection site)-dark-bright (consecutive sites) appears on the first side of the consecutive sites. Among them, the next detection site in the extending direction of the second detection site can be a detection site in the same first repeating unit where the second detection site is located, or a detection site in another adjacent first repeating unit. When the next detection site in the extending direction of the second detection site is a detection site in another adjacent first repeating unit, the second detection site is disposed on the first side of the consecutive sites in the first repeating unit, and a dark region is provided in the extending direction of the second detection site. At this time, there are no other detection sites on the first side of the first repeating unit. Exemplarily, the distance between the next detection site in the extending direction of the second detection site and the second detection site = the first distance × 3. That is, a dark region with a length of the first distance is provided in the extending direction of the second detection site.

[0164] In one embodiment, the distance between the next detection site in the extending direction of the second detection site and the second detection site is greater than the first distance, and the distance between the next detection site in the extending direction of the third detection site and the third detection site is greater than the first distance. Among them, the next detection site in the extending direction of the second detection site can be a detection site in the same first repeating unit where the second detection site is located, or a detection site in another adjacent first repeating unit; similarly, the next detection site in the extending direction of the third detection site can be a detection site in the same first repeating unit where the third detection site is located, or a detection site in another adjacent first repeating unit. When the next detection site in the extending direction of the second detection site is a detection site in an adjacent first repeating unit, and the next detection site in the extending direction of the third detection site is a detection site in another adjacent first repeating unit, the first repeating unit is composed of the second detection site, the consecutive sites, and the third detection site, forming an arrangement of second detection site-consecutive sites-third detection site, and a dark region is provided in the extending direction of each of the second detection site and the third detection site.

[0165] Such as Figure 15As shown, in a first repeating unit of the first marking line 1101, the detection sites with openings are schematically shown as solid circles, and the dotted circles represent blank area sites, which do not exist on the solid-phase substrate. This embodiment lists them for schematically showing the setting of the detection sites. At this time, in the optical image obtained by optical imaging of the solid-phase substrate, the area corresponding to the solid circle appears as a bright area, and the area between the solid circles, i.e., the area where the dotted circle is located, appears as a dark area on the optical image. The first repeating unit of the first marking line 1101 is arranged in sequence from the first side to the second side as blank site - detection site A11 - blank site - detection site A12 - detection site A13 - blank site - blank site - detection site A14 or detection site A11 - blank site - detection site A12 - detection site A13 - blank site - blank site - detection site A14 - blank site, and in the first marking line 1101 formed by the detection sites and the blank sites, the distance between adjacent sites is equal. It should be understood that one of the blank sites near the first side and the blank site near the second side comes from an adjacent another first repeating unit. Thus, a first repeating unit of the first marking line 1101 forms a band of dark - bright - dark - long bright (continuous sites) - long dark (continuous dark area) - bright - dark, making the dark and bright intervals arranged in the first direction show higher regularity, which helps to improve the recognizability of the first marking line 1101.

[0166] In an embodiment of the present application, the second repeating unit includes at least one group of continuous sites, the continuous sites include at least two fourth detection sites, and the distance between the two fourth detection sites is the fourth distance. Exemplarily, the continuous sites include two fourth detection sites. In some embodiments, the second repeating unit further includes a fifth detection site arranged on the first side of the continuous sites, and the fifth distance between the fourth detection site adjacent to the fifth detection site in the continuous sites and the fifth detection site is greater than the fourth distance. Thus, an interval without detection sites is formed between the continuous sites and the fourth detection sites, and this area appears as a signal-free area, such as a fluorescent dark area, in the image obtained by optical detection.

[0167] In some embodiments, the fifth distance ≥ the fourth distance × 2. This situation can enhance the brightness and darkness difference between the continuous sites and the fifth detection site, which helps to recognize the second marking line 1102 of the FOV 10. Exemplarily, the fifth distance = the fourth distance × 3.

