Spatial omics chip and manufacturing method therefor, and synthesis method for spatial position tag sequence and sample intake chip thereof

Abstract

A spatial omics chip and a manufacturing method therefor, and a synthesis method for a spatial position tag sequence and a sample intake chip thereof. The spatial omics chip comprises: a first substrate (10), hydrophobic regions (20), which are arranged on the same side of the first substrate (10), and a plurality of hydrophilic regions (30), which are distributed in a periodic array and are spaced apart from each other, wherein each hydrophilic region (30) is surrounded by the hydrophobic regions (20), and each hydrophilic region (30) comprises a hydrophilic structure (31), the hydrophilic structure (31) being configured to be connected to a coding probe (40).

Description

Spatial omics chip and manufacturing method thereof, spatial position tag sequence synthesis method and sampling chip thereof

[0001] This disclosure claims priority to the Chinese patent application filed with the China Patent Office on April 28, 2023, with application number 202310485391.X and invention name “Spatial omics chip and its manufacturing method, spatial position label sequence synthesis method and its injection chip”, the content of which should be understood as incorporated into this disclosure by reference. Technical Field

[0002] The embodiments of the present disclosure relate to, but are not limited to, spatial omics technology, and in particular to a spatial omics chip and a manufacturing method thereof, a synthesis method of spatial position tag sequences and a sampling chip thereof. Background Art

[0003] Traditional transcriptomics generally studies the overall RNA expression information of the target biological tissue. The research results reflect the overall transcriptome expression of the biological tissue, but ignore the gene expression specificity of different cells in the tissue. The emergence of single-cell transcriptomics technology can better distinguish the differences in RNA expression between different cells, overcoming the problem of distinguishing and detecting cell heterogeneity in the same tissue. However, the spatial position information between cells is closely related to the physiological and pathological conditions of biological tissues or organs. Traditional transcriptomics and single-cell transcriptomics technologies are unable to reveal the spatial heterogeneity information of multicellular tissues. Therefore, their application in the research of tissue and organ development and the occurrence and development mechanism of diseases such as tumors is limited to a certain extent.

[0004] In recent years, the rise of spatial transcriptomics (ST) technology has simultaneously addressed two key needs in biotechnology applications: the differentiated detection of cellular gene expression heterogeneity and the annotation of spatial location information. Since its initial publication in Science in 2016, ST technology has evolved into a variety of different technical implementations, which can be broadly categorized into two types: ST based on fluorescent probes and imaging, and ST based on in situ capture and sequencing. ST based on fluorescent probes and imaging enables true in situ RNA detection, but requires the support of high-tech microscopic imaging techniques and complex image analysis algorithms. Furthermore, its gene detection throughput is limited, and tissue clearing, probe, and enzyme penetration methods require further exploration. Therefore, commercial application remains a long way off. ST technology, based on in situ capture and sequencing, primarily utilizes specific tag sequences to capture and spatially tag transcripts in situ. Transcriptome expression information is then obtained through library construction and sequencing. This technology offers high sensitivity and resolution, is simple to operate, and has a short testing cycle. Currently, ST technology platforms based on in situ capture and sequencing include 10×Genomics-Visium, Slide-seq, Seq-scope, and Stereo-seq. Among these, 10×Genomics' Visium platform has been successfully commercialized and is the most widely used. The Stereo-seq platform, developed jointly by BGI-Shenzhen Institute of Life Sciences in China and several other institutions, also offers strong competitive advantages due to its ultra-high resolution and large sample coverage.

[0005] Summary of the Invention

[0006] The following is an overview of the subject matter described in detail herein. This overview is not intended to limit the scope of protection of the present disclosure.

[0007] An embodiment of the present disclosure provides a spatial omics chip, which includes: a first substrate, a hydrophobic region arranged on the same side of the first substrate, and a plurality of hydrophilic regions distributed in a periodic array and spaced from each other, each of the hydrophilic regions is surrounded by the hydrophobic region, and the hydrophilic region includes a hydrophilic structure; the hydrophilic structure is configured to be connected to a coding probe.

[0008] In an exemplary embodiment of the present disclosure, each of the hydrophilic areas may include a plurality of the hydrophilic structures distributed in a periodic array, the diameters of the different hydrophilic structures are all d1, and the distance between two adjacent hydrophilic structures in each of the hydrophilic areas is 0.25d1 to 1.5d1.

[0009] In an exemplary embodiment of the present disclosure, a diameter d1 of the hydrophilic structure may be 2 μm to 2.5 μm, and a height of the hydrophilic structure may be 5 μm to 10 μm.

[0010] In an exemplary embodiment of the present disclosure, the hydrophobic region may include a plurality of hydrophobic structures, and at least one hydrophobic structure may be provided between two adjacent hydrophilic regions; the diameters of the different hydrophobic structures are all d2, and the distance between two adjacent hydrophobic structures may be 0.25d2 to 1.5d2, and d2 may be 2 μm to 2.5 μm;

[0011] The distance between the adjacent hydrophobic structures and the adjacent hydrophilic structures is d3.

[0012] In an exemplary embodiment of the present disclosure, d1=d2, and d3 may be 0.25d1 to 1.5d1.

[0013] In an exemplary embodiment of the present disclosure, a plurality of the hydrophilic regions may be distributed in a periodic array on the first substrate, and a distance between centers of two adjacent hydrophilic regions is L1, and L1 may be no less than 1.5d1.

[0014] In an exemplary embodiment of the present disclosure, L1 may be 4.446d1 to 9d1. In an exemplary embodiment of the present disclosure, the hydrophilic structure may be connected to the first substrate through a carbon-hydrogen bond.

[0015] In an exemplary embodiment of the present disclosure, the surface of the hydrophilic structure contains active groups that can be connected to the coding probe, and the active groups may include any one or more of amino groups, thiol groups, carboxyl groups, ester groups and acid anhydrides; each of the hydrophobic structures contains hydrophobic groups, and the hydrophobic groups include any one or more of long-chain hydrocarbon groups and fluorocarbon groups with a carbon chain length ≥ 7.

[0016] In an exemplary embodiment of the present disclosure, the spatial omics chip may further include the coding probe connected to the hydrophilic structure; the coding probe may include any one or more of a chemical probe, a polypeptide probe, a fluorescent probe and a nucleic acid probe.

[0017] In an exemplary embodiment of the present disclosure, the encoding probe may be configured to capture an mRNA sample and mark the spatial location of the captured mRNA sample.

[0018] In an exemplary embodiment of the present disclosure, the coding probe includes a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence, and a Poly(dT) sequence, and the sequencing primer binding sequence, the spatial position tag sequence, the UMI sequence, and the Poly(dT) sequence are connected in sequence;

[0019] in,

[0020] The sequencing primer binding sequence is configured to complementarily pair with the primer during sequencing;

[0021] The spatial position tag sequence is configured to mark the spatial position of the captured mRNA sample, and the spatial position tag sequences of the coding probes connected to the hydrophilic structures located in the same hydrophilic region are the same, and the spatial position tag sequences of the coding probes connected to the hydrophilic structures located in different hydrophilic regions are different; the spatial position tag sequence includes a spatial position subtag sequence;

[0022] The UMI sequence is configured to specifically label different mRNA samples;

[0023] The Poly(dT) sequence is configured to capture the mRNA sample.

[0024] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence contains a linking group, and the sequencing primer binding sequence is connected to the hydrophilic structure through the linking group, and the linking group may include at least one of a carboxyl group, a thiol group, and an amino group.

[0025] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence and the hydrophilic structure may be linked together through at least one of a reaction between an amino group and a carboxyl group, a reaction between a sulfhydryl group and a sulfhydryl group, and a reaction between an acid anhydride and an amino group.

[0026] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence and the hydrophilic structure may be linked together via at least one of an amide group and a disulfide bond.

[0027] In an exemplary embodiment of the present disclosure, the spatial omics chip may include a plurality of sub-chips distributed at intervals, the plurality of sub-chips are distributed in a periodic array on the first substrate, and the sub-chips include a plurality of the hydrophilic areas.

[0028] The present disclosure also provides a rotary step-by-step sampling chip, which is configured to perform multi-step sampling into the hydrophilic region of the spatial omics chip provided in the above embodiment of the present disclosure. The rotary step-by-step sampling chip comprises: a second substrate and a plurality of flow channels arranged on the same side of the second substrate and spaced apart, the flow channels having a plurality of through holes penetrating the second substrate and distributed in a periodic array, each of the hydrophilic regions of the spatial omics chip corresponds to a through hole, the distance between the centers of two adjacent through holes is the same as the distance between the centers of two adjacent hydrophilic regions corresponding to the two through holes, and the through holes are configured to perform step-by-step sampling into each hydrophilic structure of the hydrophilic region corresponding thereto.

[0029] In an exemplary embodiment of the present disclosure, the area of ​​the through hole is smaller than or equal to the area of ​​the corresponding hydrophilic region.

