Space omics analysis chips and their analysis methods

Abstract

This invention provides a space omics analysis chip, comprising a substrate and a cover plate. The substrate includes a first region; the cover plate includes a recessed region; the substrate and the cover plate are mated; the orthographic projections of the first region and the recessed region on the substrate overlap, and the mating of the first region and the recessed region forms a cavity; the substrate includes a control electrode array located in the first region, the control electrode array being used to control the reaction solution; a local area within the recessed region is used to accommodate a slice to be analyzed; the control electrode array is positioned corresponding to the accommodating area of ​​the slice to be analyzed; the cover plate includes a liquid inlet located in the area outside the accommodating area of ​​the slice to be analyzed within the recessed region, for inputting the reaction solution and a separating liquid; the cover plate also includes an auxiliary contact structure located in the accommodating area of ​​the slice to be analyzed, for assisting the reaction solution in contacting the slice to be analyzed. This space omics analysis chip reduces the crosstalk between droplets at different points, helping to improve the accuracy of space omics analysis results.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and molecular diagnostics, specifically relating to a space omics analysis chip and its analysis method. Background Technology

[0002] High-throughput spatial omics technologies that have been developed recently, such as 10x Genomics Visium and GeoMx DSP, can perform sequencing analysis on tissue cells in different locations, or obtain gene expression and spatial distribution data in situ simultaneously. The analysis of this type of data can reflect the influence of the relative spatial location of cells and the microenvironment on cell activity, and has broad application prospects in the fields of cancer, immunity, neurology, and development.

[0003] Existing space omics technologies are generally based on various chip platforms, which have complex chip structures and operations, high costs, and significant sample loss during analysis, thus limiting the application of space omics. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a space omics analysis chip, comprising a substrate and a cover plate;

[0005] The substrate includes a first region; the cover plate includes a recessed region; the substrate and the cover plate are mated; the orthographic projections of the first region and the recessed region on the substrate overlap, and the first region and the recessed region are mated to form a cavity;

[0006] The substrate includes a control electrode array located in the first region, the control electrode array being used to control the reaction solution;

[0007] The local area within the recessed region is used to accommodate the slice to be analyzed;

[0008] The control electrode array corresponds to the location of the accommodating region of the slice to be analyzed;

[0009] The cover plate includes a liquid inlet located in the recessed area outside the accommodating area of ​​the slice to be analyzed, for introducing the reaction solution and the isolation solution;

[0010] The cover plate also includes an auxiliary contact structure located in the accommodating area of ​​the slice to be analyzed, which is used to assist the reaction solution in contacting the slice to be analyzed.

[0011] Optionally, the cover plate further includes a second base.

[0012] The auxiliary contact structure includes a plurality of protrusions formed on the second substrate;

[0013] The plurality of protrusions correspond one-to-one with the positions of the control electrodes in the control electrode array;

[0014] The top of the protrusion is used to contact the slice to be analyzed and provide support for it.

[0015] Optionally, the maximum radial dimension of the top support surface of the protrusion is the same as the maximum radial dimension of the control electrode;

[0016] The height of the protrusion ranges from 0.2 to 0.5 mm.

[0017] Optionally, the shape of the protrusion includes cylindrical, prismatic, frustum-shaped, or frustum-shaped;

[0018] The protrusion is made of the same material as the second substrate.

[0019] Optionally, the cover plate further includes a second base.

[0020] The auxiliary contact structure includes an intermediate layer, the slice to be analyzed is used to be disposed on the second substrate, and the intermediate layer is used to be disposed on the side of the slice to be analyzed that is opposite to the second substrate;

[0021] The intermediate layer has multiple through holes;

[0022] The plurality of through holes correspond one-to-one with the positions of the control electrodes in the control electrode array.

[0023] Optionally, the maximum radial dimension of the through hole is the same as the maximum radial dimension of the control electrode;

[0024] The thickness of the intermediate layer ranges from 0.8 to 1.2 mm.

[0025] Optionally, the substrate further includes a first substrate, electrode traces, a first insulating layer, a second insulating layer, and a hydrophobic layer; the electrode traces, the first insulating layer, the second insulating layer, and the hydrophobic layer are at least distributed in the first region;

[0026] The electrode traces, the first insulating layer, the control electrode array, the second insulating layer, and the hydrophobic layer are sequentially stacked on the first substrate;

[0027] Each control electrode in the control electrode array is connected to the electrode trace through a via formed in the first insulating layer.

[0028] Optionally, the substrate further includes a plurality of first connecting electrodes, a plurality of second connecting electrodes, a plurality of first liquid storage electrodes, and a plurality of second liquid storage electrodes, located in the first region and on the same film layer as the control electrode array;

[0029] The plurality of first connecting electrodes extend out one-to-one along each row of the control electrode array;

[0030] The plurality of second connecting electrodes extend out one-to-one along each column direction of the control electrode array;

[0031] The plurality of first liquid storage electrodes are respectively distributed one-to-one at the ends of each first connecting electrode that are away from the control electrode array;

[0032] The plurality of second liquid storage electrodes are respectively distributed one-to-one at the ends of each second connecting electrode that are away from the control electrode array;

[0033] The first connecting electrode, the second connecting electrode, the first liquid storage electrode, and the second liquid storage electrode are respectively connected to the electrode traces through vias formed in the first insulating layer.

[0034] Optionally, the liquid inlet is a through hole formed in the second substrate;

[0035] The liquid inlet includes multiple first liquid inlets, multiple second liquid inlets, and a third liquid inlet;

[0036] The positions of the plurality of first liquid inlets correspond one-to-one with the positions of the plurality of first liquid storage electrodes;

[0037] The positions of the plurality of second liquid inlets correspond one-to-one with the positions of the plurality of second liquid storage electrodes;

[0038] The plurality of first inlets and the plurality of second inlets are used to input the reaction solution marked with a spatial barcode;

[0039] The third inlet is located on a different side of the accommodating area of ​​the slice to be analyzed, and is used to input the isolation fluid.