[0168] In some embodiments, the second repeating unit includes a fifth detection site disposed on a first side of a continuous site and a sixth detection site disposed on a second side of the continuous site. The fifth distance between the fourth detection site adjacent to the fifth detection site in the continuous site and the fifth detection site is greater than the fourth distance, and the sixth distance between the fourth detection site adjacent to the sixth detection site in the continuous site and the sixth detection site is greater than the fourth distance. It should be understood that the first side and the second side are opposite sides based on a reference object. Taking the second repeating unit located on the vertical axis as an example, the first side of the continuous site refers to the side above the continuous site, and correspondingly, the second side of the continuous site refers to the side below the continuous site. Thus, a section without detection sites is formed between the continuous site and the fifth detection site, and between the continuous site and the sixth detection site. This region appears as a dark area in the optical image obtained by optical detection, thereby forming a stripe of bright (fourth detection site)-dark-bright (continuous site)-dark-bright (fifth detection site), making the dark and bright intervals arranged in the second direction more regular, which helps to improve the recognizability of the second marking line 1102. In one embodiment, when the sixth distance ≥ 2 × the fourth distance, the brightness difference between the continuous site and the fifth detection site can be enhanced, which helps to improve the recognition accuracy of the marking unit 11. Exemplarily, the sixth distance = 2 × the fourth distance.

[0169] In the embodiments of the present invention, the fifth distance and the sixth distance may be the same or different. In one embodiment, the fifth distance and the sixth distance are not equal. Thus, the fifth detection site - continuous site (fourth detection site) - sixth detection site is arranged asymmetrically, which is beneficial to distinguishing the direction of the marking 110 and realizing the regional recognition of the detection unit 10A through this difference.

[0170] In one embodiment, the second repeating unit includes a set of consecutive sites, a fifth detection site disposed on the first side of the consecutive sites, and the distance between the next detection site in the extending direction of the fifth detection site and the fifth detection site is greater than the fourth distance. At this time, a section without detection sites is formed on each side of the fifth detection site, so that the first side of the consecutive sites presents a strip of dark-(fifth detection site)-dark-bright (consecutive sites). Among them, the next detection site in the extending direction of the fifth detection site may be a detection site in the same second repeating unit where the fifth detection site is located, or a detection site in another adjacent second repeating unit. When the next detection site in the extending direction of the fifth detection site is a detection site in another adjacent second repeating unit, the fifth detection site is disposed on the first side of the consecutive sites in the second repeating unit, and a dark region is disposed in the extending direction of the fifth detection site. At this time, no other detection sites are provided on the first side of the second repeating unit. Exemplarily, the distance between the next detection site in the extending direction of the fifth detection site and the fifth detection site = the first distance × 3. That is, a dark region with a length of the first distance is disposed in the extending direction of the fourth detection site.

[0171] In one embodiment, the distance between the next detection site in the extending direction of the fifth detection site and the fifth detection site is greater than the fourth distance, and the distance between the next detection site in the extending direction of the sixth detection site and the sixth detection site is greater than the fourth distance. Among them, the next detection site in the extending direction of the fifth detection site may be a detection site in the same second repeating unit where the fifth detection site is located, or a detection site in another adjacent second repeating unit; similarly, the next detection site in the extending direction of the sixth detection site may be a detection site in the same second repeating unit where the sixth detection site is located, or a detection site in another adjacent second repeating unit. When the next detection site in the extending direction of the fifth detection site is a detection site in an adjacent second repeating unit, and the next detection site in the extending direction of the sixth detection site is a detection site in another adjacent second repeating unit, the second repeating unit is composed of the fifth detection site, the consecutive sites, and the sixth detection site, forming an arrangement of the fifth detection site-consecutive sites-sixth detection site, and a dark region is disposed in the extending direction of each of the fifth detection site and the sixth detection site. Exemplarily, in adjacent second repeating units, the distance between adjacent fifth detection sites and sixth detection sites = the fourth distance × 3. That is, a dark region with a length of the first distance is disposed in the extending direction of each of the fifth detection site and the sixth detection site.

[0172] Such as Figure 16As shown, in a second repeating unit of the second marking line 1102, the detection sites with openings are schematically shown as solid circles, and the dotted circles represent blank site regions, which do not exist on the solid-phase substrate. This embodiment lists them for the purpose of schematically showing the setting of the detection sites. At this time, in the optical image obtained by optical imaging of the solid-phase substrate, the regions corresponding to the solid circles appear as bright regions, and the regions between the solid circles, i.e., the regions where the dotted circles are located, appear as dark regions in the optical image. The second repeating unit of the second marking line 1102 is arranged in sequence from the first side to the second side as blank site - detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18 - blank site, or detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18 - blank site - blank site, or blank site - blank site - detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18, and in the second repeating unit formed by the detection sites and the blank sites, the distances between adjacent sites are all equal. It should be understood that one of the blank sites near the first side and the blank site near the second side comes from an adjacent other first repeating unit. Thus, a second repeating unit of the second marking line 1102 forms a band of dark - bright - long dark (continuous dark region) - long bright (continuous sites) - dark - bright - dark, making the dark and bright intervals in the second direction arranged more regularly, which helps to improve the recognizability of the second marking line 1102.

