A microarray method for tissue samples

Abstract

The present invention is applicable to the field of biological materials, and provides a method for microarraying of tissue samples, comprising the following steps: placing a microwell array substrate modified with a high-density functional coating and a tissue in a slicer, wherein the functional coating in the microwell is used to adhere the tissue array to the side wall or bottom of the microwell, attaching the tissue slice to the surface of the microwell array substrate, removing the tissue outside the microwell array, and the tissue at the microwell is trapped in situ in the microwell array to obtain a high-throughput tissue microarray. The tissue microarraying method kit mainly comprises a microwell array substrate modified with a high-density functional coating, and the kit of the present invention can divide the tissue sample into independent micron-level tissue blocks, realize high-throughput arraying of the tissue sample, avoid cross-contamination of nucleic acid, protein and other information between tissue microarrays, simplify the operation steps while improving the efficiency of tissue segmentation, and reduce the instrument cost of the entire process.

Description

Technical Field

[0001] The invention belongs to the field of biological materials, and in particular relates to a microarray method for tissue samples. Background Art

[0002] Tissue sectioning refers to a method of processing biological tissue into thin slices and attaching them to the surface of a glass slide to be suitable for microscopic observation. This tissue sample processing technology is an important means for the development of histology and identification of spatial structures, and has been widely used in biology, medicine, agronomy and other fields. There is obvious heterogeneity between tissues, and the relationship between the genome, proteome, cell phenotype and other information of the tissue and its spatial position in the tissue section directly maps the growth, health and pathological state of the tissue. Therefore, it is particularly important to develop technology that can obtain information such as genes and proteins in tissue sections in situ.

[0003] Traditional methods for measuring information in tissues are based on tissue blocks, which reflect the average level of information such as the genome, transcriptome, and proteome of the entire tissue. At present, there are many methods for constructing single-cell arrays, which disperse cell clusters in tissues into free single cells, and then use devices to arrange the free cells. For example, single-cell sequencing is based on free cells after tissue digestion. This sequencing result is not suitable for reflecting the spatial location information of tissue gene expression. The method currently widely used to obtain micron-level target tissue arrays in situ is laser capture microdissection (LCM). This technology can obtain target tissue microarrays in situ without destroying the spatial structure of the tissue, and can also mark the original position of the obtained tissue. This technology has a high operating resolution and can even extract single target cells of interest in the tissue in situ. However, the LCM operating system generally relies on large-scale precision machinery and equipment, with high instrument and operating costs, and the technology requires sampling of target cells one by one, and the throughput of obtaining target cells is low. Summary of the invention

[0004] The purpose of the embodiments of the present invention is to provide a microarray method for tissue samples, aiming to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for microarraying of tissue samples, comprising the following steps:

[0007] Step 1: preparing a microwell array substrate modified with a high-density functional coating, wherein the functional coating in the microwell adheres the tissue to the side wall or bottom of the microwell;

[0008] Step 2: attach the tissue slices to the surface of the microwell array substrate, and the tissues at the microwells are retained in situ in the microwell array to obtain a high-throughput tissue microarray.

[0009] Furthermore, in step 2, after the tissue slices are attached to the surface of the microwell array substrate, pressure is applied to the tissue slices by an instrument to split the tissue slices or to make the tissue slices sink, and the tissue slices on the surface of the substrate outside the microwell array are removed by the instrument.

[0010] Furthermore, the specific preparation operation of the microwell array substrate modified with a high-density functional coating is: using photolithography technology to prepare a photoresist array, then etching the surface of the photoresist array by an etching method to obtain a microwell structure array, removing the photoresist, using a high-density functional coating modification technology to modify the surface in the microwell, then using photolithography technology to form photoresist on the surface of the functional coating, using chemical modification technology to hydrophobically modify the surface outside the microwell array, and removing the photoresist on the surface of the microwell.

[0011] Furthermore, the specific preparation operation of the microwell array substrate modified with a high-density functional coating can be: spin-coating a layer of photoresist on the substrate surface, performing a photolithography process based on a transparent dot matrix photolithography mask to leave a photoresist array, and then 4 F 8 , SF 6 , O 2 The surface of the photoresist array is bombarded with plasma to obtain a micro-well structure array with vertical edges, and then O 2 The micro-well array substrate is bombarded with plasma, and the surface in the micro-well is modified by using high-density functional coating modification technology. Then a layer of photoresist is spin-coated on the surface. Based on a non-transparent dot matrix photolithography mask, the photoresist on the surface outside the micro-well is removed, and then O 2 In order to plasma bombard the microwell array substrate, the chemical modification technology is used to perform hydrophobic modification on the surface outside the microwell array.