[0040] Optionally, each control electrode in the control electrode array includes multiple sub-control electrodes; the multiple sub-control electrodes are arranged in an array.

[0041] Each of the control electrodes is used to control one reaction droplet.

[0042] Optionally, the first connecting electrode includes a plurality of sub-connecting electrodes; the plurality of sub-connecting electrodes are arranged in an array or in a straight line;

[0043] The second connecting electrode includes a plurality of sub-connecting electrodes; the plurality of sub-connecting electrodes are arranged in an array or in a straight line.

[0044] Optionally, the substrate further includes a flow guide dam disposed on the first substrate, and the orthographic projection of the flow guide dam on the first substrate is located on opposite sides of each of the first connecting electrodes and each of the second connecting electrodes along their width direction;

[0045] The width direction of the first connecting electrode and the second connecting electrode is perpendicular to their extension direction;

[0046] The diversion dam is made of insulating material.

[0047] Optionally, the guide dam is strip-shaped, and the extension lengths of the first connecting electrode and the second connecting electrode are respectively equal to the length of the guide dam;

[0048] The height of the side surface of the diversion dam away from the first substrate is greater than the height of the side surfaces of the first connecting electrode and the second connecting electrode away from the first substrate.

[0049] Optionally, the cross-sectional shape of the diversion dam perpendicular to its length direction includes a rectangle or a trapezoid.

[0050] The present invention also provides an analysis method for the above-mentioned space omics analysis chip, comprising: placing the slice to be analyzed in the slice to be analyzed receiving area within the recessed area of ​​the cover plate, and then aligning the substrate with the cover plate and sealing its four edges; the first area of ​​the substrate and the recessed area of ​​the cover plate are aligned to form a cavity;

[0051] Isolation fluid is introduced into the cavity through the inlet;

[0052] The reaction solution is introduced into the cavity through the inlet, and the reaction solution is distributed to each site of the slice to be analyzed corresponding to each control electrode under the drive of the control electrode array; the auxiliary contact structure of the cover plate assists the reaction solution in contact with the slice to be analyzed;

[0053] Operate the control electrode array to combine the reaction solutions corresponding to each site of the slice to be analyzed;

[0054] Extract the reaction solution.

[0055] The beneficial effects of this invention are as follows: The space omics analysis chip provided by this invention has a simple structure and can complete the marking of space barcodes without switching the chip cover back and forth; on the other hand, the closed chip structure reduces the evaporation loss of the reaction liquid; at the same time, with the help of the electrowetting effect of the electrodes, the droplets are confined to the electrode area, reducing the crosstalk between droplets at different points and helping to improve the accuracy of space omics analysis results.

[0056] The spatial omics analysis chip method provided by this invention uses electrowetting and microfluidic technology to uniformly distribute reaction liquid with spatial barcode markers to various regions of the slice to be analyzed. The reaction droplets capture the cellular nucleic acid at each slice site, and can simultaneously record the location information and nucleic acid information of each site, thereby realizing the spatial omics analysis of the slice to be analyzed. The spatial omics analysis results can be used for subsequent experiments and data analysis in various spatial omics fields. Attached Figure Description

[0057] Figure 1a This is a schematic diagram of the structure of a substrate in a space omics analysis chip provided in an embodiment of the present invention;

[0058] Figure 1b This is a schematic diagram of the structure of a cover plate in a spatial omics analysis chip provided in an embodiment of the present invention;

[0059] Figure 1c This is a schematic diagram of the structure of a space omics analysis chip provided in an embodiment of the present invention;

[0060] Figure 1d For along Figure 1c Schematic diagram of the structure along the AA section line;

[0061] Figure 1e For along Figure 1c Enlarged cross-sectional view of the structure along the BB section line;

[0062] Figure 2 This is a schematic diagram of the structure in an embodiment of the present invention, showing the control electrode divided into multiple sub-control electrodes;

[0063] Figure 3a This is a schematic diagram of another cover plate in the spatial omics analysis chip provided in an embodiment of the present invention;

[0064] Figure 3b This is a schematic diagram of another spatial omics analysis chip provided in an embodiment of the present invention;

[0065] Figure 3c A top view of the structure of another space omics analysis chip provided in an embodiment of the present invention;

[0066] Figure 3d For along Figure 3c Enlarged cross-sectional view of the structure along the CC section line;

[0067] Figure 4a This is a schematic diagram of the structure of another cover plate in the spatial omics analysis chip provided in an embodiment of the present invention;

[0068] Figure 4b A top view of the structure of another space omics analysis chip provided in an embodiment of the present invention;

[0069] Figure 4c For along Figure 4b Enlarged cross-sectional view of the structure along the DD section line;

[0070] Figure 5a This is a schematic diagram of the structure of another substrate in the space omics analysis chip provided in an embodiment of the present invention;

[0071] Figure 5b A schematic diagram of the structure of another space omics analysis chip provided in an embodiment of the present invention;

[0072] Figure 5c A top view of the structure of another space omics analysis chip provided in an embodiment of the present invention;

[0073] Figure 5d This is a top view of the diversion dam structure in the space omics analysis chip provided in an embodiment of the present invention;

[0074] Figure 6 This is a schematic diagram of the spatial barcode marking process of reaction droplets in transcriptome analysis according to an embodiment of the present invention;

[0075] Figure 7 This is a schematic diagram illustrating the process of linking an oligonucleotide with spatial barcode marker B to an oligonucleotide with spatial barcode marker A in an embodiment of the present invention.