[0173] According to an embodiment of the present application, the maximum diameter of the detection sites on the marking lines (including the first marking line 1101 and the second marking line 1102) may be the same as or different from the maximum diameter of the detection sites in the region outside the marking lines in the detection unit. In one embodiment, the maximum diameter of the detection sites on the marking lines (including the first marking line 1101 and the second marking line 1102) is greater than the maximum diameter of the detection sites in the region outside the marking lines in the detection unit, making the marking lines (including the first marking line 1101 and the second marking line 1102) easier to identify. The maximum diameter refers to the straight-line distance between the two farthest points within the detection site. For a circular detection site, it is the diameter of the circle.

[0174] In one embodiment, the maximum diameter on the marking lines (including the first marking line 1101 and the second marking line 1102) is 400 - 500 nm, including but not limited to 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, 500 nm, etc. The maximum diameter of the detection sites on the first marking line 1101 may be the same as or different from the maximum diameter of the detection sites on the second marking line 1102.

[0175] In one embodiment, the maximum diameter of the detection sites on the first marking line 1101 is the same as the maximum diameter of the detection sites on the second marking line 1102. Exemplarily, in the first direction and the second direction, the maximum diameter of each detection site is 450 nm.

[0176] In a second aspect, in one embodiment, a biochip is provided, which includes the solid-phase substrate of any one of the first aspects. The solid-phase substrate is used for gene sequencing. Each detection area on the solid-phase substrate is a sequencing area. Fluorescence can be collected from each opening. According to the marking unit and the position and setting characteristics of the marks in the marking unit, the FOV is calibrated to realize sequencing of the detection sites of the openings in the sequencing area. At the same time, the area ratio of the marking unit in the detection unit is reduced, which is beneficial to improving the detection throughput.

[0177] The solid-phase substrate can be a solid planar substrate made of silicon, glass wafer or other materials. Exemplarily, the surface size of the solid-phase substrate is about 83 mm * 35 mm.

[0178] The sequencing method can adopt the method of sequencing by synthesis. DNA polymerase, adapter primers and 4 types of dNTPs with base-specific fluorescent labels are simultaneously added to the reaction system (similar to the Sanger sequencing method). The 3'-OH of these dNTPs is protected by chemical methods, so only one dNTP can be added each time, which ensures that only one base will be added during the sequencing process. At the same time, after the dNTP is added to the synthesized strand, all unused free dNTPs and DNA polymerase are washed off. Then, the buffer required to excite fluorescence is added, the fluorescence signal is excited by a laser, and the fluorescence signal is recorded by an optical device. Finally, the optical signal is converted into sequencing bases by computer analysis. In the solid-phase substrate of the present invention, each detection site in the detection area is a detection site that can be excited by a laser to generate a fluorescence signal. The first marking line (including the first marking line 1101 and the second marking line 1102) and the second marking line 1102 are used to pre-calibrate the FOV to improve the accuracy of sequencing. After the fluorescence signal recording is completed, a chemical reagent is added to quench the fluorescence signal and remove the dNTP 3'-OH protecting group, so as to enable the next round of sequencing reaction.

[0179] The present invention is applicable to any optical signal-based sequencing methods such as second-generation sequencing and third-generation sequencing.

[0180] Example 1

[0181] In this embodiment, the biochip includes a plurality of rectangular detection units. Each detection unit includes a number of detection sites, and a group such as Figure 11 and Figure 12The labeled unit 11 shown, taking a detection unit 10A as an example, the labeled unit 11 includes four labels 110. Each label 110 includes a first label line 1101 arranged in the first direction and a second label line 1102 arranged in the second direction. The first label line 1101 and the second label line 1102 are perpendicular to each other and intersect, and the connecting lines of the four intersection points form a rectangle. The lengths and widths of the first label line 1101 and the second label line 1102 are the same respectively. Taking the distance between the centers of two adjacent detection sites in the first direction as l, the lengths and widths of the first label line 1101 and the second label line 1102 are: the length of the first label line 1101 is 100l, and the width is 5l; the length of the second label line 1102 is 100l, and the width is 5l. A circular opening is arranged in the first label line 1101, and the size of the circular opening is the same as the size of the detection site in the detection unit 10A, with a diameter of 300 nm. The structure of the first repeating unit constituting the first label line 1101 is as Figure 15 shown, from the first side to the second side, there are arranged in sequence a blank site - detection site A11 - blank site - detection site A12 - detection site A13 - blank site - blank site - detection site A14 or detection site A11 - blank site - detection site A12 - detection site A13 - blank site - blank site - detection site A14 - blank site, and in the first label line 1101 formed by the detection sites and the blank sites, the distances between adjacent sites are all equal. The structure of the first repeating unit constituting the second label line 1102 is as Figure 16 shown, from the first side to the second side, there are arranged in sequence a blank site - detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18 - blank site, or detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18 - blank site - blank site, or blank site - blank site - detection site A15 - blank site - blank site - detection site A16 - detection site A17 - blank site - detection site A18, and in the second repeating unit formed by the detection sites and the blank sites, the distances between adjacent sites are all equal.

