Chip substrate preparation method based on glass sheet
Patent Information
- Application Number
- CN202380080729.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-20
- Publication Date
- 2025-07-04
AI Technical Summary
Existing silicon microarray chips are expensive, and probe differences exist in tissue permeation time testing, leading to inconsistent results and affecting the stability and accuracy of spatial transcriptomics technology.
A glass slide is used as the chip substrate. Amide bonds between amino and azide groups are formed on the surface of the slide, and the bonds are reacted with probes modified at the 5' end to bind to the surface of the glass slide, thus forming a glass slide-based fluorescent chip.
It reduces the cost of chip raw materials, improves chip stability and flexibility, ensures the number and quality of probes, and enables flexible design of various tissues and stability of permeation time.
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Abstract
Description
Glass sheet-based chip substrate preparation method Technical Field
[0001] The present invention relates to the field of biotechnology, and in particular to a method for preparing a chip substrate based on a glass sheet. Background Art
[0002] The relationship between cells and their relative positions within tissue samples is crucial for understanding disease pathology. Single-cell transcriptome sequencing, while addressing cellular heterogeneity and enabling the identification of key cells involved in disease, cannot accurately pinpoint their spatial locations, leading to limited insights into cellular function.
[0003] In recent years, research on spatial transcriptomics has gradually gained favor among researchers. It not only provides data such as the transcriptome of the research object, but also locates its spatial position in the tissue. This is of great significance to research in many fields such as cancer pathogenesis, neuroscience, and developmental biology. Spatial transcriptomics refers to the generation of complete transcriptome data from a complete tissue sample, which can locate and distinguish the active expression of functional genes in specific tissue regions, providing valuable information for research and diagnosis. As a breakthrough technology, spatial transcriptomics allows scientists to detect all gene activities in a tissue sample and map the location where the activity occurs. This technology has already led to many new discoveries that will help scientists better understand diseases and biological processes.
[0004] BGI currently offers two products for its spatiotemporal omics technology, Stereo-seq: the STOmics Gene Expression Kit-S1 (Stereo Chip, henceforth referred to as the SC chip) and the STOmics Fluorescent Kit-F1 (Fluorescent Chip, henceforth referred to as the FC chip). The FC chip is made from BGI's proprietary silicon microarray chips, which boast an array resolution of 500 nanometers. Very few photolithography machines can achieve such high precision, resulting in high silicon wafer costs, accounting for 82.4% of the total material cost. However, the FC chip's lower price tag keeps its profit margin low. Before using the SC chip for tissue transcript capture, tissue permeabilization conditions must be tested using the FC chip. When using the FC chip for tissue permeabilization time testing, four F1 chips are typically used for time-lapse testing. In some cases, permeabilization time results obtained using the FC chip are inconsistent with those obtained using the SC chip. This may be due to probe variations between the four F1 chips.
[0005] Summary of the Invention
[0006] The purpose of the present invention is to provide a method for preparing a spatiotemporal fluorescence chip based on a glass slide, so as to reduce the cost of chip raw materials, while ensuring the quantity and quality of probes on the chip and improving the stability of chip use.
[0007] In a first aspect, the present invention provides a method for preparing a chip substrate based on a glass sheet, the method comprising the following steps:
[0008] 1) Make the glass surface carry amino groups;
[0009] 2) reacting a coupling reagent with an azide group with the amino group on the surface of the glass slide to form an amide bond.
[0010] In a second aspect, the present invention provides a chip substrate prepared by the method described in the first aspect, wherein the chip substrate comprises a glass sheet having amino groups on its surface, and a coupling reagent having an azide group forms an amide bond with the amino group.
[0011] In a third aspect, the present invention provides a chip preparation method, which, based on the method steps described in the first aspect, further comprises:
[0012] 3) reacting the probe modified at the 5' end with the azide group of the coupling reagent to bind the probe to the surface of the glass slide.
[0013] In a fourth aspect, the present invention provides a chip prepared using the method described in the third aspect, wherein the chip comprises a glass sheet having amino groups on its surface, a coupling reagent with an azide group forms an amide bond with the amino group, and a probe modified at the 5' end reacts with the azide group to bind the probe to the surface of the glass sheet.
[0014] By adopting the above scheme, the beneficial effects of the present invention are:
[0015] 1) Reduce chip raw material costs: The present invention uses common, inexpensive, and readily available glass slides instead of silicon wafers to reduce chip raw material costs. The price of 50 pcs of a common 7.5 cm x 2.5 cm glass slide on the market is 30 yuan, reducing the chip raw material cost to 1 / 1000 of the existing silicon wafer.