[0076] Figure 8 This is a schematic diagram of the spatial barcode marking process of reaction droplets in spatial genome analysis according to an embodiment of the present invention;

[0077] Figure 9 This is a schematic diagram of the spatial barcode marking process of reaction droplets in spatial proteomics analysis according to an embodiment of the present invention.

[0078] The reference numerals in the attached figures are:

[0079] 1. Substrate; 101. First region; 11. Control electrode; 110. Sub-control electrode; 12. First substrate; 13. Electrode trace; 14. First insulating layer; 15. Second insulating layer; 16. Hydrophobic layer; 17. First connecting electrode; 170. Sub-connecting electrode; 18. Second connecting electrode; 19. First liquid storage electrode; 10. Second liquid storage electrode; 102. Flow dam; 2. Cover plate; 201. Recessed area; 100. Cavity; 200. Reception area for the slice to be analyzed; 21. Liquid inlet; 20. Second substrate; 211. First liquid inlet; 212. Second liquid inlet; 213. Third liquid inlet; 22. Protrusion; 23. Intermediate layer; 230. Through hole. Detailed Implementation

[0080] To enable those skilled in the art to better understand the technical solution of the present invention, the following describes in further detail a space omics analysis chip and its analysis method in conjunction with the accompanying drawings and specific embodiments.

[0081] DNA (deoxyribonucleic acid) is the genetic material of living organisms. Genetic information is transmitted sequentially from DNA to RNA and proteins through the central dogma, regulating cellular life activities from different angles and ultimately influencing the phenotypic characteristics of the organism. However, the base sequence of DNA itself generally does not completely determine the final phenotype. Epigenetic modifications, transcription, translation, and the cellular microenvironment can also cause differences in cellular metabolites, further affecting phenotypic characteristics. Therefore, the rapid development of various omics research systems based on sequencing and mass spectrometry technologies, including genomics, transcriptomics, epigenomics, proteomics, and metabolomics, enables the analysis of the regulatory mechanisms of complex physiological processes at the molecular level, providing favorable prerequisites for basic research related to diseases and pharmaceutical development. However, single-omics data mainly rely on sequencing analysis of large numbers of mixed cells. While this can obtain the average genetic characteristics of these cells, it cannot reveal the genotypic characteristics of cells within the cell population, thus failing to reflect cellular heterogeneity. Furthermore, cellular gene expression is spatially specific, meaning that cells in different tissue sites exhibit expression differences due to variations in the microenvironment and intercellular interactions. Therefore, the analysis of mixed cell samples loses spatial information associated with expression, which is detrimental to the study of complex regulatory mechanisms.

[0082] High-throughput spatial omics technologies that have been developed recently, such as 10x Genomics Visium and GeoMx DSP, can perform sequencing analysis on tissue cells in different locations, or obtain gene expression and spatial distribution data in situ simultaneously. The analysis of this type of data can reflect the influence of the relative spatial location of cells and the microenvironment on cell activity, and has broad application prospects in the fields of cancer, immunity, neurology, and development.

[0083] Existing microfluidic chips used for space omics analysis have complex structures and operations. For example, the DBiT-Seq platform (Liu et al., 2020, Cell 183, 1665–1681) requires two different cover plates to be used with the substrate to assign barcodes for recording spatial information. At the same time, the chip needs to be placed in a certain humidity environment to prevent solution evaporation. Liquid leakage and crosstalk may also occur between the chip channels, which further increases the manufacturing and use costs of the chip.

[0084] To address the issues of complex structures and high costs associated with current space omics analysis chips, as well as significant sample loss during analysis, this invention provides a space omics analysis chip, such as... Figure 1a , Figure 1b , Figure 1c , Figure 1d and Figure 1e As shown, the device includes a substrate 1 and a cover plate 2. The substrate 1 includes a first region 101. The cover plate 2 includes a recessed region 201. The substrate 1 and the cover plate 2 are mated together. The orthographic projections of the first region 101 and the recessed region 201 on the substrate 1 overlap, and the mating of the first region 101 and the recessed region 201 forms a cavity 100. The substrate 1 includes an array of control electrodes 11 located in the first region 101, which is used to control the reaction solution. A local area within the recessed region 201 is used to accommodate the slice to be analyzed. The array of control electrodes 11 corresponds to the accommodating area 200 of the slice to be analyzed. The cover plate 2 includes a liquid inlet 21 located in the area outside the accommodating area 200 of the slice to be analyzed within the recessed region 201, which is used to input the reaction solution and the isolation solution. The cover plate also includes an auxiliary contact structure located in the accommodating area 200 of the slice to be analyzed, which is used to assist the reaction solution in contacting the slice to be analyzed.

[0085] The tissue section to be analyzed can be a tissue section from any part or location of a biological organism. The cavity 100 provides a space for the reaction solution to contact and react with the tissue section to be analyzed. The cover plate 2 can be made of plastic, such as easily processed materials like PDMS or PC.

[0086] Optionally, both the cover plate 2 and the substrate 1 are rectangular plates; the length × width × thickness of the cover plate 2 is 80mm × 80mm × 1.5mm; the length × width × thickness of the substrate 1 is 80mm × 80mm × 3mm. The cavity 100 can be a cuboid shape, and the length × width × height of the cavity 100 can be 70mm × 70mm × 1mm. Of course, the cavity 100 can also be a cylinder, prism, or other shapes. The cover plate 2 and the substrate 1 can also be other shapes, which are not limited here.

[0087] Optionally, the area of ​​the receiving area 200 of the slice to be analyzed on the cover plate 2 is approximately the same as the overall area of ​​the array of control electrodes 11. For example, the receiving area 200 of the slice to be analyzed is a cuboid area recessed on the cover plate 2, and the length × width × height of the cuboid area can be 28mm × 28mm × 0.1mm.