[0012] Furthermore, the functional coating is one of a hydrogel coating, a molecular brush coating and a polymer coating.

[0013] Furthermore, the hydrogel coating is a hydrogel group with high-density carboxyl groups as branch chains. The specific operation of obtaining the hydrogel coating is as follows: first, the substrate is treated with oxygen plasma to graft hydroxyl groups on its surface, 3-aminopropyltrimethoxysilane is grafted by vapor deposition technology, the substrate surface is incubated with a triethylamine solution dissolved in anhydrous dichloroethane, and 2-bromoisobutyryl bromide is added to graft the polymerization initiator to the amino group, and its monomers hydroxyethyl methacrylate and methyldiethanolamine are added under a nitrogen atmosphere, and copper chloride is added after degassing to prevent oxidation. The polymerization temperature is maintained at 37°C, and the surface is cleaned after the monomers are polymerized for 1-9 hours to obtain a substrate surface with a high density of carboxyl groups.

[0014] Furthermore, the chemical modification technology is a gas phase grafting technology or a liquid phase grafting technology.

[0015] Furthermore, the specific operation of the liquid phase grafting technology is: immersing the micro-well substrate and the fluorination reagent in the organic reagent, controlling the grafting temperature to be 25-35° C., and the reagent content ratio of the fluorination reagent to the organic reagent to be 1-20%.

[0016] Furthermore, the C 4 F 8 The flow rate is 2-10sccm, SF 6 The flow rate is 4-10sccm, O 2 The flow rate is 2-5sccm and the total gas pressure is 10-50mToor.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1. The preparation process of the present invention is simple, and high-throughput microarraying of tissue sections can be achieved through readily available microwell array substrates and instruments, effectively reducing the instrument cost required for in situ tissue segmentation;

[0019] 2. The present invention prepares a hydrogel functional coating with high group density, which is a key factor in adhering tissue to the microwell array;

[0020] 3. The present invention prepares a highly hydrophobic surface outside the microwell array, which is a key factor in separating the tissue from the outer surface of the microwell;

[0021] 4. The present invention can confine tissues to a micrometer-level space, ensuring that the information of genes, proteins, etc. of tissues in each microarray does not contaminate each other;

[0022] 5. The size, quantity and arrangement of the microwell array of the present invention can be adjusted as needed, and even tissue sections can be segmented and confined into cell microarrays at the single cell level. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a diagram of a microwell array used to achieve tissue microarray in the present invention.

[0024] Figure 2 Microscopic images of tissue microarrays of different microwell sizes in the present invention.

[0025] Figure 3 This is a microscope cross-sectional view of the tissue microarray in the present invention. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] The specific implementation of the present invention is described in detail below in conjunction with specific embodiments.

[0028] An embodiment of the present invention provides a method for microarraying of tissue samples, comprising the following steps:

[0029] Step 1: preparing a microwell array substrate modified with a high-density functional coating, wherein the functional coating in the microwell adheres the tissue to the side wall or bottom of the microwell;

[0030] Step 2: attach the tissue slices to the surface of the microwell array substrate, and the tissues at the microwells are retained in situ in the microwell array to obtain a high-throughput tissue microarray.

[0031] In the embodiment of the present invention, preferably, the tissue microarray method kit mainly comprises a microwell array substrate modified with a high-density functional coating. The kit of the present invention can be used to segment tissue samples into independent micron-level tissue blocks, thereby realizing high-throughput arraying of tissue samples. At the same time, the method avoids cross-contamination of nucleic acid, protein and other information between tissue microarrays, simplifies the operation steps while improving the efficiency of tissue segmentation, and reduces the instrument cost of the entire process.

[0032] As a preferred embodiment of the present invention, in the step 2, after the tissue slices are attached to the surface of the microwell array substrate, pressure is applied to the tissue slices by an instrument to divide the tissue slices or to make the tissue slices sink.

[0033] As a preferred embodiment of the present invention, in the step 2, after the tissue slices are attached to the surface of the micro-well array substrate, the tissue slices on the surface of the substrate outside the micro-well array are removed by a tool.

[0034] As a preferred embodiment of the present invention, the tool used in the process of removing tissue outside the micro-well array is a sticky substrate.

[0035] As a preferred embodiment of the present invention, the tool used in the process of removing tissue outside the micro-well array is an adhesive tape.

[0036] As a preferred embodiment of the present invention, the adhesive tape is a hard or soft adhesive tape with adhesiveness.