[0016] 2) Reducing reagent costs: The present invention replaces the flow-type reaction with an immersion-type reaction, which can achieve the reuse of reaction reagents, thereby further reducing reagent costs;
[0017] 3) Improved result stability: The slide FC chip can achieve different permeabilization time gradients for the same tissue on the same slide, with better stability;
[0018] 4) Higher flexibility: The present invention can test a variety of tissues on a glass slide, and the patch position can be freely designed, so the size and quantity of the tissues can be flexible and varied. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] In order to more clearly illustrate the technical solutions in the specific embodiments of the present invention, the drawings in the specific embodiments are briefly introduced below.
[0020] FIG1 shows the poly T structure at the 3' end of the probe coupled to the surface of the glass sheet in the present invention.
[0021] FIG2 is a schematic diagram of the process of preparing a chip using a glass slide in Example 1 of the present invention.
[0022] FIG3 is a schematic diagram of probe detection (binding of poly T and polyA-cy5) in Example 1 of the present invention.
[0023] FIG4 is a fluorescence microscope photographic result in Example 2 of the present invention.
[0024] FIG5 is a schematic diagram of the permeabilization process in Example 3 of the present invention.
[0025] FIG6 is a fluorescence microscope photographic result in Example 3 of the present invention. DETAILED DESCRIPTION
[0026] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.
[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of the present invention belongs. Before describing the present invention in detail, the following definitions are provided for a better understanding of the present invention.
[0028] As used herein, the term "glass sheet" refers to a rigid or flexible glass material having a length, width, and thickness.
[0029] In this article, the term "probe" refers to a nucleic acid sequence (DNA or RNA) with a known sequence and a detection label that is complementary to the target gene. Probes with known target nucleotide sequences are immobilized on the surface of a gene chip, and nucleic acid sequences are determined by hybridization with the nucleic acid probes.
[0030] In a first aspect, the present invention provides a method for preparing a chip substrate based on a glass sheet, the method comprising the following steps:
[0031] 1) Make the glass surface carry amino groups;
[0032] 2) reacting a coupling reagent with an azide group with the amino group on the surface of the glass slide to form an amide bond.
[0033] In one embodiment, in step 1), the glass slide is directly immersed in the reagent having an amino group.
[0034] The reagent with an amino group is selected from the group consisting of isopropylamine, ethylenediamine, aniline, lysine, polylysine, arginine, leucine, and tryptophan; and the coupling reagent is selected from the group consisting of N-hydroxysuccinimide active ester, imide ester, fluorophenyl ester, STP ester, maleimide, and SMCC.
[0035] In a second aspect, the present invention provides a chip substrate prepared by the method described in the first aspect, wherein the chip substrate comprises a glass sheet having amino groups on its surface, and a coupling reagent having an azide group forms an amide bond with the amino group.
[0036] In a third aspect, the present invention provides a chip preparation method, which, based on the method steps described in the first aspect, further comprises:
[0037] 3) reacting the probe with the 5'-end modification with the azide group of the coupling reagent to bind the probe to the surface of the glass sheet, wherein the 5'-end modification of the probe is selected from: DBCO dibenzocyclooctyne and thiol.
[0038] In one embodiment, a glass slide is immersed in a probe solution containing a 5'-end modified oligonucleotide chain.
[0039] In one embodiment, the 3' end of the probe is a poly-T structure.
[0040] In a fourth aspect, the present invention provides a chip prepared using the method described in the third aspect, wherein the chip comprises a glass sheet having amino groups on its surface, a coupling reagent with an azide group forms an amide bond with the amino group, and a probe modified at the 5' end reacts with the azide group to bind the probe to the surface of the glass sheet.
[0041] The glass sheet refers to a rigid or flexible glass material having length, width and thickness, and can be a common glass sheet in daily life. In one embodiment, the glass sheet is a glass slide.
[0042] In the present invention, reagents with amino groups, such as polylysine, can generate strong adhesion through the interaction between its polycations and anions on the glass. Common molecular weights of polylysine are 70,000-150,000, 150,000-300,000, and >300,000. The larger the molecular weight, the stronger the adhesion. While polylysine adheres to the glass sheet, the amino groups on its surface also successfully bind to the glass sheet.
[0043] In this invention, the coupling reagent serves as a crucial "bridge," tightly binding the probe in step 3) to the amino group in step 1). The ester-activated crosslinker and labeled compound in the coupling reagent react with the amino groups on the glass surface under physiological to weakly alkaline conditions (i.e., pH 7.2-9), forming a stable amide bond. This successfully binds the coupling reagent to the glass, exposing its azide group.