[0088] Optionally, such as Figure 1eAs shown, the substrate 1 further includes a first substrate 12, electrode traces 13, a first insulating layer 14, a second insulating layer 15, and a hydrophobic layer 16; the electrode traces 13, the first insulating layer 14, the second insulating layer 15, and the hydrophobic layer 16 are at least distributed in the first region 101; the electrode traces 13, the first insulating layer 14, the array of control electrodes 11, the second insulating layer 15, and the hydrophobic layer 16 are sequentially stacked on the first substrate 12; each control electrode 11 in the array of control electrodes 11 is connected to the electrode traces 13 through vias formed in the first insulating layer 14.

[0089] The first substrate 12 can be made of glass or other rigid materials. The electrode traces 13 and the control electrode 11 use the same conductive material, such as Mo / Al or other materials with good electrical properties. The electrode traces 13 are used to input electrical signals to the control electrode 11, thereby creating an electric field on the control electrode 11. This allows for morphological control of the reaction droplet P on the control electrode 11, facilitating contact and reaction between the reaction droplet P and the analyte slice Q, and ultimately enabling the analysis of the analyte slice Q by the space omics analysis chip. The hydrophobic layer 16 is in direct contact with the reaction liquid, ensuring that the reaction droplet P is well controlled within the corresponding region of the control electrode 11.

[0090] Optionally, the control electrodes 11 in the array of control electrodes 11 are rectangular blocks, with a length × width × thickness of 2mm × 2mm × 0.1mm, and a spacing of 0.2mm between adjacent edges of any two adjacent control electrodes 11. It should be noted that the control electrodes 11 can also be other shapes such as circular blocks or regular polygonal blocks.

[0091] Optionally, the number of control electrodes 11 in the control electrode array can change as the measurement sites of the slice to be analyzed change. For example, the control electrode array can be a 12×12 array, or a smaller array (such as an 8×8 or 6×6 array) or a larger array (such as a 16×16 or 20×20 array).

[0092] Optionally, such as Figure 2 As shown, each control electrode 11 in the array of control electrodes 11 includes multiple sub-control electrodes 110; the multiple sub-control electrodes 110 are arranged in an array; each control electrode 11 is used to control a reaction droplet P.

[0093] Each control electrode 11 is divided into multiple sub-control electrodes 110. With this configuration, the driving of the reaction liquid can be changed from being controlled by a single control electrode 11 to being controlled by multiple sub-control electrodes 110. This increases the flexibility of manipulating the reaction droplet P and makes it easier to more effectively limit the movement range of the reaction droplet P when needed. At the same time, it can also increase the number of analytical sites on the slice to be analyzed, thereby improving the accuracy of the analysis results of the slice to be analyzed by the spatial omics analysis chip.

[0094] Optionally, such as Figure 1a As shown, the substrate 1 also includes a plurality of first connecting electrodes 17, a plurality of second connecting electrodes 18, a plurality of first liquid storage electrodes 19, and a plurality of second liquid storage electrodes 10, located in the first region 101 and on the same film layer as the control electrode 11 array; the plurality of first connecting electrodes 17 extend out along each row direction of the control electrode 11 array respectively; the plurality of second connecting electrodes 18 extend out along each column direction of the control electrode 11 array respectively; the plurality of first liquid storage electrodes 19 are distributed at the ends of each first connecting electrode 17 away from the control electrode 11 array respectively; the plurality of second liquid storage electrodes are distributed at the ends of each second connecting electrode 18 away from the control electrode 11 array respectively; the first connecting electrodes 17, second connecting electrodes 18, first liquid storage electrodes 19, and second liquid storage electrodes 10 are connected to electrode traces 13 through vias formed in the first insulating layer 14.

[0095] The first liquid storage electrode 19 and the second liquid storage electrode 10 are used to store the input reaction droplets; the first connecting electrode 17 and the second connecting electrode 18 are used to transport the reaction droplets stored above the first liquid storage electrode 19 and the second liquid storage electrode 10 to the corresponding areas of the control electrode 11 array, respectively. There can be one or more electrode traces 13, and all electrode traces 13 input the same electrical signal during the spatial omics analysis chip analysis process. The first connecting electrode 17, the second connecting electrode 18, the first liquid storage electrode 19, the second liquid storage electrode 10, and the control electrode 11 array can be formed through a single patterning process; the electrode traces 13 can also be formed through a single patterning process.

[0096] Optionally, the first connecting electrode 17 includes a plurality of sub-connecting electrodes 170; the plurality of sub-connecting electrodes 170 are arranged in an array or in a straight line; the second connecting electrode 18 includes a plurality of sub-connecting electrodes 170; the plurality of sub-connecting electrodes 170 are arranged in an array or in a straight line. The first connecting electrode 17 and the second connecting electrode 18, which are composed of a plurality of sub-connecting electrodes 170, can control the reaction liquid in the corresponding regions of the first liquid storage electrode 19 and the second liquid storage electrode 10 to be uniformly distributed to the corresponding region of each control electrode 11 in the array of control electrodes 11, thereby achieving uniform distribution of the reaction liquid to each site of the slice to be analyzed.

[0097] Optionally, each sub-connecting electrode 170 has the same shape and size. The sub-connecting electrode 170 is rectangular, for example, its length × width × thickness is 2mm × 2mm × 0.1mm. The interval between adjacent edges of any two adjacent sub-connecting electrodes 170 on the first connecting electrode 17 or the second connecting electrode 18 is 0.2mm. The first liquid storage electrode 19 and the second liquid storage electrode 10 have the same shape and size. For example, both the first liquid storage electrode 19 and the second liquid storage electrode 10 are rectangular, and their length × width × thickness is 4mm × 4mm × 0.1mm.