[0030] In an exemplary embodiment of the present disclosure, the rotary step-by-step sampling chip may include an effective sampling area, the rotary step-by-step sampling chip rotates around its center at least once according to a set angle, and at least part of the through holes of the rotary step-by-step sampling chip before and after at least one rotation are completely overlapped, and the completely overlapping through holes form the effective sampling area. When the spatial omics chip and the rotary step-by-step sampling chip are matched and used, the orthographic projection of the through holes in the effective sampling area on the first substrate overlaps with the orthographic projection of the hydrophilic area of ​​the spatial omics chip on the first substrate.

[0031] In an exemplary embodiment of the present disclosure, the effective sample injection area may be a regular polygon having n sides, where n is an integer ≥ 3;

[0032] The distance between the center lines of two adjacent flow channels satisfies: L3 = L2 × sinθ;

[0033] Wherein, L3 is the distance between two adjacent flow channels;

[0034] L2 is the distance between the centers of two adjacent through holes;

[0035] θ is the rotation angle of the rotary step-by-step sampling chip.

[0036] The present disclosure also provides a method for synthesizing a spatial position tag sequence, the method comprising:

[0037] The subspace position label sequence is injected multiple times through the through holes of the rotary step-by-step sampling chip provided in the embodiment of the present disclosure, and the rotary step-by-step sampling chip is rotated around its center by a set angle between two adjacent injections, and at least part of the through holes of the rotary step-by-step sampling chip before and after the rotation are completely overlapped, and the subspace position label sequence is injected into the hydrophilic area of ​​the spatial omics chip through the through holes in the overlapping area to form the spatial position label sequence;

[0038] The subspace position label sequences of the multiple through-hole injections on the same flow channel are the same, and the subspace position label sequences of the through-hole injections on different flow channels are different.

[0039] In an exemplary embodiment of the present disclosure, injecting the subspace position tag sequence into the hydrophilic area of ​​the spatial omics chip through the through-holes in the overlapping area may include:

[0040] The rotary step-by-step sampling chip is laminated with the spatial omics chip provided in the above embodiment of the present disclosure, so that the hydrophilic region of the spatial omics chip corresponds to the through hole of the flow channel of the rotary step-by-step sampling chip, and the hydrophilic structure of the hydrophilic region is connected to the nucleic acid sequence;

[0041] Different subspace position label sequences are injected into different flow channels of the rotary step-by-step sampling chip respectively. The subspace position label sequences enter the hydrophilic area of ​​the spatial omics chip through the through holes on the flow channels and connect with the nucleic acid sequences on the hydrophilic structure.

[0042] The present disclosure also provides a method for manufacturing the spatial omics chip provided in the above embodiment of the present disclosure, the manufacturing method comprising:

[0043] Covering the first substrate with a first photomask having a first light-transmitting area and a first light-shielding area, wherein the first light-transmitting area completely overlaps with an orthographic projection of a region on the first substrate where the hydrophilic structure is to be formed;

[0044] Under light conditions, using a hydrophilic monomer as a raw material, a graft polymerization reaction is performed on the first substrate in an area completely overlapping with the orthographic projection of the first light-transmitting area to form a hydrophilic polymer, wherein the hydrophilic polymer includes an active group capable of connecting to the coding probe, and the hydrophilic polymer forms the hydrophilic structure;

[0045] Using a second photomask having a second light-transmitting area and a second light-shielding area to cover the first substrate, wherein the second light-transmitting area completely overlaps with an orthographic projection of a region on the first substrate where the hydrophobic structure is to be formed;

[0046] Under light conditions, a hydrophobic monomer is used as a raw material to perform a graft polymerization reaction in an area on the first substrate that completely overlaps with the orthographic projection of the second light-transmitting area to form a hydrophobic polymer, wherein the hydrophobic polymer contains the hydrophobic group, the hydrophobic polymer forms a hydrophobic structure, and the hydrophobic structure forms the hydrophobic area.

[0047] In an exemplary embodiment of the present disclosure, the method for manufacturing the spatial omics chip may further include sequentially connecting a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence and a Poly (dT) sequence at the end of the hydrophilic structure away from the first substrate.

[0048] In an exemplary embodiment of the present disclosure, the spatial position tag sequence can be formed using the synthesis method of the spatial position tag sequence provided in the above embodiment of the present disclosure.

[0049] Other features and advantages of the present disclosure will be described in the following description, and in part will become more apparent from the description, or understood by practicing the present disclosure. Other advantages of the present disclosure can be realized and obtained by the solutions described in the description and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] The accompanying drawings are used to provide an understanding of the technical solution of the present disclosure and constitute a part of the specification. Together with the embodiments of the present disclosure, they are used to explain the technical solution of the present disclosure and do not constitute a limitation to the technical solution of the present disclosure.

[0051] FIG1A is a top view of a spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0052] FIG1B is a front view of the spatial omics chip shown in FIG1A ;

[0053] FIG2 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0054] FIG3 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0055] FIG4 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0056] FIG5 is a schematic structural diagram of a hydrophilic region of a spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0057] FIG6 is a schematic structural diagram of a hydrophilic region of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0058] FIG7 is a schematic structural diagram of a hydrophilic region of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0059] FIG8 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0060] FIG9 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure;

[0061] FIG10 is a top view of a rotary step-by-step sampling chip provided by an exemplary embodiment of the present disclosure;

[0062] FIG11 is a top view of another rotary step-by-step sampling chip provided by an exemplary embodiment of the present disclosure;

[0063] FIG12 is a process flow chart of a method for forming a coding sequence provided by an exemplary embodiment of the present disclosure;

[0064] FIG13 is a process flow chart of another method for forming a coding sequence provided by an exemplary embodiment of the present disclosure;

[0065] FIG14 is a top view of a first photomask provided in accordance with an exemplary embodiment of the present disclosure;

[0066] FIG15 is a top view of a second photomask provided in accordance with an exemplary embodiment of the present disclosure;

[0067] FIG16 is a reaction mechanism diagram of the graft polymerization reaction on the surface of the first substrate.

[0068] The meanings of the marking symbols in the accompanying drawings are: 10-first substrate; 20-hydrophobic area; 21-hydrophobic structure; 30-hydrophilic area; 31-hydrophilic structure; 40-coding probe; 50-sub-chip; 51-sequencing primer binding sequence; 52-spatial position label sequence; 521-first sub-spatial position label sequence; 522-second sub-spatial position label sequence; 523-third sub-spatial position label sequence; 53-UMI sequence; 54-Poly (dT) sequence; 70-second substrate; 80-flow channel; 90-through hole; 100-effective sample injection area; 110-first background layer; 111-first light-transmitting area; 1111-first light-transmitting hole; 112-first light-shielding area; 120-second background layer; 121-second light-transmitting area; 1211-second light-transmitting hole; 122-second light-shielding area; 130-photomask. DETAILED DESCRIPTION

[0069] To make the objectives, technical solutions and advantages of the present disclosure more clearly understood, the embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings. It should be noted that, unless there is a conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other in any manner.

[0070] The embodiments herein can be implemented in a variety of different forms. A person skilled in the art can easily understand that the implementation and content can be transformed into various forms without departing from the purpose and scope of the present disclosure. Therefore, the present disclosure should not be interpreted as being limited to the contents described in the following embodiments. In the absence of conflict, the embodiments and features in the embodiments of the present disclosure can be combined with each other in any manner.

[0071] The scales of the figures in this disclosure can be used as a reference for actual processes, but are not limited to such. For example, the width-to-length ratio of the channel, the thickness and spacing of the various film layers, and the width and spacing of the various signal lines can be adjusted according to actual needs. The number of pixels in the display substrate and the number of sub-pixels in each pixel are not limited to the numbers shown in the figures. The figures described in this disclosure are merely schematic structural diagrams, and one embodiment of this disclosure is not limited to the shapes or values ​​shown in the figures.

[0072] In this specification, for convenience, words and phrases indicating orientation or positional relationships, such as "middle," "upper," "lower," "front," "back," "vertical," "horizontal," "top," "bottom," "inside," and "outside," are used to illustrate the positional relationships of constituent elements with reference to the accompanying drawings. This is merely for the purpose of facilitating the description of this specification and simplifying the description, and does not indicate or imply that the device or element referred to must have a specific orientation, be constructed, or operate in a specific orientation. Therefore, they should not be construed as limitations on the present disclosure. The positional relationships of constituent elements may be appropriately changed depending on the direction in which each constituent element is described. Therefore, the present disclosure is not limited to the words and phrases described in the specification and may be appropriately replaced according to the circumstances.

[0073] In this specification, unless otherwise specified or limited, the terms "disposed" and "connected" should be understood broadly. For example, they can refer to fixed, removable, or integral connections; mechanical or electrical connections; direct connections, indirect connections through an intermediary, or internal communication between two components. Those skilled in the art will understand the specific meanings of these terms in this disclosure based on the specific circumstances.