[0037] As a preferred embodiment of the present invention, the instrument used in the process of removing tissue outside the micro-well array is a sharp instrument.

[0038] As a preferred embodiment of the present invention, the instrument used in the process of removing tissue outside the micro-well array is a hard sharp instrument.

[0039] As a preferred embodiment of the present invention, the material of the microwell array substrate is organic and inorganic materials.

[0040] In the embodiment of the present invention, preferred materials include glass, silicon, and polymer.

[0041] As a preferred embodiment of the present invention, the shapes of the micro-wells in the micro-well array are regular or irregular.

[0042] In the embodiment of the present invention, preferred are, for example, a cylinder, a cuboid, and a cube.

[0043] As a preferred embodiment of the present invention, the method for preparing the micro-well array includes any method that can construct a morphological structure.

[0044] As a preferred embodiment of the present invention, the volume range of the micro-well is 0.1fm 3 -1cm 3 .

[0045] As a preferred embodiment of the present invention, the micro-well array comprises at least one micro-well.

[0046] As a preferred embodiment of the present invention, the spacing width between the micro-wells ranges from 0.1 nm to 1000 μm.

[0047] As a preferred embodiment of the present invention, the diameter of the micro-well ranges from 0.1 nm to 1000 μm.

[0048] As a preferred embodiment of the present invention, the height of the micro-well ranges from 0.1 nm to 1000 mm.

[0049] As a preferred embodiment of the present invention, the type of the tissue is treated or untreated tissue.

[0050] As a preferred embodiment of the present invention, the preparation process of the micro-well array substrate modified with a high-density functional coating is as follows: using photolithography technology to realize the preparation of a photoresist array, then etching the surface of the photoresist array by an etching method to obtain a micro-well structure array, removing the photoresist, and using a high-density functional coating modification technology to modify the surface in the micro-well.

[0051] As a preferred embodiment of the present invention, the preparation process of the microwell array substrate modified with a high-density functional coating can be described as follows: using photolithography technology to realize the preparation of a photoresist array, then etching the surface of the photoresist array by an etching method to obtain a microwell structure array, removing the photoresist, using a high-density functional coating modification technology to modify the surface in the microwell, then using photolithography technology to form a photoresist on the surface of the functional coating, using chemical modification technology to perform hydrophobic modification on the surface outside the microwell array, and removing the photoresist on the surface of the microwell.

[0052] As a preferred embodiment of the present invention, the angle between the side surface and the bottom surface of the micro-well structure of the obtained micro-well structure array is 30-150°.

[0053] As a preferred embodiment of the present invention, the obtained micro-well structure array is a micro-well structure with sharp edges.

[0054] As a preferred embodiment of the present invention, the obtained micro-well structure array is a micro-well structure with vertical edges.

[0055] As a preferred embodiment of the present invention, the etching method for preparing the micro-well array is a deep etching method or a soft etching method.

[0056] As a preferred embodiment of the present invention, the deep etching method for preparing the micro-well array is plasma bombardment.

[0057] As a preferred embodiment of the present invention, the deep etching method for preparing the micro-well array is that plasma bombards the substrate in a vertical direction.

[0058] As a preferred embodiment of the present invention, the plasma gas in the plasma bombardment of the deep etching method includes C 4 F 8 , SF 6 , O 2 Or any combination of the three gases.

[0059] As a preferred embodiment of the present invention, the specific preparation operation of the micro-well array substrate modified with a high-density functional coating can be: spin-coating a layer of photoresist on the surface of the substrate, performing a photolithography process based on a transparent dot matrix photolithography mask to leave a photoresist array, and then 4 F 8 , SF 6 , O 2 The surface of the photoresist array is bombarded with plasma to obtain a micro-well structure array with vertical edges, and then O 2 The micro-well array substrate is bombarded with plasma, and the surface in the micro-well is modified by using high-density functional coating modification technology. Then a layer of photoresist is spin-coated on the surface. Based on a non-transparent dot matrix photolithography mask, the photoresist on the surface outside the micro-well is removed, and then O2 In order to plasma bombard the microwell array substrate, the chemical modification technology is used to perform hydrophobic modification on the surface outside the microwell array.

[0060] As a preferred embodiment of the present invention, the photoresist is a positive photoresist.

[0061] As a preferred embodiment of the present invention, the photoresist is a negative photoresist.

[0062] As a preferred embodiment of the present invention, the C 4 F 8 The flow rate is 2-10sccm, SF 6 The flow rate is 4-10sccm, O 2 The flow rate is 2-5sccm and the total gas pressure is 10-50mToor.