[0098] In this embodiment, the hydrophobic layer 16, in conjunction with the control electrode 11, enables electrowetting control of the reaction solution. The first connecting electrode 17, the second connecting electrode 18, the first reservoir electrode 19, the second reservoir electrode 10, and the control electrode 11 control the input and output of the reaction solution, achieving microfluidic control of the reaction solution. Through electrowetting and microfluidic technology, the reaction solution with spatial barcode markings is evenly distributed to various sites on the slice to be analyzed, thereby enabling spatial omics analysis of the slice.

[0099] Optionally, such as Figure 1b As shown, the cover plate 2 includes a second base 20, and the liquid inlet 21 is a through hole opened in the second base 20; the liquid inlet 21 includes a plurality of first liquid inlets 211, a plurality of second liquid inlets 212 and a third liquid inlet 213; the plurality of first liquid inlets 211 correspond one-to-one with the positions of a plurality of first liquid storage electrodes 19; the plurality of second liquid inlets 212 correspond one-to-one with the positions of a plurality of second liquid storage electrodes 10; the plurality of first liquid inlets 211 and the plurality of second liquid inlets 212 are used to input the reaction liquid marked with spatial barcode; the third liquid inlet 213 is located on a different side of the accommodating area 200 of the slice to be analyzed, and is used to input the isolation liquid.

[0100] Optionally, six (e.g., A1 to A6) first liquid inlets 211 correspond one-to-one with the positions of six first liquid storage electrodes 19 arranged along the row direction of the control electrode 11 array; six (e.g., B1 to B6) second liquid inlets 212 correspond one-to-one with the positions of six second liquid storage electrodes 10 arranged along the column direction of the control electrode 11 array; a single third liquid inlet 213 is located on the other side of the control electrode 11 array. After the reaction solution is input through the first liquid inlet 211 and the second liquid inlet 212, it is stored in the corresponding areas of the first liquid storage electrodes 19 and the second liquid storage electrodes 10, respectively; then, under the action of the electric field after the first connecting electrode 17, the second connecting electrode 18 and the control electrode 11 array are energized, the reaction solution is uniformly distributed to each site corresponding to the control electrode 11 in the control electrode 11 array via the first connecting electrode 17 and the second connecting electrode 18, thereby achieving uniform distribution of the reaction solution to each site of the slice to be analyzed.

[0101] Optionally, the first liquid inlet 211, the second liquid inlet 212, and the third liquid inlet 213 have the same specifications. For example, all three liquid inlets 21 are through holes with a diameter of 2 mm and a depth of 2 mm, which are opened in the second substrate 20.

[0102] Optionally, such as Figure 3a , Figure 3b , Figure 3c and Figure 3d As shown, the auxiliary contact structure includes multiple protrusions 22, which are formed on the second substrate 20. The multiple protrusions 22 correspond one-to-one with the positions of the control electrodes 11 in the array of control electrodes 11. The top of the protrusion 22 is used to contact the slice to be analyzed and provide support for it.

[0103] In this way, when the reaction solution passes through the protrusion 22, it is easier to make full contact with the section to be analyzed at that location, while reducing the probability of contact between the reaction solution and other parts of the section to be analyzed. At the same time, the interval between adjacent protrusions 22 can also limit the reaction droplets, improve or avoid analytical crosstalk between adjacent sites on the section to be analyzed, and make the analysis results more accurate.

[0104] Optionally, the maximum radial dimension of the top support surface of the protrusion 22 is the same as the maximum radial dimension of the control electrode 11; the height of the protrusion 22 ranges from 0.2 to 0.5 mm. Optionally, the area of ​​the top support surface of the protrusion 22 is the same as the area of ​​the control electrode 11 facing the cover plate 2.

[0105] Optionally, the protrusion 22 may be cylindrical, prismatic, frustum-shaped, or truncated pyramidal; the protrusion 22 and the second base 20 may be made of the same material. The protrusion 22 may be achieved by injection molding or molding.

[0106] If protrusion 22 is cylindrical, the diameter of the cylinder is the same as the diagonal dimension of control electrode 11 (e.g., 2 mm). The slice to be analyzed can be placed over the surface of protrusion 22.

[0107] Optionally, such as Figure 4a , Figure 4b and Figure 4c As shown, the auxiliary contact structure includes an intermediate layer 23, on which the slice to be analyzed is disposed on the second substrate 20. The intermediate layer 23 is disposed on the side of the slice to be analyzed that is away from the second substrate 20. Multiple through holes 230 are provided on the intermediate layer. The multiple through holes 230 correspond one-to-one with the positions of the control electrodes 11 in the array of control electrodes 11.

[0108] By setting an intermediate layer 23 between the slice to be analyzed and the substrate 1, the reaction droplets in the cavity 100 between the slice to be analyzed and the substrate 1 can be physically separated. At the same time, the array of through holes 230 on the intermediate layer 23 corresponding to the array of control electrodes 11 can ensure that the reaction droplets and the exposed sites of the slice to be analyzed at the through holes 230 can make full contact, avoiding the reaction liquid from contaminating other areas of the slice to be analyzed other than the exposed sites. This improves or avoids analytical crosstalk between adjacent sites on the slice to be analyzed, making the analytical results more accurate.

[0109] Optionally, the area of ​​the intermediate layer 23 can be the same as the area of ​​the accommodating region 200 of the slice to be analyzed. For example, if the intermediate layer 23 is rectangular, the area of ​​the intermediate layer 23 is 28mm × 28mm.

[0110] Optionally, the maximum radial dimension of the through hole 230 is the same as the maximum radial dimension of the control electrode 11; the thickness of the intermediate layer 23 ranges from 0.8 to 1.2 mm. Optionally, the cross-sectional area of ​​the through hole 230 is the same as the area of ​​the control electrode 11 facing the cover plate 2.