[0074] In the description of this disclosure, "parallel" means approximately parallel or almost parallel. For example, the angle formed by two straight lines is greater than -10° and less than 10°, and thus also includes the angle of greater than -5° and less than 5°. In addition, "perpendicular" means approximately perpendicular. For example, the angle formed by two straight lines is greater than 80° and less than 100°, and thus also includes the angle of greater than 85° and less than 95°.

[0075] In the description of the present disclosure, ordinal numbers such as “first” and “second” are provided to avoid confusion among constituent elements, rather than to limit the quantity.

[0076] In this specification, "membrane" and "layer" can be interchanged. For example, "hydrophilic layer" can sometimes be replaced with "hydrophilic membrane".

[0077] At present, ST technology platforms based on in situ capture and sequencing, such as the 10×Genomics-Visium technology platform, still have problems such as low efficiency in capturing sample mRNA, cross-contamination caused by lateral diffusion between adjacent areas, and complex chip processing and preparation methods and harsh conditions. In addition, most technology platforms require in situ decoding and sequencing of spatial position tag sequences in advance before starting the processing of sample tissues and subsequent capture and sequencing, which further increases the cost and process complexity of spatial transcriptome analysis. The current solution is to prepare a chip substrate (such as a glass slide, etc.) with an array of micron-scale partitioned micropores or microwell reaction chambers on the surface, and then place the coded microspheres or microcarriers connected to a unique nucleic acid molecule identifier into it, and capture the mRNA sample after tissue permeabilization, and finally complete the library construction and sequencing. During the tissue permeabilization process, the sample surface can be covered with a porous membrane to reduce lateral contamination. However, the above two solutions are still not essentially different from the current technology platforms. Both rely on the assembly of microspheres or microcarriers on the surface of a porous substrate for capture and sequencing. They face problems such as limited microsphere or microcarrier drop rate and complex processes. In addition, they are not very effective in improving the capture efficiency of mRNA samples, avoiding cross-contamination, and simplifying chip processing methods.

[0078] Therefore, it is extremely important to develop a spatial omics chip that has high capture efficiency for mRNA samples, is not prone to cross-contamination between adjacent sample areas, is easy to prepare, and is convenient and quick to operate.

[0079] An embodiment of the present disclosure provides a spatial omics chip, which includes: a first substrate, a hydrophobic region arranged on the same side of the first substrate, and a plurality of hydrophilic regions distributed in a periodic array and spaced from each other, each of the hydrophilic regions is surrounded by the hydrophobic region, and the hydrophilic region includes a hydrophilic structure; the hydrophilic structure is configured to be connected to a coding probe.

[0080] FIG1A is a top view of a spatial omics chip provided by an exemplary embodiment of the present disclosure; FIG1B is a front view of the spatial omics chip shown in FIG1A .

[0081] As shown in FIG1 , the spatial omics chip comprises: a first substrate 10 , a hydrophobic region 20 disposed on the same side of the first substrate 10 , and a plurality of hydrophilic regions 30 distributed in a periodic array and spaced apart from each other;

[0082] The main function of the first substrate 10 is to provide a base material support for the growth arrangement of the hydrophobic region 20 and the hydrophilic region 30, and subsequently serve as a base to carry the product after tissue permeabilization to complete the capture reaction;

[0083] The main function of the hydrophobic region 20 is to isolate all the periodically arranged hydrophilic regions 30 to prevent cross contamination between adjacent hydrophilic regions 30 caused by lateral flow of the sample. The hydrophobic region 20 can be formed of a hydrophobic material.

[0084] Each hydrophilic region 30 is surrounded by a hydrophobic region 20 and includes a hydrophilic structure 31 configured to connect to the coding probe 40. The hydrophilic region 30 may be formed of a hydrophilic material, and one end of the hydrophilic structure 31 may be connected to the first substrate 10.

[0085] In the description of the present disclosure, "surrounding" can be understood as partial surrounding or full surrounding. In some embodiments, the surrounding can be full surrounding, that is, the entire periphery of the hydrophilic area is surrounded by the hydrophobic area, and the hydrophobic area surrounding the hydrophilic area is a closed ring. In some embodiments, the surrounding can be partial surrounding, that is, part of the periphery of the hydrophilic area is surrounded by the hydrophobic area, and the hydrophobic area surrounding the hydrophilic area is not closed. For example, it can be a ring with an opening or multiple independent hydrophobic areas forming a surround of the hydrophilic area, as shown in Figure 1A. In an exemplary embodiment of the present disclosure, the coding probe may include any one or more of a chemical probe, a polypeptide probe, a fluorescent probe, and a nucleic acid probe (for example, a genomic probe, a cDNA probe, a cRNA probe, and an artificially synthesized oligonucleotide probe, etc.).

[0086] Exemplarily, the coding probe 40 can be configured to capture an mRNA sample and mark the spatial position of the captured mRNA sample; the main function of the hydrophilic structure 31 is to connect the coding probe to achieve the capture of mRNA. The hydrophilic structure 31 has a side surface and an end face away from the first substrate 10, and both the end face and the side surface can be connected to the coding probe 40.

[0087] In an exemplary embodiment of the present disclosure, the spatial omics chip may be a proteomic chip, a transcriptomic chip, a genomic chip, a methylomic chip, a metabolomic chip, or the like.

[0088] The spatial omics chip of the disclosed embodiment adopts a three-dimensional hydrophilic structure to form a hydrophilic area. The end face and side surface of the hydrophilic structure can be connected to the coding probe, which can increase the contact area between the spatial omics chip and the test object, thereby improving the test efficiency. For example, the contact area between the spatial omics chip and, for example, tissue permeabilization products can be increased, thereby improving the capture efficiency of samples such as mRNA. In addition, the spatial omics chip of the disclosed embodiment can increase the number of hydrophilic structures in the hydrophilic area by adjusting parameters such as the diameter and arrangement of the hydrophilic structure in the hydrophilic area, thereby increasing the total surface area of ​​the hydrophilic structure in the hydrophilic area and obtaining a higher sample mRNA capture efficiency. In addition, each hydrophilic area is surrounded by a hydrophobic area. On the one hand, the tissue permeabilization product in the hydrophobic area will not enter the interior of the hydrophilic area. On the other hand, the mRNA sample captured in the hydrophilic area will not flow laterally, thereby reducing cross contamination between adjacent sample areas.

[0089] In an exemplary embodiment of the present disclosure, the hydrophilic structure may extend in a direction perpendicular to the first substrate.

[0090] In an exemplary embodiment of the present disclosure, each hydrophilic region may include multiple hydrophilic structures distributed in a periodic array. The multiple hydrophilic structures in the hydrophilic region are arranged in a periodic array to effectively increase the contact area between the hydrophilic region and the tissue permeabilization product, thereby improving the capture efficiency of sample mRNA.

[0091] In an exemplary embodiment of the present disclosure, the diameters of the different hydrophilic structures are all d1, and the distance between two adjacent hydrophilic structures in each hydrophilic area can be 0.25d1 to 1.5d1, for example, it can be 0.25d1, 0.5d1, 0.75d1, d1, 1.25d1, 1.5d1.

[0092] In an exemplary embodiment of the present disclosure, a diameter d1 of the hydrophilic structure may be 2 μm to 2.5 μm, and a height of the hydrophilic structure may be 5 μm to 10 μm.

[0093] In an exemplary embodiment of the present disclosure, as shown in FIG1A , the hydrophobic region 20 may include a plurality of hydrophobic structures 21. The first substrate may have only one continuous hydrophobic region, which includes all of the hydrophobic structures; or, as shown in FIG1 , the first substrate may have multiple connected hydrophobic regions, each of which surrounds a hydrophilic region, and each of which includes multiple hydrophobic structures surrounding the hydrophilic region.

[0094] In an exemplary embodiment of the present disclosure, the diameters of the different hydrophobic structures are all d2, and the distance between two adjacent hydrophobic structures may be 0.25d2 to 1.5d2.

[0095] In an exemplary embodiment of the present disclosure, d2 may be 2 μm to 2.5 μm.

[0096] In an exemplary embodiment of the present disclosure, the diameter d2 of all the hydrophobic structures 21 is the same as the diameter d1 of all the hydrophilic structures 31 , and the distance between adjacent hydrophobic structures 21 and hydrophilic structures 31 may be 0.25d1 to 1.5d1.

[0097] In an exemplary embodiment of the present disclosure, as shown in FIG1A , a plurality of the hydrophilic areas may be distributed in a periodic array on the first substrate, and the distance between the centers of two adjacent hydrophilic areas is L1, that is, the length of the arrangement period of the hydrophilic areas, and L1 may be no less than 1.5d1. For example, L1 may be greater than 4d1. For another example, L1 may be from 4.446d1 to 9d1. For another example, L1 may be 4.446d1 (the hydrophilic area may be an equilateral triangle), 4.5d1 (the hydrophilic area may be a square), 6d1 (the hydrophilic area may be a regular hexagon), or 9d1 (the hydrophilic area may be a regular hexagon). In an exemplary embodiment of the present disclosure, at least one hydrophobic structure 21 may be provided between two adjacent hydrophilic areas 30, that is, the minimum distance between the edges of two adjacent hydrophilic areas 30 may be 2d1.