[0182] Example 2

[0183] In this example, the size of the biochip is the same as that in Example 1, including a plurality of rectangular detection units the same as those in Example 1. Each detection unit includes a number of detection sites, and a group such as Figure 11 and Figure 12The labeled unit 11 shown, taking a detection unit 10A as an example, the labeled unit 11 includes four labels 110. Each label 110 includes a first label line 1101 arranged in the first direction and a second label line 1102 arranged in the second direction. The first label line 1101 and the second label line 1102 are perpendicularly intersected, and the connection lines of the four intersection points form a rectangle. The lengths and widths of the first label line 1101 and the second label line 1102 are respectively the same. Taking the spacing between the centers of two adjacent detection sites in the first direction as l, the lengths and widths of the first label line 1101 and the second label line 1102 are: the length of the first label line 1101 is 100l, and the width is 5l; the length of the second label line 1102 is 100l, and the width is 5l. As Figure 9 shown, the first label line 1101 and the second label line 1102 are grooves formed on the surface of the solid-phase substrate, and there is a continuous region in the groove that can bind the recognizable label. For example, the groove is a surface-treated groove, and the surface-treated groove is integrally combined with a nucleic acid sample, and the density of the nucleic acid sample is more than 10 times the density of the detection sites in the sequencing unit 1. During the sequencing process, the nucleic acid sample can bind to the nucleic acid substrate with a recognizable label, so that the first label line 1101 and the second label line 1102 in the label 110 present continuous high-strength signals.

[0184] Comparative example

[0185] In this comparative example, the size of the biochip is the same as that in Example 1, and it includes a plurality of rectangular detection units that are the same as those in Example 1 (except for the labels, the settings, sizes, and morphologies of the detection sites in other regions are the same). As Figure 1 - 3 shown in FIGS. 10 and 17, each detection unit is provided with a trackline. Each trackline includes a first trackline 102 arranged in the first direction (or X direction) and a second trackline 103 arranged in the second direction (or Y direction). The first trackline 102 and the second trackline 103 are perpendicularly intersected, and the width of the trackline is the same as the width of the label line in Example 1 and Example 2. The first trackline 102 and the second trackline 103 divide the rectangular detection unit into a plurality of detection areas 101. A circular opening structure is provided in the trackline. The pore diameter of the sites on the first trackline 102 is 450 nm, and the pore diameter of the sites on the second trackline 103 is 450 nm.

[0186] In the sample detection area 101, the detection site 105 is set as a circular opening structure with a pore diameter of 300 nm. The spacing between adjacent detection sites in the X direction is 750 nm, and in the Y direction, the spacing between adjacent detection sites is 1300 nm. On the first trackline 102, the spacing between adjacent sites is 750 nm, and on the second trackline 103, the spacing between adjacent sites is 1300 nm. The vertical distance between the boundary line of the first trackline 102 and the boundary line of the adjacent detection area 101 is 1500 nm, and the vertical distance h between the boundary line of the second trackline 103 and the boundary line of the adjacent detection area 101 is 2100 nm. The pore diameter of the sites on the first trackline 102 is 450 nm, and the pore diameter of the sites on the second trackline 103 is 450 nm.

[0187] The chips provided in Example 1 and Example 1 and the comparative example were used for sequencing by synthesis under the same conditions, and Q30, Mapped rate, Error, Output Reads, etc. were statistically analyzed during the sequencing. Among them,

[0188] Q30 refers to the proportion of the accuracy rate of the sequenced bases being 99.9%; for example, Q30≥85 means that the proportion of the accuracy rate of the measured bases being 99.9% is greater than or equal to 85%;

[0189] Mapped rate represents the alignment rate, which refers to the percentage of nucleic acid fragments that can be aligned with the sample nucleic acid sequence;

[0190] Error represents the sequencing error rate;

[0191] Output Reads represents the amount of Reads data output per unit area. The more Reads output per unit area, the higher the surface sequencing throughput.