[0111] Optionally, the cross-sectional shape of the through hole 230 includes circles, rectangles, regular polygons, etc.

[0112] Optionally, the intermediate layer 23 can be made of inert polymer plastics such as PC, PMMA, or PDMS. The intermediate layer 23 can be encapsulated with the second substrate 20 around its perimeter by means of surface plasma treatment, UV curing adhesive bonding, or other methods. Through holes 230 in the intermediate layer 23 can be formed by drilling.

[0113] Optionally, such as Figure 5a , Figure 5b , Figure 5c and Figure 5d As shown, the substrate 1 also includes a flow dam 102 disposed on the first substrate 12, and the orthographic projection of the flow dam 102 on the first substrate 12 is located on opposite sides of each first connecting electrode 17 and each second connecting electrode 18 along their width direction; the width direction of the first connecting electrode 17 and the second connecting electrode 18 is perpendicular to their extension direction; the flow dam 102 is made of insulating material.

[0114] Optionally, the flow guide dam 102 is strip-shaped, with the extension lengths of the first connecting electrode 17 and the second connecting electrode 18 being equal to the length of the flow guide dam 102; the height of the surface of the flow guide dam 102 away from the first substrate 12 is greater than the height of the surfaces of the first connecting electrode 17 and the second connecting electrode 18 away from the first substrate 12. This configuration creates a groove-shaped structure between the first connecting electrode 17 and the flow guide dam 102 located on opposite sides of its width direction, and simultaneously creates a groove-shaped structure between the second connecting electrode 18 and the flow guide dam 102 located on opposite sides of its width direction.

[0115] Optionally, the cross-sectional shape of the diversion dam 102 perpendicular to its length direction includes a rectangle or a trapezoid.

[0116] The flow guide dam 102 can guide and limit the flow of the reaction liquid, ensuring that the reaction droplets passing through each first connecting electrode 17 and each second connecting electrode 18 are of uniform size. This ensures that the processing conditions of each point in each region of the slice to be analyzed are consistent, eliminates the influence of non-parallel reaction systems of each reaction droplet, reduces the possibility of cross-contamination of the reaction liquid, and improves the accuracy of analysis of each point in the slice to be analyzed.

[0117] Optionally, the width of the diversion dam 102 ranges from 1.5 to 1.8 mm, and the height ranges from 0.5 to 0.8 mm.

[0118] Optionally, the flow guide dam 102 is made of silicon oxide or silicon nitride. Optionally, the flow guide dam 102 is fabricated by a process of film formation, exposure, development, and dry etching.

[0119] This space omics analysis chip has several advantages. First, its simple structure eliminates the need to switch chip covers repeatedly to mark spatial barcodes. Second, its enclosed chip structure reduces evaporation loss of the reaction solution. Third, the electrowetting effect of the electrodes confines droplets within the electrode region, reducing crosstalk between droplets and improving the accuracy of space omics analysis results.

[0120] Based on the above structure of the space omics analysis chip, this embodiment of the invention also provides an analysis method for the space omics analysis chip, including: preparing a substrate and a cover plate respectively;

[0121] Step S1: Place the slice to be analyzed in the slice receiving area within the recessed area of ​​the cover plate, then align the substrate with the cover plate and seal its four edges; the first area of ​​the substrate aligns with the recessed area of ​​the cover plate to form a cavity.

[0122] In this step, the assembled cover plate and the perimeter of the substrate are sealed and encapsulated using methods such as UV-curing adhesive, double-sided adhesive, or plasma treatment. Plasma treatment causes the substrate material to form functional groups under plasma conditions, which then react with the cover plate to achieve bonding and encapsulation. Details will not be elaborated further.

[0123] Step S2: Inject isolation fluid into the cavity through the inlet.

[0124] In this step, an oil phase is introduced into the cavity from the third inlet to seal the reaction environment and separate the reaction liquid (i.e., to separate the interval area between the control electrodes) until the cavity is full. The oil phase component can be an inert fluoroalkane.

[0125] Step S3: The reaction solution is introduced into the cavity through the inlet. Driven by the control electrode array, the reaction solution is distributed to each site of the slice to be analyzed corresponding to each control electrode. The auxiliary contact structure of the cover plate assists the reaction solution in contact with the slice to be analyzed.

[0126] In this step, for example, a pipette tip or syringe is used to introduce the reaction solution containing surfactant components and oligonucleotides with spatial barcode markers A1-A6 into six first reservoir electrodes through six first inlet ports. Driven by the first connecting electrode and control electrode, the solution splits into droplets and is evenly distributed to various sites on the slide to be analyzed. The surfactant promotes cell lysis at each site on the slide, and the oligonucleotides with spatial barcode markers A1-A6 are used to hybridize and capture mRNA (messenger ribonucleic acid) in the cells. The reaction flow is as follows: Figure 6 As shown.

[0127] After the previous reaction is completed, the reaction solution containing oligonucleotides (with biotin labeling) bearing spatial barcodes B1 to B6 is introduced into six second reservoir electrodes through six second inlets in the same manner. Driven by the second connecting electrode and the control electrode, the solution is distributed to the predetermined sites on the slide to be analyzed and merged with the droplets from the previous step. The purpose of this step is to link the oligonucleotides bearing spatial barcode B with the oligonucleotides bearing spatial barcode A, so that the aforementioned mRNA (messenger ribonucleic acid) carries the specific AB spatial barcode label. The droplet flow is as follows: Figure 7 As shown.

[0128] Step S4: Operate the control electrode array to combine the reaction solutions corresponding to each site of the slice to be analyzed.

[0129] In this step, after the reaction in the above steps is completed, the control electrode is operated to merge all the droplets.