[0098] In exemplary embodiments of the present disclosure, the hydrophilic structure and the hydrophobic structure can each independently include any one or more three-dimensional structures such as pillars, protrusions, and depressions. Figure 2 is a top view of another spatial omics chip provided in exemplary embodiments of the present disclosure; Figure 3 is a top view of yet another spatial omics chip provided in exemplary embodiments of the present disclosure; and Figure 4 is a top view of yet another spatial omics chip provided in exemplary embodiments of the present disclosure.

[0099] As shown in FIG1A and FIG2 to FIG4, the first substrate may be a rhombus, a regular polygon, for example, a regular hexagon; in other exemplary embodiments, the first substrate may also be other polygons, for example, an equilateral triangle, a rectangle, a square, etc., or may be a circle or an irregular shape.

[0100] In an exemplary embodiment of the present disclosure, the hydrophilic area may be a regular polygon or a combination of regular polygons. For example, the hydrophilic area may be an equilateral triangle, a square, a regular pentagon, a regular hexagon, etc. For another example, the hydrophilic area may be a rhombus formed by a combination of two equilateral triangles. In other embodiments, the hydrophilic area may also be a rhombus, a rectangle, a circle or an irregular shape. Figure 5 is a structural schematic diagram of a hydrophilic area of ​​a spatial omics chip provided in an exemplary embodiment of the present disclosure; Figure 6 is a structural schematic diagram of a hydrophilic area of ​​another spatial omics chip provided in an exemplary embodiment of the present disclosure; Figure 7 is a structural schematic diagram of a hydrophilic area of ​​another spatial omics chip provided in an exemplary embodiment of the present disclosure. In the spatial omics chips shown in Figures 5, 6 and 7, the hydrophilic areas are respectively an equilateral triangle (equilateral triangle), a rhombus formed by a combination of two equilateral triangles, and a regular pentagon.

[0101] As shown in FIG1A and FIG2 to FIG4, each side of the regular polygonal hydrophilic area 30 may have 2 or 3 hydrophilic columns; in other exemplary embodiments, each side of the regular polygonal hydrophilic area 30 may have more hydrophilic columns, for example, 4, 5, etc.

[0102] In the spatial omics chip shown in Figure 1A, the four sides of the diamond-shaped first substrate 10 can all be 6 mm long, and the minimum internal angle can be 60°; the adjacent 7 hydrophilic columns form a regular hexagonal hydrophilic area 30, and all the hydrophilic areas are arranged periodically with the same size and shape; the diameters of all the hydrophobic columns and all the hydrophilic columns are d1, and the distances between adjacent hydrophobic columns and hydrophilic columns, the distances between two adjacent hydrophobic columns, and the distances between two adjacent hydrophilic columns are all 0.5d1, so the length of the longest diagonal of each hydrophilic area is 4d1, which is comparable to the average size of mammalian cells, and can achieve single-cell level resolution; there is a hydrophobic column between each two adjacent hydrophilic areas 30 in the direction extending along the same diagonal line, that is, the minimum distance between the edges of two adjacent hydrophilic areas 30 is 2d1, and the distance L1 between the centers of two adjacent hydrophilic areas 30 is 6d1, so the distribution density of the hydrophilic areas 30 on the surface of the first substrate 10 for detection resolution is approximately 5132 / mm 2 Up to 8018 pieces / mm 2 The detection resolution of the spatial omics chip is 12μm.

[0103] In the spatial omics chip shown in Figures 2 and 4, the hydrophilic area 30 is a regular hexagon, and each hydrophilic area 30 includes 19 hydrophilic columns. The diameters of all hydrophobic columns and all hydrophilic columns are d1, and the distances between adjacent hydrophobic columns and hydrophilic columns, the distances between two adjacent hydrophobic columns, and the distances between two adjacent hydrophilic columns are all 0.5d1. The distance L1 between the centers of two adjacent hydrophilic areas 30 is 9d. If d1 is 2μm, the detection resolution of this spatial omics chip is 18μm. In hydrophilic areas of the same size, the more hydrophilic columns there are, the lower the detection resolution of the spatial omics chip, which can meet the test requirements of lower resolution. In an exemplary embodiment of the present disclosure, the hydrophilic structure can be connected to the first substrate via a carbon-hydrogen bond.

[0104] In an exemplary embodiment of the present disclosure, the first electrode plate may be a silicon wafer, a glass sheet, or a plastic sheet. The plastic sheet may be made of, but not limited to, polymethyl methacrylate (PMMA), polycarbonate (PC), and cyclic olefin copolymer (COC). These materials can be surface-modified and grafted to form hydrophilic or hydrophobic structure arrays to meet the requirements of subsequent spatial omics analysis and testing, such as spatial transcriptomics.

[0105] In an exemplary embodiment of the present disclosure, the surface of the hydrophilic structure comprises active groups capable of connecting to the coding probes, and the active groups are used for subsequent connection with the coding probes, thereby connecting the coding probes to the hydrophilic structure.

[0106] The active group may include any one or more of an amino group, a thiol group, a carboxyl group, an ester group and an acid anhydride; each of the hydrophobic structures does not carry the active group, and each of the hydrophobic structures contains a hydrophobic group, and the hydrophobic group includes any one or more of a long-chain hydrocarbon group with a carbon chain length ≥ 7 and a fluorocarbon group.

[0107] In an exemplary embodiment of the present disclosure, the spatial omics chip may further include a coding probe connected to the hydrophilic structure; the coding probe includes a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence and a Poly (dT) sequence.

[0108] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence, the spatial location tag sequence, the UMI sequence, and the Poly(dT) sequence are connected in sequence.

[0109] As shown in FIG1A , along the direction away from the hydrophilic structure, the coding probe includes a sequencing primer binding sequence (Partial Read 1), a spatial position tag sequence (Spatial Barcode), a UMI sequence, and a Poly (dT) sequence in sequence.

[0110] The sequencing primer binding sequence is used for complementary pairing with a primer during on-machine sequencing, and the sequencing primer binding sequence of the coding probes connected to all the hydrophilic structures on the first substrate is the same.

[0111] The spatial position label sequence is used to mark the specific spatial position of the mRNA sample captured subsequently; and the spatial position label sequences of the coding probes connected to the hydrophilic structures located in the same hydrophilic region are the same, indicating that the mRNA samples with the same nucleic acid sequence structure are derived from the same cell; the spatial position label sequences of the coding probes connected to the hydrophilic structures located in different hydrophilic regions are different, indicating that the mRNA samples with different nucleic acid sequence structures are derived from different cells, thereby distinguishing the spatial positions of different cells. Therefore, the spatial position label sequence is a particularly important sequence in the spatial transcriptome sequencing analysis process.

[0112] The UMI sequence is used to specifically label different mRNA samples within the same cell so as to quantify all different mRNA samples, and the UMI sequences of the coding probes connected to all the hydrophilic structures on the first substrate are different.

[0113] The Poly(dT) sequence is used to capture the mRNA sample, and is a sequence consisting of consecutive T bases.

[0114] In an exemplary embodiment of the present disclosure, the end surface and the side surface of the hydrophilic structure are both connected with the coding probe, which is beneficial to improving the capture efficiency of the mRNA sample.

[0115] In an exemplary embodiment of the present disclosure, the end of the sequencing primer binding sequence that is connected to the hydrophilic structure (typically the 5' end) carries a linking group, which may include a carboxyl group, a sulfhydryl group, and an amino group. The linking group is used to react with the active group on the hydrophilic structure, thereby achieving the connection between the sequencing primer binding sequence and the hydrophilic structure.

[0116] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence and the hydrophilic structure may be linked together through at least one of a reaction between an amino group and a carboxyl group, a reaction between a sulfhydryl group and a sulfhydryl group, and a reaction between an acid anhydride and an amino group.

[0117] In an exemplary embodiment of the present disclosure, the sequencing primer binding sequence and the hydrophilic structure may be linked together via at least one of an amide group and a disulfide bond. For example, the active group on the hydrophilic structure may be an amino group, and the linking group on the sequencing primer binding sequence may be a carboxyl group. The sequencing primer binding sequence and the hydrophilic structure may be linked via an amide group formed by the reaction of the amino group and the carboxyl group.

[0118] FIG8 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure; FIG9 is a top view of another spatial omics chip provided by an exemplary embodiment of the present disclosure.

[0119] In an exemplary embodiment of the present disclosure, as shown in FIG8 and FIG9 , the spatial omics chip may include a plurality of sub-chips 50 distributed at intervals, the plurality of sub-chips 50 are distributed in a periodic array on the first substrate 10 , and the sub-chips 50 include a plurality of hydrophilic regions 30 .