[0044] In the present invention, the probe solution contains an oligonucleotide chain with a modification at the 5' end. The chemical group at the 5' end tightly binds to the azide group of the coupling reagent through a click chemistry reaction, ultimately successfully binding the probe with the poly T tail to the glass surface.
[0045] Example
[0046] Example 1: Development of a process flow for a fluorescent chip based on a glass sheet as a carrier
[0047] This example mainly demonstrates the development of the process flow and principle verification, and performs probe quality testing. The steps are as follows:
[0048] 1) Use the tooling slot to partition a slide. The partition numbers are shown in Figure 2. The main purpose is to set up non-probe areas (non-reaction areas) to prove that the probe connection in the reaction area is successful;
[0049] 2) Add an amino group-containing reagent dropwise to areas B1, B2, C1, C2, D1, and D2. Add tryptophan to B1 and B2, arginine to C1 and C2, and polylysine to D1 and D2. Incubate at room temperature for 10 minutes. Add molecular-grade water to A1 and A2 as a blank control.
[0050] 3) Use a pipette to aspirate the reagents from step 2) and add coupling reagents dropwise to the six regions (B1, B2, C1, C2, D1, and D2). Add SMCC to B1, C1, and D1, and add N-hydroxysuccinimide (NHS) active ester to B2, C2, and D2. Incubate for 30 minutes. No reagents should be added to regions A1 and A2.
[0051] 4) Use a pipette to aspirate the reagents from step 3) and dropwise add probe solutions to the six areas (B1, B2, C1, C2, D1, and D2). Add the solution of the probe modified with 5'-terminal DBCO dibenzocyclooctyne to B1, C1, and D1, and the solution of the probe modified with 5'-terminal thiol to B2, C2, and D2. Incubate for 3 hours. No reagents are added to A1 and A2.
[0052] 5) At this point, the six regions B1, B2, C1, C2, D1, and D2 have been coupled to oligonucleotide chain probes with a poly T structure at the 3' end;
[0053] 6) Add polyA-cy5 solution (1 μM polyA-cy5 primer dissolved in 5xSSC solution) to each of the eight regions A1, A2, B1, B2, C1, C2, D1, and D2 and allow to react for 20 minutes. If the probe is successfully seeded, the polyA-cy5 will bind to the poly T on the probe and emit fluorescence. The principle diagram is shown in Figure 3.
[0054] 7) The slide was photographed under a fluorescence microscope. The results showed that the negative controls A1 and A2 were significantly darker than other areas. The six active areas B1, B2, C1, C2, D1, and D2 had significantly stronger luminescence, with signal values ranging from 2.5w to 3.5w. The edge boundary between each active area and the inactive area was clear, indicating that the probe coupling was successful and the effect was good.
[0055] Example 2: Application of glass slide-based fluorescent chip to mouse brain tissue
[0056] This example mainly applies the fluorescent chip in Example 1 to rat brain tissue. The reagents used in the experiments in this example are all "STOmics Fluorescent Reagent Set-F1" kit (commercially available).
[0057] The experimental process is as follows:
[0058] 1) Remove the tissue. Remove fresh OCT-embedded mouse brain tissue from the -80°C freezer and equilibrate it in a cryostat.
[0059] 2) Microtome adjustment. Pre-cool the cryostat cabinet (-20°C) and the specimen head (-10°C to -15°C, adjusted according to the actual operation process);
[0060] 3) Sectioning and mounting. Repair the tissue block and remove the excess OCT embedding medium around the tissue block; use OCT to fix the tissue block to the sample holder; use a microtome to cut the tissue into 10 μm thickness and mount it on the glass slide FC chip, respectively, in the B1, C1, and D1 areas;
[0061] 4) Baking. After the patch is applied, quickly bake the patch at 37°C for 3 minutes.
[0062] 5) Fixation. Place the dried chip in a -20°C pre-cooled methanol solution for 40 minutes.
[0063] 6) Permeabilization. Remove the fixed tissue chip from the methanol, dry the methanol on the back and surrounding areas of the chip with lint-free paper, add permeabilization reagent, and incubate at 37°C for 10 minutes.
[0064] 7) RT. Aspirate the permeabilization reagent from the chip surface, add RT MIX dropwise, and incubate at 42°C for 3 h.
[0065] 8) Tissue removal: Aspirate the RT MIX and tissue removal reagent from the chip surface and incubate at 37°C for 1 hour.
[0066] 9) Fluorescence microscopy. After adding SRE, take fluorescence photos using a 10x magnification or a microscope selected based on tissue size, using the CY3 or TRITC channel, with an exposure time of approximately 1 s.
[0067] The results are shown in Figure 4 . The fluorescence of the mouse brain tissue in the three regions was obvious and the morphology was clear, indicating that the probe capture effect was good.