[0130] Step S5: Extract the reaction solution.

[0131] In this step, the merged droplets are moved to any one of the reservoir electrodes, and the aqueous phase solution in the droplets is removed by means of a syringe or other means; or all the solution in the cavity is aspirated, and after the solution is separated into layers, the aqueous phase solution is taken (that is, the droplets after the reaction are removed from the oil phase); the collected reaction solution is used to capture the biotin-labeled oligonucleotides with spatial barcodes that have hybridized with the mRNA using streptavidin-labeled magnetic beads, and can then be used for subsequent routine operations, such as transcriptome library preparation, for next-generation sequencing.

[0132] In this embodiment, the analysis process of the spatial omics analysis chip used for spatial genome (targeted capture sequence) analysis is as follows: (1) Prepare the slice to be analyzed and place it on the cover plate to accommodate the slice to be analyzed. Then, the cover plate and the substrate are bonded together by UV curing adhesive, double-sided adhesive or plasma treatment.

[0133] (2) Introduce the oil phase, which is used to seal the reaction environment and separate the droplet units, into the cavity through the third inlet. Then, add the reaction solution, such as surfactant and protease, for lysis through any of the first or second inlets. Driven by the connected electrodes and control electrodes, the reaction droplets are evenly distributed to each analysis site of the slide to be analyzed. After the reaction has been going on for a period of time, the liquid in the cavity is emptied and the oil phase is added again until the cavity is full. The purpose of this step is to perform preliminary tissue cell lysis and also to avoid the reaction solution from affecting the subsequent PCR reaction (nucleic acid amplification reaction).

[0134] (3) Using a pipette tip or syringe, the targeted primer pair solution with spatial barcode markers (A1-A6) is introduced into six first reservoir electrodes through six first inlet ports (each first reservoir electrode has a different spatial barcode sequence). Driven by the first connecting electrode and the control electrode, the solution splits into droplets and is evenly distributed to various sites on the slide to be analyzed, performing one round of PCR reaction (nucleic acid amplification reaction). The primers here are used to amplify specific sequences in the genome of tissue cells; the reaction procedure is as follows: Figure 8 As shown;

[0135] (4) After the previous reaction is completed, the reaction solution containing oligonucleotides (with biotin label) with spatial barcode markers B1 to B6 is distributed to each point of the slice to be analyzed in the same way. The oligonucleotides with spatial barcode marker B are linked with the oligonucleotides with spatial barcode marker A, so that the aforementioned amplified sequence is marked with specific AB spatial barcode markers.

[0136] (5) After the reaction is complete, operate the control electrode to combine all droplets and move them to any one of the storage electrodes. The aqueous solution is then removed by means of a syringe or other means; or all the solution in the cavity is aspirated and the aqueous solution is taken after the solution is separated into layers. The collected reaction solution is used to capture biotin-labeled oligonucleotides with streptavidin-labeled magnetic beads. After that, subsequent routine operations can be performed, such as the preparation of targeted capture sequencing libraries for next-generation gene sequencing.

[0137] In this embodiment, the analysis process of the spatial omics analysis chip used for spatial epigenomics (methylation) analysis is the same as in the above embodiments. The same fragment amplification method is used to enrich the targeted fragments, and spatial barcode marking is performed with the help of the spatial omics analysis chip. The difference is that after obtaining the amplified DNA (deoxyribonucleic acid) fragments, the subsequent methylation transformation and library preparation experiments are carried out using methylation library preparation kits for next-generation sequencing.

[0138] In this embodiment, spatial proteomics analysis can also be performed using this spatial omics analysis chip. The labeling process for the reaction droplets is as follows: Figure 9 As shown. The main experimental procedure is similar to the above embodiment, but the reactants are different. The reaction droplets introduced through the six first inlets mainly contain antibodies labeled with oligonucleotides bearing spatial barcodes A1 to A6. These antibodies can specifically bind to the antigen proteins to be analyzed within the cells, thus capturing the proteins. The reaction droplets introduced through the six second inlets mainly contain oligonucleotides labeled with biotin bearing spatial barcodes B1 to B6, which are used to further ligate and form spatial barcode labels AB. After the reaction, the labeled proteins are collected using streptavidin magnetic beads and can then be used in subsequent mass spectrometry analysis for proteomics analysis.

[0139] The spatial omics analysis chip provided in this embodiment uses electrowetting and microfluidic technology to uniformly distribute the reaction solution with spatial barcode markers to various regions of the slice to be analyzed. The reaction droplets capture the cellular nucleic acid at each slice site, and can simultaneously record the location information and nucleic acid information of each site, thereby realizing the spatial omics analysis of the slice to be analyzed. The spatial omics analysis results can be used for subsequent experiments and data analysis in various spatial omics fields.