[0120] The spatial omics chip shown in Figure 8 integrates four diamond-shaped sub-chips 50 on a diamond-shaped first substrate. The spatial omics chip shown is a four-flux spatial omics chip; in the extension direction of the edge of the diamond-shaped sub-chip 50, the distance between two adjacent sub-chips 50 can be 2mm, and the distance between the sub-chip 50 and the edge of the first substrate can be 1mm.

[0121] The spatial omics chip shown in FIG9 integrates seven regular hexagonal sub-chips 50 on a regular hexagonal first substrate. The spatial omics chip shown is a 7-flux spatial omics chip; the minimum distance between two adjacent sub-chips 50 can be 1.5 mm.

[0122] The embodiment of the present disclosure further provides a rotary step-by-step sampling chip, which is configured to be able to perform step-by-step sampling on the hydrophilic structures in the hydrophilic region of the spatial omics chip provided in the embodiment of the present disclosure.

[0123] In the description of the present disclosure, "step-by-step injection" refers to injection in multiple times (at least twice).

[0124] FIG10 is a top view of a rotary step-by-step sampling chip provided by an exemplary embodiment of the present disclosure; FIG11 is a top view of another rotary step-by-step sampling chip provided by an exemplary embodiment of the present disclosure.

[0125] As shown in FIG10 and FIG11, the rotary step-by-step sampling chip includes: a second substrate 70 and a plurality of flow channels 80 arranged on the same side of the second substrate 70 and spaced apart, which are the first flow channel A1, the second flow channel A2, the third flow channel A3, ..., the nth flow channel A n The flow channel 80 has a plurality of through holes 90 that penetrate the second substrate 70 and are distributed in a periodic array. Each hydrophilic area 30 of the spatial omics chip corresponds to a through hole 90. The distance between the centers of two adjacent through holes 90 is the same as the distance between the centers of two adjacent hydrophilic areas 30 corresponding to the two through holes 90. The through holes 90 are configured to inject samples into the hydrophilic areas 30 corresponding to them in steps.

[0126] In an exemplary embodiment of the present disclosure, the through-hole is configured to inject sample only into the hydrophilic area corresponding thereto, but not into the hydrophobic area.

[0127] In an exemplary embodiment of the present disclosure, the rotary step-by-step sampling chip may include an effective sampling area 100, and the rotary step-by-step sampling chip rotates at least once around its center according to a set angle. At least part of the through holes of the rotary step-by-step sampling chip before and after at least one rotation can be completely overlapped, and the through holes that can be completely overlapped form the effective sampling area 100.

[0128] In the description of the present disclosure, "through holes completely overlap" means that the through holes before and after rotation have a common overlapping area. For example, the center of the through hole after rotation is the same as that of the through hole before rotation.

[0129] In an exemplary embodiment of the present disclosure, when the spatial omics chip is matched with the rotary step-by-step sampling chip, the orthographic projection of the through hole in the effective sampling area on the second substrate overlaps with the orthographic projection of the hydrophilic area of ​​the spatial omics chip on the second substrate, which can be completely overlapped or with an overlapping area. If the spatial omics chip contains a sub-chip, the effective sampling area 100 can correspond to the sub-chip of the spatial omics chip, that is, the effective sampling area 100 has the same shape and size as the sub-chip, and the orthographic projection of the through hole in the effective sampling area on the second substrate overlaps with the orthographic projection of the hydrophilic area of ​​the sub-chip on the second substrate; if the spatial omics chip does not contain a sub-chip, the effective sampling area 100 can correspond to the spatial omics chip, that is, the effective sampling area 100 has the same shape and size as the spatial omics chip, and the orthographic projection of the through hole in the effective sampling area on the second substrate overlaps with the orthographic projection of the hydrophilic area of ​​the spatial omics chip on the second substrate.

[0130] In an exemplary embodiment of the present disclosure, the set angle of rotation of the rotary step-by-step sampling chip can be set according to the desired shape of the effective sampling area. For example, it can be the minimum inner angle of a rhombus, the central angle of a regular polygon, etc. For another example, it can be 30°, 45°, 60°, 90°, etc.

[0131] In an exemplary embodiment of the present disclosure, the effective sample injection area may be a regular polygon having n sides, where n is an integer ≥ 3;

[0132] The distance between the center lines of two adjacent flow channels satisfies: L3 = L2 × sinθ;

[0133] Wherein, L3 is the distance between two adjacent flow channels;

[0134] L2 is the distance between the centers of two adjacent through holes;

[0135] θ is the rotation angle of the rotary step-by-step sampling chip.

[0136] For example, if the effective sample injection area 100 is a square, the effective sample injection area 100 is obtained by rotating the rotary step-by-step sample injection chip once, and the rotation angle is 90°;

[0137] For example, if the effective sampling area 100 is a regular polygon with n sides, and n is an even number, the effective sampling area 100 is obtained by rotating the rotary step-by-step sampling chip n / 2 times, and the rotation angle is the central angle θ of the regular polygon.

[0138] In an exemplary embodiment of the present disclosure, the second substrate may be a silicon wafer or a glass wafer, and may be rectangular or in other shapes. The flow channels may be parallel to each other and extend in a direction parallel to the long sides of the rectangular second substrate. The diameter of the hydrophilic structure is d1, and the length of the second substrate may be 3×749×6×d1, and the width may be 499×5.196×d1, with both length and width rounded to millimeters (mm) and the thickness being 2 mm.

[0139] When the diameter d1 of all hydrophilic structures of the spatial omics chip is 2 μm, 500 flow channels can be evenly distributed in the extension direction of the short side of the rectangular second substrate, and the distance between the center lines of two adjacent flow channels is 5.196×d1 (i.e., the arrangement period length of the hydrophilic area × sin30°, d1 is the diameter of the hydrophilic structure). The length of each flow channel is the length of the second substrate, and the through holes are distributed in the middle 1 / 3 of the flow channel (within the rectangular box in Figure 10). The number of through holes on a flow channel is 749 and 750 alternating cycles. For example, the first flow channel A1 is 750 (or 749), the second flow channel A2 is 749 (or 750), the third flow channel A3 is 750 (or 749), the fourth flow channel is 749 (or 750)... and so on. When the sample flows in the flow channel, it can be injected downward through these through holes to achieve sampling. The through-holes 90 located in the effective sample inlet area 100 correspond exactly to a hydrophilic area on the surface of the spatial omics chip substrate. Therefore, all samples passing through the through-holes can enter a unique hydrophilic area. That is, the distance between two adjacent through-holes on the same flow channel is the same as the arrangement period length between the hydrophilic areas of the spatial omics chip provided in the embodiment of the present disclosure, which is 6d1.

[0140] In an exemplary embodiment of the present disclosure, the diameter of the through hole is slightly smaller than the diameter of the hydrophilic area. For example, the hydrophilic area is a rhombus formed by 7 hydrophilic structures, and the diameter of the hydrophilic area is 4d1. Then the diameter of the hole can be 3×d1 to ensure the accuracy of all injections.

[0141] In an exemplary embodiment of the present disclosure, the area of ​​the through hole may be smaller than or equal to the area of ​​the corresponding hydrophilic region.

[0142] The present disclosure also provides a method for synthesizing a spatial position tag sequence, the method comprising:

[0143] The subspace position label sequence is injected multiple times through the through holes of the rotary step-by-step sampling chip provided in the embodiment of the present disclosure, and the rotary step-by-step sampling chip is rotated around its center by a set angle between two adjacent injections, and at least part of the through holes of the rotary step-by-step sampling chip before and after the rotation are completely overlapped, and the subspace position label sequence is injected into the hydrophilic area of ​​the spatial omics chip through the through holes in the overlapping area to form the spatial position label sequence;

[0144] The subspace position label sequences of the multiple through-hole injections on the same flow channel are the same, and the subspace position label sequences of the through-hole injections on different flow channels are different.

[0145] The method for synthesizing the spatial position tag sequence of the embodiment of the present disclosure can use multiple injections and connections of sub-space position tag sequences with known sequences to form a spatial position tag sequence. Therefore, the sequence of the synthesized spatial position tag sequence is also known. When using a spatial omics chip connected to this spatial position tag sequence for spatial omics analysis, there is no need to sequence and decode the spatial position tag information in advance through complex in situ sequencing and other methods, which can effectively simplify the spatial transcriptome analysis process and reduce detection costs.

[0146] In an exemplary embodiment of the present disclosure, injecting the subspace position tag sequence into the hydrophilic area of ​​the spatial omics chip through the through-holes in the overlapping area may include:

[0147] The rotary step-by-step sampling chip is laminated with the spatial omics chip provided in the above embodiment of the present disclosure, so that the hydrophilic region of the spatial omics chip corresponds to the through hole of the flow channel of the rotary step-by-step sampling chip, and the hydrophilic structure of the hydrophilic region is connected to the nucleic acid sequence (for example, the sequencing primer binding sequence of the coding probe);

[0148] Different subspace position tag sequences are injected into different flow channels of the rotary step-by-step sampling chip respectively. The subspace position tag sequences enter the hydrophilic area of ​​the spatial omics chip through the through holes on the flow channels and connect with the nucleic acid sequence connected to the hydrophilic structure (for example, the sequencing primer binding sequence, or the subspace position tag sequence that is already connected to the sequencing primer binding sequence).