[0068] Example 3: Application of glass slide-based fluorescent chip to mouse spleen and heart
[0069] This example mainly applies the FC chip with a glass slide as the carrier to the spleen and heart of mice. The reagents used in the experiment in this example are all "STOmics Fluorescent Reagent Set-F1" kit (commercially available). The experimental process is as follows:
[0070] 1) Chip preparation: Soak the entire glass slide in isopropylamine reagent for 10 minutes, then in imidoester reagent for 30 minutes, and then in probe solution for 3 hours.
[0071] 2) Tissue removal. Remove fresh mouse spleen and heart tissue embedded in OCT from the -80°C freezer and equilibrate in a cryostat.
[0072] 3) Microtome adjustment. Pre-cool the cryostat cabinet (-20°C) and the specimen head (-10°C to -15°C, adjusted according to the actual operation process);
[0073] 4) Sectioning and mounting. Repair the tissue block and remove the excess OCT embedding medium around the tissue block; use OCT to fix the tissue block to the sample holder; use a microtome to cut the tissue into 10μm thickness and mount it on the glass slide FC chip;
[0074] 5) Baking. After the patch is applied, quickly bake the patch at 37°C for 3 minutes.
[0075] 6) Fixation. Place the dried chip in a -20°C pre-cooled methanol solution for 40 minutes.
[0076] 7) Permeabilization. Remove the fixed tissue chip from the methanol, dry the methanol on the back and surrounding areas of the chip with lint-free paper, and add the permeabilization reagent. The permeabilization times for the spleen were 3 minutes, 6 minutes, 12 minutes, and 16 minutes, respectively; the permeabilization times for the heart were 3 minutes, 6 minutes, 12 minutes, and 16 minutes, respectively (Figure 5).
[0077] 8) RT. Aspirate the permeabilization reagent from the chip surface, add RT MIX dropwise, and incubate at 42°C for 3 h.
[0078] 9) Tissue removal: Aspirate the RT MIX and tissue removal reagent from the chip surface and incubate at 37°C for 1 hour.
[0079] 10) Fluorescence microscopy. After adding SRE, take fluorescence photos using a 10x magnification or a microscope selected based on tissue size, using the CY3 or TRITC channel, with an exposure time of approximately 1 s.
[0080] The results are shown in Figure 6. From the spleen results (4 on the left), 12 minutes of permeabilization time was the best, and the effects of different permeabilization times were significantly different; from the heart results (4 on the right), 6 minutes of permeabilization time was the best, and the effects of different permeabilization times were significantly different.
[0081] In this embodiment, two types of eight tissues are attached to a glass slide FC chip. Compared with other chips, the chip of the present invention has high flexibility and clear and intuitive data results.
[0082] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.
Claims
1. A method for preparing a chip substrate based on a glass sheet, characterized in that: The method comprises the following steps: 1) Make the glass surface carry amino groups; 2) reacting a coupling reagent with an azide group with the amino group on the surface of the glass sheet to form an amide bond.
2. The method according to claim 1, characterized in that In step 1), the glass slide is directly immersed in the reagent having an amino group.
3. The method according to claim 2, characterized in that The reagent having an amino group is selected from the group consisting of isopropylamine, ethylenediamine, aniline, lysine, polylysine, arginine, leucine and tryptophan.
4. The method according to any one of claims 1 to 3, characterized in that The coupling reagent is selected from the group consisting of: N-hydroxysuccinimide active ester, imide ester, fluorophenyl ester, STP ester, maleimide and SMCC.
5. A chip substrate prepared by the method according to any one of claims 1 to 4, characterized in that: The chip substrate comprises a glass sheet, the surface of the glass sheet carries amino groups, and a coupling reagent with an azide group forms an amide bond with the amino group.
6. A chip preparation method, characterized in that: The method further comprises, based on the method steps described in any one of claims 1 to 4: 3) reacting the probe modified at the 5' end with the azide group of the coupling reagent to bind the probe to the surface of the glass slide.
7. The method according to claim 6, characterized in that The glass slide is immersed in a probe solution containing a 5'-end modified oligonucleotide chain.
8. The method according to claim 6 or 7, characterized in that: The 5' end modification of the probe is selected from: DBCO dibenzocyclooctyne and thiol.
9. The method according to any one of claims 6 to 8, characterized in that: The 3' end of the probe is a poly-T structure.
10. A chip prepared by the method according to any one of claims 6 to 9, characterized in that: The chip comprises a glass sheet, the surface of which carries amino groups, a coupling reagent with an azide group forms an amide bond with the amino group, and a probe modified at the 5' end reacts with the azide group to bind the probe to the surface of the glass sheet.