[0140] It is understood that the above embodiments are merely exemplary implementations used to illustrate the principles of the present invention, and the present invention is not limited thereto. For those skilled in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A space omics analysis chip, comprising a substrate and a cover plate; The substrate includes a first region; the cover plate includes a recessed region; the substrate and the cover plate are mated; the orthographic projections of the first region and the recessed region on the substrate overlap, and the first region and the recessed region are mated to form a cavity; The substrate includes a control electrode array located in the first region, the control electrode array being used to control the reaction solution; The local area within the recessed region is used to accommodate the slice to be analyzed; The control electrode array corresponds to the location of the accommodating region of the slice to be analyzed; The cover plate includes a liquid inlet located in the recessed area outside the accommodating area of ​​the slice to be analyzed, for introducing the reaction solution and the isolation solution; The cover plate also includes a second base. The liquid inlet is a through hole formed in the second substrate; the liquid inlet includes a plurality of first liquid inlets, a plurality of second liquid inlets, and a third liquid inlet; The plurality of first inlets and the plurality of second inlets are used to input the reaction solution marked with a spatial barcode; The third inlet is located on a different side of the accommodating area of ​​the slice to be analyzed, and the third inlet is used to input the isolation liquid into the cavity; The isolation fluid is used to seal the reaction environment and separate the interval area between the control electrodes, and the isolation fluid fills the cavity. Its features are, The cover plate also includes an auxiliary contact structure located in the accommodating area of ​​the slice to be analyzed, which is used to assist the reaction solution in contacting the slice to be analyzed; The auxiliary contact structure includes a plurality of protrusions formed on the second substrate; The plurality of protrusions correspond one-to-one with the positions of the control electrodes in the control electrode array; The top of the protrusion is used to contact the slice to be analyzed and to provide support for it; Alternatively, the auxiliary contact structure includes an intermediate layer, the slice to be analyzed is disposed on the second substrate, and the intermediate layer is disposed on the side of the slice to be analyzed that is opposite to the second substrate; The intermediate layer has multiple through holes; The plurality of through holes correspond one-to-one with the positions of the control electrodes in the control electrode array.

2. The space omics analysis chip according to claim 1, characterized in that, The maximum radial dimension of the top support surface of the protrusion is the same as the maximum radial dimension of the control electrode; The height of the protrusion ranges from 0.2 to 0.5 mm.

3. The space omics analysis chip according to claim 2, characterized in that, The shape of the protrusion includes cylindrical, prismatic, frustum-shaped, or frustum-shaped; The protrusion is made of the same material as the second substrate.

4. The space omics analysis chip according to claim 1, characterized in that, The maximum radial dimension of the through hole is the same as the maximum radial dimension of the control electrode; The thickness of the intermediate layer ranges from 0.8 to 1.2 mm.

5. The space omics analysis chip according to claim 1, characterized in that, The substrate further includes a first substrate, electrode traces, a first insulating layer, a second insulating layer, and a hydrophobic layer; the electrode traces, the first insulating layer, the second insulating layer, and the hydrophobic layer are at least distributed in the first region; The electrode traces, the first insulating layer, the control electrode array, the second insulating layer, and the hydrophobic layer are sequentially stacked on the first substrate; Each control electrode in the control electrode array is connected to the electrode trace through a via formed in the first insulating layer.

6. The space omics analysis chip according to claim 5, characterized in that, The substrate further includes a plurality of first connecting electrodes, a plurality of second connecting electrodes, a plurality of first liquid storage electrodes, and a plurality of second liquid storage electrodes, located in the first region and on the same film layer as the control electrode array; The plurality of first connecting electrodes extend out one-to-one along each row of the control electrode array; The plurality of second connecting electrodes extend out one-to-one along each column direction of the control electrode array; The plurality of first liquid storage electrodes are respectively distributed one-to-one at the ends of each first connecting electrode that are away from the control electrode array; The plurality of second liquid storage electrodes are respectively distributed one-to-one at the ends of each second connecting electrode that are away from the control electrode array; The first connecting electrode, the second connecting electrode, the first liquid storage electrode, and the second liquid storage electrode are respectively connected to the electrode traces through vias formed in the first insulating layer.

7. The space omics analysis chip according to claim 6, characterized in that, The positions of the plurality of first liquid inlets correspond one-to-one with the positions of the plurality of first liquid storage electrodes; The positions of the plurality of second liquid inlets correspond one-to-one with the positions of the plurality of second liquid storage electrodes.

8. The space omics analysis chip according to claim 5, characterized in that, Each control electrode in the control electrode array includes multiple sub-control electrodes; the multiple sub-control electrodes are arranged in an array. Each of the control electrodes is used to control one reaction droplet.

9. The space omics analysis chip according to claim 6, characterized in that, The first connecting electrode includes a plurality of sub-connecting electrodes; the plurality of sub-connecting electrodes are arranged in an array or in a straight line; The second connecting electrode includes a plurality of sub-connecting electrodes; the plurality of sub-connecting electrodes are arranged in an array or in a straight line.

10. The space omics analysis chip according to claim 6, characterized in that, The substrate further includes a flow guide dam disposed on the first substrate, and the orthographic projection of the flow guide dam on the first substrate is located on opposite sides of each of the first connecting electrodes and each of the second connecting electrodes along their width direction. The width direction of the first connecting electrode and the second connecting electrode is perpendicular to their extension direction; The diversion dam is made of insulating material.

11. The space omics analysis chip according to claim 10, characterized in that, The guide dam is strip-shaped, and the extension lengths of the first connecting electrode and the second connecting electrode are respectively equal to the length of the guide dam; The height of the side surface of the diversion dam away from the first substrate is greater than the height of the side surfaces of the first connecting electrode and the second connecting electrode away from the first substrate.

12. The space omics analysis chip according to claim 11, characterized in that, The cross-sectional shape of the diversion dam perpendicular to its length direction includes rectangular or trapezoidal.

13. An analysis method for a space omics analysis chip as described in any one of claims 1-12, characterized in that, include: The slice to be analyzed is placed in the slice receiving area within the recessed area of ​​the cover plate, and then the substrate and the cover plate are aligned and their four edges are sealed and encapsulated; the first area of ​​the substrate and the recessed area of ​​the cover plate are aligned to form a cavity; Isolation fluid is introduced into the cavity through the third inlet; The reaction solution is introduced into the cavity through the first and second inlets. Driven by the control electrode array, the reaction solution is distributed to each site of the slice to be analyzed corresponding to each control electrode. The auxiliary contact structure of the cover plate assists the reaction solution in contacting the slice to be analyzed. Operate the control electrode array to combine the reaction solutions corresponding to each site of the slice to be analyzed; Extract the reaction solution.

Images