[0149] In an exemplary embodiment, the spatial position label sequence can be divided into two segments for connection, wherein the first segment is the first sub-spatial position label sequence 521 (Spatial Barcode 1), and the second segment is the second sub-spatial position label sequence 522 (Spatial Barcode 2). Figure 12 is a process flow diagram of a method for forming a coding sequence provided by an exemplary embodiment of the present disclosure. As shown in Figure 12, after the sequencing primer binding sequence 51 is linked to the hydrophilic structure 31, the first subspatial position tag sequence 521 (Spatial Barcode 1) is first connected to the 3' end of the sequencing primer binding sequence 51 through the Spatial Barcode 1 adapter structure under the action of DNA ligase. Then, the second subspatial position tag sequence 522 (Spatial Barcode 2) is also connected to the 3' end of the first subspatial position tag sequence 521 through the Spatial Barcode 2 adapter structure under the action of DNA ligase, thereby forming a complete spatial position tag sequence 52. Finally, with the help of a composite adapter structure composed of a UMI sequence 53 and a Poly (dT) sequence 54, the UMI sequence 53 and the Poly (dT) sequence 54 are connected to the ends of the spatial position tag sequence 52 to form a complete coding probe sequence. In the above synthesis process, the first subspace position label sequence 521 and the second subspace position label sequence 522 of the spatial position label sequence 52 are actually the same group of sequences. The purpose of describing the first subspace position label sequence 521 and the second subspace position label sequence 522 separately is only to distinguish the injection sequence. The specific method involved in this injection sequence is the rotary step-by-step injection method.

[0150] The rotary step-by-step sampling method utilizes a rotary step-by-step sampling chip to achieve the connection of the first subspace position label sequence after the first sample addition, and then rotates around the center of the rotary step-by-step sampling chip to a set angle, for example, it can be rotated 60° clockwise, and then a second sample addition is performed to achieve the connection of the second subspace position label sequence. After two sample additions and connections, the spatial position label sequence is synthesized.

[0151] The following uses the example of using the rotary step-by-step sampling chip shown in Figure 10 to step-by-step sample injection into the spatial omics chip shown in Figure 1A or Figure 2 to illustrate the synthesis process of the spatial position label sequence. It should be emphasized that the rotary step-by-step sampling chip needs to be tightly covered on the surface of the spatial omics chip provided in the embodiment of the present disclosure as above when in use, so as to smoothly complete the injection; and, each hydrophilic area corresponds to a through hole, so that the through hole can only inject samples into the hydrophilic area corresponding to it, and cannot inject samples into the hydrophilic area that does not correspond to it. For example, each hydrophilic area can be located below a through hole and connected, and the projection of each through hole on the first substrate can also fall within the range of the projection of the hydrophilic area corresponding to the through hole on the first substrate, and even the center of the projection of each through hole on the first substrate can overlap with the center of the projection of the hydrophilic area corresponding to the through hole on the first substrate. Assume that the rotary step-by-step sampling chip has 500 flow channels, and during the first injection, the first subspace position label sequence (including the first label sequence B1, the first label sequence B2, the first label sequence B3, ..., the first label sequence B 500 ), these 500 first tag sequences are all different from each other; after the DNA polymerase ligation reaction is completed, the rotary step-by-step sampling chip is rotated 60° clockwise around its center, and then the second sampling is performed. At this time, 500 second subspace position tag sequences (including second tag sequence C1, second tag sequence C2, second tag sequence C3, ..., second tag sequence C1) with known sequences and different from each other are continuously introduced through 500 flow channels. 500 ).

[0152] It should be emphasized that the second subspace position label sequences introduced into each flow channel after rotation are respectively the same as the first subspace position label sequences introduced into the flow channel before rotation, that is, the second label sequence C1 is the same as the first label sequence B1, the second label sequence C2 is the same as the first label sequence B2, the second label sequence C3 is the same as the first label sequence B3, ..., the second label sequence C 500 With the first tag sequence B 500The same means that 500 different known label sequences are introduced twice before and after the rotation, and two connection reactions are carried out. The first label sequence and the second label sequence (or the first subspace position label sequence and the second subspace position label sequence) are used here only to distinguish the order of injection. The rotary step-by-step injection chip completes two injections before and after the rotation. The hydrophilic areas corresponding to the intersection of the injection holes realize the connection of the first label sequence and the second label sequence in sequence, that is, the connection synthesis of the spatial position label sequence is realized on each hydrophilic area of ​​the spatial omics chip, and then the UMI sequence and the first Poly (dT) sequence are connected to the spatial position label sequence in sequence, thereby obtaining a spatial omics chip with a coding probe connected to the hydrophilic structure. This chip has both mRNA sample capture function and sample spatial position labeling function, which can meet the subsequent spatial transcriptome sequencing and analysis needs.

[0153] The above-mentioned method for synthesizing spatial position label sequences for rotary step-by-step sampling is as follows: if the first substrate of the spatial omics chip is a rhombus, with a side length of 6 mm and a minimum internal angle of 60°, the diameters d1 and d2 of the hydrophilic structure and the hydrophobic structure are both 2 μm, and there are 500 flow channels on the rotary step-by-step sampling chip, then 250,000 (500×500) different coding probes can be arranged and combined to form, theoretically, 250,000 cell samples can be detected simultaneously, with a detection resolution of 12 μm. Moreover, since both the first label sequence and the second label sequence are known sequences, the spatial position label sequence formed by the arrangement and combination of the two is also known. When using a spatial omics chip connected to this spatial position label sequence for spatial omics analysis, there is no need to sequence and decode the spatial position label information in advance through complex in situ sequencing and other methods, which can effectively simplify the spatial transcriptome analysis process and reduce detection costs.

[0154] In an exemplary embodiment of the present disclosure, a spatial position label sequence with higher discrimination and precision can be obtained by adjusting the number of step-by-step injections (or the number of rotations of the rotary step-by-step injection chip), thereby more effectively avoiding erroneous analysis results caused by errors in spatial position label sequence testing or accidental similarity in sequence combinations during library construction and sequencing, thereby meeting the needs of sample testing with higher precision and accuracy.

[0155] For example, the number of step-by-step injections can be three, and in this case, the number of rotations of the rotary step-by-step injection chip is two. The shape of the spatial omics chip caused by the adjustment method changes, and a spatial omics chip as shown in Figure 3 can be obtained. As shown in Figure 3, the effective injection area formed after the rotary step-by-step injection chip rotates twice at an angle of 60° can be a regular hexagon; compared to the spatial omics chip shown in Figure 1A obtained by a single rotation, the shape, size, and spacing of the hydrophilic area formed by the hydrophilic structure remain unchanged. If the diameter d1 of the hydrophilic structure is 2 μm, the detection resolution of the spatial omics chip shown in Figure 3 remains unchanged relative to the spatial omics chip shown in Figure 1A, and is 12 μm. However, it is worth noting that the number of flow channels on the rotary step-by-step injection chip can only be set to an odd number. This is because when the number of injections is odd, the number of flow channels on the rotary step-by-step injection chip should also be odd; when the number of injections is even, the number of flow channels on the rotary step-by-step injection chip should also be even. If the number of injections and the number of flow channels on the rotary step-by-step injection chip are not matched according to the above rules, the through-holes on each flow channel after the next rotation will not fall precisely at the intersection of the two flow channels formed after the previous rotation, which will cause the subsequent injection to fail. In addition, the number and position distribution of the through-holes on each flow channel also need to be adjusted accordingly, and the structure of the photomask related to the synthesis of hydrophilic and hydrophobic structures should also be changed accordingly.

[0156] Since the number of injections has been changed from one to three, the synthesis method of the coding probe sequence is also different from the process flow shown in Figure 12. Figure 13 is a process flow chart of another method for forming a coding sequence provided by an exemplary embodiment of the present disclosure. As shown in Figure 13, the spatial position label sequence of the coding probe sequence is divided into a first subspace position label sequence 521 (Spatial Barcode 1), a second subspace position label sequence 522 (Spatial Barcode 2), and a third subspace position label sequence 523 (Spatial Barcode 3), which are connected in sequence, and the connection of other parts has no significant changes.

[0157] The present disclosure also provides a method for manufacturing the spatial omics chip provided in the above embodiment of the present disclosure, the manufacturing method comprising:

[0158] forming a hydrophilic region including a hydrophilic structure on the first substrate;

[0159] A hydrophobic area entirely surrounding the hydrophilic area is formed on the first substrate.