[0192] The data of the biochip of Example 1 are shown in Table 1 below:

[0193] Table 1

[0194] Number Mapped rate Error Q30 Output Reads Example 1 V02 86.99％ 0.36％ 86.8％ 2472M Example 2 V03 89.78％ 0.27％ 89.3％ 2761M Comparative Example V04 89.54％ 0.28％ 89.1％ 2753M

[0195] Combining the data in Tables 1 and 2, it can be seen that compared with the trackline markings of the comparative example, in the key data of biochip sequencing of Example 1 and Example 2, the Mapped rate has increased, the Error has decreased, and the Q30 has increased. This shows that when using the biochips marked with Example 1 and Example 2 of the present application for sequencing, the marking lines (including the first marking line 1101 and the second marking line 1102) have better recognizability and are convenient for algorithm calculation, which can effectively improve the accuracy of sequencing. At the same time, the Output Reads corresponding to Example 1 and Example 2 are higher than those of Comparative Example 1, indicating that when using the biochips marked with Example 1 and Example 2 of the present application for sequencing, the sequencing throughput can be improved, which may be attributed to the reduction in the area ratio of the marking unit provided in the embodiments of the present application in the detection unit.

[0196] Figure 18 Fig. shows the Q30 data statistical curve of Example 1 (V03) and Comparative Example 1 (V02). As can be seen from the figure, both Example 1 (V03) and the comparative example (V02) have a relatively high Q30, and the Q30 of Example 1 (V03) is better than that of the comparative example.

[0197] Figure 19 Fig. shows the error rate data statistical curve of Example 1 (V03) and Comparative Example 1 (V02). As can be seen from the figure, compared with the comparative example (V02), Example 1 (V03) has a lower error rate.

[0198] Figure 20 Fig. shows the Q30 data statistical curve of Example 2 (V04) and Comparative Example 1 (V02). As can be seen from the figure, both Example 2 (V04) and the comparative example (V02) have a relatively high Q30, and the Q30 of Example 1 (V03) is better than that of the comparative example.

[0199] Figure 21 Fig. shows the error rate data statistical curve of Example 2 (V04) and Comparative Example 1 (V02). As can be seen from the figure, compared with the comparative example (V02), Example 2 (V04) has a lower error rate.

[0200] The above uses specific examples to elaborate on the present invention, which is only used to help understand the present invention and is not intended to limit the present invention. For those skilled in the technical field to which the present invention belongs, based on the idea of the present invention, several simple deductions, deformations or substitutions can also be made.

Claims

1. A solid-phase substrate, characterized in that, One surface of the solid-phase substrate is provided with multiple groups of regularly arranged marking units. Each group of the marking units includes at least three marks arranged in a preset manner, and each mark includes a first marking line in a first direction and a second marking line in a second direction and intersecting with the first marking line.

2. The solid-phase substrate according to claim 1, wherein, At least one detection area is provided on the solid-phase substrate, and at least one group of the marking units is provided in each detection area.

3. The solid-phase substrate according to claim 1 or 2, characterized in that, The detection area includes one or more detection units, and one group of the marking units is provided in each detection unit.

4. The solid-phase substrate according to claim 3, wherein, The detection area includes multiple detection rows arranged along the first direction; Optionally, the distance between adjacent detection rows is equal; Optionally, multiple equally spaced detection sites are arranged in the same row; Optionally, the detection sites are micron pores or nano pores, or the detection sites are nano-scale or micron-scale protrusions; Optionally, the sizes of the detection sites in the detection area are the same; Optionally, in two adjacent rows, except for at least one of the two detection sites at both ends of the row, the detection sites on one row are all located on the midline of the connection line between two adjacent detection sites on the other row.

5. The solid-phase substrate according to claim 3 or 4, wherein The detection unit includes a rectangular area block in the central area of the detection unit; Optionally, the marking units are arranged within the rectangular area block; Optionally, in a group of the marking units, the area of the region enclosed by the connections of the marks accounts for 50% or more of the total area of the rectangular area block; Optionally, in a group of the marking units, the area of the region enclosed by the connections of the marks accounts for 80% or more of the total area of the rectangular area block.