[0063] As a preferred embodiment of the present invention, the functional coating is a high-density group coating.

[0064] As a preferred embodiment of the present invention, the functional coating is one of a hydrogel coating, a molecular brush coating and a polymer coating.

[0065] As a preferred embodiment of the present invention, the hydrogel coating is a hydrogel group with high-density carboxyl groups as branches.

[0066] As a preferred embodiment of the present invention, the carboxyl-branched hydrogel group is obtained by copolymerization.

[0067] As a preferred embodiment of the present invention, the specific operation of obtaining the hydrogel coating is as follows: first, the substrate is treated with oxygen plasma to graft hydroxyl groups on its surface, 3-aminopropyltrimethoxysilane is grafted by vapor deposition technology, the substrate surface is incubated with a triethylamine solution dissolved in anhydrous dichloroethane, and 2-bromoisobutyryl bromide is added to graft the polymerization initiator to the amino group, and its monomers hydroxyethyl methacrylate and methyldiethanolamine are added under a nitrogen atmosphere, and copper chloride is added after degassing to prevent oxidation. The polymerization temperature is maintained at 37°C, and the surface is cleaned after the monomers are polymerized for 1-9 hours to obtain a substrate surface with a high density of carboxyl groups.

[0068] As a preferred embodiment of the present invention, the chemical modification technology is a gas phase grafting technology or a liquid phase grafting technology.

[0069] As a preferred embodiment of the present invention, the specific operation of the liquid phase grafting technology is: immersing the microwell substrate and the fluorination reagent in the organic reagent, controlling the grafting temperature to 25-35°C, and the reagent content ratio of the fluorination reagent to the organic reagent is 1-20%, so as to fully improve the grafting density.

[0070] As a preferred embodiment of the present invention, the fluorination agent is 1H,1H,2H,2H-perfluorooctyltrichlorosilane.

[0071] As a preferred embodiment of the present invention, the organic reagent is a reagent having a surface tension lower than 30 mN / m.

[0072] As a preferred embodiment of the present invention, the organic reagent is acetone, ethanol, or tetrahydrofuran.

[0073] In the embodiment of the present invention, preferably, the reagent content is 1H,1H,2H,2H-perfluorooctyltrichlorosilane / acetone (v / v)=3-5%, which fully increases the grafting density.

[0074] In embodiments of the present invention, for example, animal, plant, microbial, frozen, paraffin-fixed tissue types. The tape comprises a hard or soft surface with stickiness, for example, a transparent tape and a double-sided tape. The method disclosed herein includes using the method disclosed herein for any type of biomics testing and analysis.

[0075] Example 1

[0076] Preparation of micro-well substrate: Place a glass plate with a uniform chromium film and photoresist layer under a mask composed of a transparent array and expose it to UV light for 10 seconds. Then, soak the substrate in photoresist developer and chromium etching solution for 30 seconds to obtain a glass surface with a patterned photoresist array. 4 F 8 , SF 6 , O 2 The surface of the photoresist array is bombarded by plasma to obtain a micro-well structure array with vertical edges.

[0077] Example 2

[0078] Modification of microwell surface groups: First, the substrate is treated with oxygen plasma to graft hydroxyl groups on its surface, and 3-aminopropyltrimethoxysilane is grafted by vapor deposition technology at a grafting temperature of 70°C. The substrate surface is incubated with a triethylamine solution dissolved in anhydrous dichloroethane, and 2-bromoisobutyryl bromide is added to graft the polymerization initiator to the amino group. Its monomers hydroxyethyl methacrylate and methyldiethanolamine are added in a nitrogen atmosphere, and copper chloride is added after degassing to prevent oxidation. The polymerization temperature is maintained at 37°C, and the surface is cleaned after the monomers are polymerized for 8 hours to obtain a substrate surface with a high density of carboxyl groups.

[0079] Example 3

[0080] Modification of surface groups outside the microwell array: Spin a layer of photoresist on the surface, remove the photoresist on the surface outside the microwell based on a non-transparent dot matrix photolithography mask, and then 2In order to plasma bombard the microwell array substrate, 1H,1H,2H,2H-perfluorooctyltrichlorosilane was immersed in acetone using liquid phase grafting technology, the grafting temperature was 30°C, the reagent content was 1H,1H,2H,2H-perfluorooctyltrichlorosilane / acetone (v / v) = 5%, and then the photoresist in the microwell was removed to obtain the target microwell array substrate, wherein the microwell height was 40μm and the diameter was 20-70μm ( Figure 1 ).