[0160] In an exemplary embodiment of the present disclosure, forming a hydrophilic area including a hydrophilic structure on the first substrate may include:

[0161] Covering the first substrate with a first photomask having a first light-transmitting area and a first light-shielding area, wherein the first light-transmitting area completely overlaps with an orthographic projection of a region on the first substrate where the hydrophilic structure is to be formed;

[0162] Under illumination conditions, a hydrophilic monomer is used as a raw material to carry out a graft polymerization reaction in an area on the first substrate that completely overlaps with the orthographic projection of the first light-transmitting area to form a hydrophilic polymer, and the hydrophilic polymer contains an active group that can be connected to the coding probe, and the hydrophilic polymer forms the hydrophilic structure.

[0163] In an exemplary embodiment of the present disclosure, forming a hydrophobic region entirely surrounding the hydrophilic region on the first substrate may include:

[0164] Using a second photomask having a second light-transmitting area and a second light-shielding area to cover the first substrate, wherein the second light-transmitting area completely overlaps with an orthographic projection of a region on the first substrate where the hydrophobic structure is to be formed;

[0165] Under light conditions, a hydrophobic monomer is used as a raw material to perform a graft polymerization reaction in an area on the first substrate that completely overlaps with the orthographic projection of the second light-transmitting area to form a hydrophobic polymer, wherein the hydrophobic polymer contains the hydrophobic group, the hydrophobic polymer forms a hydrophobic structure, and the hydrophobic structure forms the hydrophobic area.

[0166] In the manufacturing method of the spatial omics chip provided by the above-mentioned exemplary embodiment of the present disclosure, the hydrophilic structure and the hydrophobic structure are synthesized using hydrophilic monomers and hydrophobic monomers as raw materials, respectively, and are generated on the surface of the first substrate under the irradiation of ultraviolet light or visible light with the help of two photomasks with complementary light-transmitting structures. This manufacturing method is efficient and simple, and the conditions are mild and controllable.

[0167] Figure 14 is a top view of a first photomask provided in an exemplary embodiment of the present disclosure. As shown in Figure 14, the first photomask comprises a first light-transmitting region 111 and a first light-blocking region 112 disposed on a first background layer 110. For example, the first light-transmitting region 111 may include a plurality of first light-transmitting apertures 1111 extending through the first background layer 110. The projections of the first light-transmitting apertures 1111 on the first substrate completely overlap with the projections of the hydrophilic structures on the first substrate. The first light-transmitting region 111 is where the hydrophilic structures are located, while the hydrophobic structures are located on the first light-blocking region 112, both being in a light-blocking state.

[0168] Figure 15 is a top view of a second photomask provided in an exemplary embodiment of the present disclosure. As shown in Figure 15 , the second photomask comprises a second light-transmitting region 121 and a second light-blocking region 122 disposed on a second background layer 120. For example, the second light-transmitting region 121 may include a plurality of second light-transmitting apertures 1211 extending through the second background layer 120. The projections of the second light-transmitting apertures 1211 on the first substrate completely overlap with the projections of the hydrophobic structure on the first substrate. The second light-transmitting region 121 is where the hydrophobic structure is located, while the hydrophilic structure is located on the second light-blocking region 122, both being in a light-blocking state.

[0169] In an exemplary embodiment of the present disclosure, the shape and size of the first photomask and the second photomask can be the same as the shape and size of the first substrate, the size and distribution spacing of the first light-transmitting holes are the same as the size and distribution spacing of the hydrophilic structure on the surface of the first substrate, and the size and distribution spacing of the second light-transmitting holes are the same as the size and distribution spacing of the hydrophobic structure on the surface of the first substrate.

[0170] In an exemplary embodiment of the present disclosure, when the first substrate is a silicon wafer or a glass wafer, the method for manufacturing the spatial omics chip may further include: before forming the hydrophilic area and the hydrophobic area on the surface of the first substrate, modifying the first substrate with a silane coupling agent having a saturated or unsaturated hydrocarbon group, so as to introduce carbon-hydrogen bonds (CH) on the surface of the first substrate.

[0171] In an exemplary embodiment of the present disclosure, the silane coupling agent may include, but is not limited to, methyltriethoxysilane, octadecyltrimethoxysilane, γ-methacryloxypropyltriethoxysilane, vinyltrimethoxysilane, and the like.

[0172] When the first substrate is a plastic sheet such as polymethyl methacrylate, polycarbonate, or cycloolefin copolymer, there is no need to modify the first substrate.

[0173] In the above-described manufacturing method, the growth of the hydrophilic and hydrophobic structures on the surface of the first substrate primarily utilizes the principle of ultraviolet / visible light-induced graft polymerization on the surface of the first substrate material containing CH. Figure 16 illustrates the reaction mechanism of the graft polymerization reaction on the surface of the first substrate.

[0174] As shown in FIG16 , under the excitation of ultraviolet / visible light, the photoinitiator captures H atoms on the surface of the first substrate 10 containing CH, thereby forming a large number of active free radicals on the surface of the first substrate 10. These active free radicals can further initiate the hydrophilic monomer or hydrophobic monomer in the reaction system to undergo a graft polymerization reaction on the surface of the first substrate 10. With the assistance of the two photomasks 130 described above, a hydrophilic structure or a hydrophobic structure can be formed on the surface of the first substrate 10 after the graft polymerization is completed. The reaction system for preparing the hydrophilic structure and the hydrophobic structure is the same except for the different reactive monomers. The remaining components include solvents (including but not limited to water, ketones such as acetone, alcohols such as ethanol, esters such as ethyl acetate, ethers such as dioxane, and amides such as N,N-dimethylformamide), initiators (including but not limited to benzophenone and its derivatives, thioxanthones, and anthraquinones), and crosslinkers (including but not limited to unsaturated dienes such as divinylbenzene and acrylates such as polyethylene glycol dimethacrylate).

[0175] In exemplary embodiments of the present disclosure, the hydrophilic monomer may have an olefin group and a hydrophilic group, and the hydrophilic group may include any one or more of an amino group, a carboxyl group, and an ester group. For example, the hydrophilic monomer includes, but is not limited to, acrylic acid, mercapto polyethylene glycol acrylate, vinyl acetate, and 2-aminoethyl methacrylate. After homopolymerization to form a hydrophilic structure, such monomers retain reactive groups on the surface of the hydrophilic structure that are subsequently reactive with the coding probe.

[0176] The hydrophilic monomer may also contain only a hydrophilic structure and an olefin group, but does not contain an active group having subsequent reaction activity with the coding probe, for example, polyethylene glycol methacrylate, etc.; however, such monomers need to be copolymerized with other olefins with active groups (such as maleic anhydride, etc.) to ensure that the surface of the hydrophilic structure formed after polymerization can retain active groups having subsequent reaction activity with the coding probe.

[0177] In an exemplary embodiment of the present disclosure, the hydrophobic monomer may have an olefin group and a hydrophobic group, and the hydrophobic group may include any one or more of a long-chain hydrocarbon group and a fluorocarbon group. The carbon chain length of the long-chain hydrocarbon group may be ≥7, for example, it may be 8 to 20, or it may be 12 to 18. For example, the hydrophobic monomer includes, but is not limited to: dodecyl methacrylate, heptadecyl methacrylate, trifluoroethyl acrylate, perfluoroalkylethyl acrylate, dodecafluoroheptyl methacrylate, etc. The hydrophobic monomer may also be copolymerized with other unsaturated olefins. It should be emphasized that after polymerization, the hydrophobic monomer and the monomers copolymerized therewith cannot have any active groups that subsequently react with the coding probe.

[0178] In an exemplary embodiment of the present disclosure, the method for manufacturing the spatial omics chip may further include sequentially connecting a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence and a Poly (dT) sequence at the end of the hydrophilic structure away from the first substrate.

[0179] In an exemplary embodiment of the present disclosure, the spatial position tag sequence can be formed using the synthesis method of the spatial position tag sequence provided in the above embodiment of the present disclosure.

[0180] In order to adapt to the detection needs of different resolutions, the resolution and accuracy of the detection can be adjusted by designing different photomask structures to adapt to different sample detection needs; the accuracy of the spatial positioning of the mRNA sample can also be increased by adjusting the number of rotary step-by-step injections during the synthesis of the spatial position label sequence to meet the detection needs of samples with higher precision requirements.

[0181] When the number of flow channels of the rotary step-by-step sampling chip and the size, quantity, and position distribution of the sampling holes on the flow channels need to be adjusted, the distribution of the light-transmitting holes of the photomask related to the synthesis of hydrophilic and hydrophobic structures should also change accordingly.

[0182] Although the embodiments disclosed in this disclosure are as described above, the contents described are merely embodiments adopted to facilitate understanding of the disclosure and are not intended to limit the disclosure. Any person skilled in the art may make any modifications and changes in the form and details of the implementation without departing from the spirit and scope of the disclosure. However, the scope of protection of the disclosure shall still be based on the scope defined by the appended claims.