6. The solid-phase substrate according to any one of claims 1-5, characterized in that, In a group of the marking units, the image enclosed by the connections of the marks is a symmetric image; Optionally, the center of the symmetric image coincides with the center of the detection unit; Optionally, each group of the marking units includes three marks arranged in a preset manner; Optionally, the image enclosed by the connections of the three marks is an isosceles triangle; Optionally, each group of the marking units includes four marks arranged in a preset manner; Optionally, the image enclosed by the connections of the four marks is a rectangle; Optionally, the detection unit includes a rectangular area block in the central area of the detection unit, and the sides of the rectangular area block are respectively parallel or perpendicular to the first direction; Optionally, the four marks are respectively arranged at the four vertices of the rectangular area block.

7. The solid-phase substrate according to any one of claims 1-6, characterized in that, The first direction and the second direction are perpendicular; Optionally, the area of the marking unit accounts for less than 1% of the area of the detection unit.

8. The solid-phase substrate according to any one of claims 1-7, characterized in that, The aspect ratio of the first marking line is 15-30:1; and / or the aspect ratio of the second marking line is 15-30:1; Optionally, the length ratio of the first marking line to the second marking line is 0.8-1.2:1; Optionally, the solid-phase substrate includes multiple detection units, and one group of the marking units is provided in each detection unit; multiple detection sites are arranged at equal intervals in the first direction in the detection unit; Optionally, taking the distance between the centers of two adjacent detection sites in the first direction as l, at least one of the following conditions is satisfied for the length and width of the first marking line and the second marking line: The length of the first marking line is 80 - 150l, The length of the second marking line is 80 - 150l, The width of the first marking line is 3 - 8l, The width of the second marking line is 3 - 8l.

9. The solid-phase substrate according to any one of claims 1-8, characterized in that, The first marking line and / or the second marking line is a groove formed on the surface, and an identifiable mark is arranged in the groove; Optionally, the solid-phase substrate includes a plurality of detection units, and each detection unit is provided with a set of the marking units; the detection unit is provided with a plurality of detection sites at equal intervals in the first direction, and the distance between adjacent identifiable marks in the groove is less than the distance between adjacent detection sites in the detection unit; Optionally, the distance between the identifiable marks in the groove is 1 / 2 or less of the distance between adjacent detection sites in the detection unit.

10. The solid-phase substrate according to any one of claims 1-8, characterized in that, The first marking line and / or the second marking line includes holes or protrusions formed on the surface of the solid-phase substrate, and at least part of the holes or the surfaces of the protrusions are provided with identifiable marks; Optionally, in the first marking line, the connecting line of the centers of the holes or the protrusions is a straight line, and the straight line is parallel to the first direction; Optionally, in the second marking line, the connecting line of the centers of the holes or the protrusions is a straight line, and the straight line is perpendicular to the first direction; Optionally, the distances between adjacent identifiable marks on the first marking line and / or the second marking line are equal respectively; Optionally, the solid-phase substrate includes a plurality of detection units, and each detection unit is provided with a set of the marking units; the detection unit is provided with a plurality of detection sites at equal intervals in the first direction, and the distance between at least part of adjacent identifiable marks is greater than the distance between adjacent two detection sites; Optionally, taking the distance between the centers of two adjacent detection sites in the first direction as l, the distance between adjacent identifiable marks is n times of l, and n is an integer greater than or equal to 1; Optionally, n is a positive integer of 2 - 4; Optionally, the identifiable marks are regularly distributed on the first marking line according to a first arrangement rule, and the identifiable marks are regularly distributed on the second marking line according to a second arrangement rule; Optionally, the identifiable marks are arranged in all the holes or on the surfaces of the protrusions of the first marking line and / or the second marking line; Optionally, the holes are at least one of circular holes, oval holes, polygonal holes and irregular holes; Optionally, the holes are at least one of circular holes and regular polygonal holes; Optionally, the shapes and sizes of the holes arranged on the surface where the first marking line and / or the second marking line are located are the same; Optionally, the protrusions are at least one of circular protrusions, oval protrusions, polygonal protrusions and irregular protrusions; Optionally, the protrusions are at least one of circular protrusions and regular polygonal protrusions; Optionally, the shapes and sizes of the protrusions provided on the surfaces where the first marking line and / or the second marking line are located are the same; Optionally, the recognizable mark is a fluorescent mark.

11. A biochip comprising the solid-phase substrate according to any one of claims 1 to 10.

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