[0081] Example 4

[0082] Microarraying of tissue slices based on microwell functional surfaces: Place a microwell array substrate modified with a high-density functional coating and tissue in a slicer. The function of the functional coating is to adhere the tissue array to the side wall or bottom of the microwell. Then, the slicing process is performed. Mouse brain tissue slices are attached to the surface of microwell array substrates of different sizes. Tape is used to remove tissue outside the microwell array. After pressing, the tissue at the microwell is trapped in situ in the microwell array to obtain a high-throughput tissue microarray ( Figure 2 ). Its cross-sectional view also shows that the tissue exists in the microwell array ( Figure 3 ).

[0083] The above are only preferred embodiments of the present invention. It should be pointed out that, for those skilled in the art, several modifications and improvements can be made without departing from the concept of the present invention. These should also be regarded as the protection scope of the present invention. These will not affect the effect of the implementation of the present invention and the practicality of the patent.

Claims

1. A method for microarraying of tissue samples, characterized in that: The following steps are involved: Step 1: preparing a microwell array substrate modified with a high-density functional coating, wherein the functional coating in the microwell adheres the tissue to the side wall or bottom of the microwell; The functional coating is one of a hydrogel coating, a molecular brush coating and a polymer coating; The hydrogel coating is a hydrogel group with high-density carboxyl groups as branch chains. The specific operation of obtaining the hydrogel coating is as follows: first, oxygen plasma is used to treat the substrate to graft hydroxyl groups on its surface, 3-aminopropyltrimethoxysilane is grafted by vapor deposition technology, and a triethylamine solution dissolved in anhydrous dichloroethane is used to incubate the substrate surface, and then 2-bromoisobutyryl bromide is added to graft the polymerization initiator to the amino group, and its monomers hydroxyethyl methacrylate and methyldiethanolamine are added under a nitrogen atmosphere, and copper chloride is added after degassing to prevent oxidation, and the polymerization temperature is maintained at 37° C. After the monomers are polymerized for 1-9 hours, the surface is cleaned to obtain a substrate surface with a high density of carboxyl groups; The specific preparation operation of the microwell array substrate modified with a high-density functional coating adopts one of the following two methods: A photoresist array is prepared by using a photolithography technique, and then the surface of the photoresist array is etched by an etching method to obtain a micro-well structure array, the photoresist is removed, and the surface in the micro-well is modified by using a high-density functional coating modification technology, and then a photoresist is formed on the surface of the functional coating by using a photolithography technique, and the surface outside the micro-well array is hydrophobically modified by using a chemical modification technology, and the photoresist on the surface of the micro-well is removed; A layer of photoresist is spin-coated on the surface of the substrate, a photolithography process is performed based on a transparent dot-matrix photolithography mask, leaving a photoresist array, and then C4F8, SF6, and O2 are used as plasma to bombard the surface of the photoresist array to obtain a micro-well structure array with vertical edges, and then O2 is used as plasma to bombard the micro-well array substrate, and a high-density functional coating modification technology is used to modify the surface in the micro-well, and then a layer of photoresist is spin-coated on the surface, and a non-transparent dot-matrix photolithography mask is used as a basis to remove the photoresist on the surface outside the micro-well, and then O2 is used as plasma to bombard the micro-well array substrate, and a chemical modification technology is used to perform hydrophobic modification on the surface outside the micro-well array; Step 2: attaching tissue slices to the surface of a microwell array substrate, and the tissue at the microwell is retained in situ in the microwell array to obtain a high-throughput tissue microarray; In the step 2, after the tissue slices are attached to the surface of the microwell array substrate, the tissue slices are divided or made to sink by applying pressure to the tissue slices by an instrument, and the tissue slices on the surface of the substrate outside the microwell array are removed by the instrument.

2. The method for microarraying of tissue samples according to claim 1, characterized in that: The chemical modification technology is a gas phase grafting technology or a liquid phase grafting technology.

3. The method for microarraying of tissue samples according to claim 2, characterized in that: The specific operation of the liquid phase grafting technology is: immersing the micro-well substrate and the fluorination reagent in the organic reagent, controlling the grafting temperature to be 25-35° C., and the reagent content ratio of the fluorination reagent to the organic reagent to be 1-20%.

4. The method for microarraying of tissue samples according to claim 1, characterized in that: The flow rate of C4F8 is 2-10 sccm, the flow rate of SF6 is 4-10 sccm, the flow rate of O2 is 2-5 sccm, and the total gas pressure is 10-50 mToor.

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