Claims

1. A spatial omics chip, comprising: A first substrate, a hydrophobic region arranged on the same side of the first substrate, and a plurality of hydrophilic regions distributed in a periodic array and spaced from each other, each of the hydrophilic regions is surrounded by the hydrophobic region, and the hydrophilic region includes a hydrophilic structure; the hydrophilic structure is configured to be connected to the coding probe.

2. The spatial omics chip according to claim 1, wherein: Each of the hydrophilic regions includes a plurality of the hydrophilic structures distributed in a periodic array, the diameters of the different hydrophilic structures are all d1, and the distance between two adjacent hydrophilic structures in each of the hydrophilic regions is 0.25d1 to 1.5d1.

3. The spatial omics chip according to claim 2, wherein: The diameter d1 of the hydrophilic structure is 2 μm to 2.5 μm, and the height of the hydrophilic structure is 5 μm to 10 μm.

4. The spatial omics chip according to claim 2 or 3, wherein: The hydrophobic area includes a plurality of hydrophobic structures, and at least one hydrophobic structure is disposed between two adjacent hydrophilic areas; the diameters of the different hydrophobic structures are all d2, and the distance between two adjacent hydrophobic structures is 0.25d2 to 1.5d2, and d2 is 2 μm to 2.5 μm; The distance between the adjacent hydrophobic structures and the adjacent hydrophilic structures is d3.

5. The spatial omics chip according to claim 4, wherein: d1=d2, d3 is 0.25d1 to 1.5d1.

6. The spatial omics chip according to claim 4, wherein: The plurality of hydrophilic areas are distributed in a periodic array on the first substrate, and the distance between the centers of two adjacent hydrophilic areas is L1, and L1 is not less than 1.5d1.

7. The spatial omics chip according to claim 6, wherein: L1 is 4.446d1 to 9d1.

8. The spatial omics chip according to any one of claims 1 to 7, wherein: The hydrophilic structure is connected to the first substrate via a carbon-hydrogen bond; the surface of the hydrophilic structure contains active groups that can connect to the coding probe, and the active groups include any one or more of amino, thiol, carboxyl, ester and anhydride; each of the hydrophobic structures contains hydrophobic groups, and the hydrophobic groups include any one or more of long-chain hydrocarbon groups and fluorocarbon groups with a carbon chain length ≥ 7.

9. The spatial omics chip according to claim 8, further comprising the coding probe connected to the hydrophilic structure; the coding probe comprises any one or more of a chemical probe, a polypeptide probe, a fluorescent probe and a nucleic acid probe.

10. The spatial omics chip according to claim 9, wherein: The encoding probe is configured to capture an mRNA sample and mark the spatial location of the captured mRNA sample.

11. The spatial omics chip according to claim 10, wherein: The coding probe includes a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence and a Poly (dT) sequence, and the sequencing primer binding sequence, the spatial position tag sequence, the UMI sequence and the Poly (dT) sequence are connected in sequence; wherein, The sequencing primer binding sequence is configured to complementarily pair with the primer during sequencing; The spatial position tag sequence is configured to mark the spatial position of the captured mRNA sample, and the spatial position tag sequences of the coding probes connected to the hydrophilic structures located in the same hydrophilic region are the same, and the spatial position tag sequences of the coding probes connected to the hydrophilic structures located in different hydrophilic regions are different; the spatial position tag sequence includes a spatial position subtag sequence; The UMI sequence is configured to specifically label different mRNA samples; The Poly(dT) sequence is configured to capture the mRNA sample.

12. The spatial omics chip according to claim 11, wherein: The sequencing primer binding sequence contains a linking group, the sequencing primer binding sequence is connected to the hydrophilic structure through the linking group, and the linking group includes at least one of a carboxyl group, a thiol group and an amino group; Alternatively, the sequencing primer binding sequence and the hydrophilic structure are connected together through at least one of an amino group and a carboxyl group reaction, a thiol group and a thiol group reaction, and an anhydride and an amino group reaction; Alternatively, the sequencing primer binding sequence and the hydrophilic structure are connected together through at least one of an amide group and a disulfide bond.

13. The spatial omics chip according to any one of claims 1 to 12, comprising a plurality of sub-chips distributed at intervals, wherein the plurality of sub-chips are distributed in a periodic array on the first substrate, and the sub-chips include a plurality of the hydrophilic areas.

14. A rotary step-by-step sampling chip, configured to step-by-step sample injection into the hydrophilic region of the spatial omics chip according to any one of claims 1 to 13, the rotary step-by-step sampling chip comprising: A second substrate and a plurality of flow channels arranged on the same side of the second substrate and distributed at intervals, the flow channels having a plurality of through holes penetrating the second substrate and distributed in a periodic array, each of the hydrophilic areas of the spatial omics chip corresponding to a through hole, the distance between the centers of two adjacent through holes being the same as the distance between the centers of two adjacent hydrophilic areas corresponding to the two through holes, and the through holes being configured to inject samples step by step into each hydrophilic structure of the hydrophilic area corresponding to the through holes.

15. The rotary step-by-step sampling chip according to claim 14, wherein: The area of ​​the through hole is smaller than or equal to the area of ​​the corresponding hydrophilic region.

16. The rotary step-by-step sampling chip according to claim 15 comprises an effective sampling area, wherein the rotary step-by-step sampling chip rotates at least once around its center according to a set angle, and at least part of the through holes of the rotary step-by-step sampling chip before and after at least one rotation completely overlap, and the completely overlapping through holes form the effective sampling area, and when the spatial omics chip and the rotary step-by-step sampling chip are matched and used, the orthographic projection of the through holes in the effective sampling area on the first substrate overlaps with the orthographic projection of the hydrophilic area of ​​the spatial omics chip on the first substrate.

17. The rotary step-by-step sampling chip according to claim 16, wherein: The effective injection area is a regular polygon with n sides, where n is an integer ≥ 3; The distance between the center lines of two adjacent flow channels satisfies: L3 = L2 × sinθ; Wherein, L3 is the distance between two adjacent flow channels; L2 is the distance between the centers of two adjacent through holes; θ is the rotation angle of the rotary step-by-step sampling chip.

18. A method for synthesizing a spatial position tag sequence, comprising: The subspace position label sequence is injected multiple times through the through holes of the rotary step-by-step sampling chip according to any one of claims 14 to 17, and the rotary step-by-step sampling chip is rotated around its center by a set angle between two adjacent injections, and at least part of the through holes of the rotary step-by-step sampling chip before and after the rotation are completely overlapped, and the subspace position label sequence is injected into the hydrophilic area of ​​the spatial omics chip through the through holes in the overlapping area to form the spatial position label sequence; The subspace position label sequences of the multiple through holes injected on the same flow channel are the same, and the subspace position label sequences of the through holes injected on different flow channels are different.

19. The synthesis method according to claim 18, wherein Injecting the subspace position tag sequence into the hydrophilic area of ​​the spatial omics chip through the through hole of the overlapping area comprises: Laminating the rotary step-by-step sampling chip with the spatial omics chip according to any one of claims 1 to 13, so that the hydrophilic area of ​​the spatial omics chip corresponds to the through hole of the flow channel of the rotary step-by-step sampling chip; Different subspace position label sequences are respectively injected into different flow channels of the rotary step-by-step sampling chip. The subspace position label sequences enter the hydrophilic area of ​​the spatial omics chip through the through holes on the flow channels and connect with the nucleic acid sequences on the hydrophilic structure.

20. A method for manufacturing a spatial omics chip according to any one of claims 1 to 13, comprising: Using a first photomask having a first light-transmitting area and a first light-shielding area to cover the first substrate, wherein the first light-transmitting area completely overlaps with an orthographic projection of a region on the first substrate where the hydrophilic structure is to be formed; Under light conditions, using a hydrophilic monomer as a raw material, a graft polymerization reaction is performed on the first substrate in an area completely overlapping with the orthographic projection of the first light-transmitting area to form a hydrophilic polymer, wherein the hydrophilic polymer comprises an active group capable of connecting to the coding probe, and the hydrophilic polymer forms the hydrophilic structure; Using a second photomask having a second light-transmitting area and a second light-shielding area to cover the first substrate, wherein the second light-transmitting area completely overlaps with the orthographic projection of the area on the first substrate where the hydrophobic structure is to be formed; Under illumination conditions, a hydrophobic monomer is used as a raw material to carry out a graft polymerization reaction in an area on the first substrate that completely overlaps with the orthographic projection of the second light-transmitting area to form a hydrophobic polymer, wherein the hydrophobic polymer contains the hydrophobic group, the hydrophobic polymer forms a hydrophobic structure, and the hydrophobic structure forms the hydrophobic area.

21. The manufacturing method according to claim 20, further comprising sequentially connecting a sequencing primer binding sequence, a spatial position tag sequence, a UMI sequence and a Poly(dT) sequence at an end of the hydrophilic structure away from the first substrate; in, The spatial position tag sequence is formed by the synthesis method of the spatial position tag sequence according to claim 19 or 20.

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