Method for reusing microarray chip
Patent Information
- Application Number
- CN202380080728.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-06-27
- Publication Date
- 2025-07-01
AI Technical Summary
In the current microarray chip fabrication process, chips that fail quality control are discarded, resulting in excessively high raw material and sequencing costs, and making effective reuse impossible.
Microarray chips that fail quality control can be treated with strong alkali or protease and then reprocessed into FC or SC chips, reducing chip raw material and sequencing costs.
Through chemical processing, substandard SC chips were successfully reused as FC or SC chips, reducing the cost of raw materials and sequencing, and improving chip utilization and economic benefits.
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Abstract
Description
Method for reusing microarray chips Technical Field
[0001] The present invention relates to the field of biotechnology, in particular to a method for reusing a microarray chip. 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] Currently, BGI's spatiotemporal omics technology, Stereo-seq, has two products: the STOmics Gene Expression Chip Set-S1 (Stereo Gene Expression Chip, hereafter referred to as the SC chip) and the STOmics Fluorescent Chip Set-F1 (Fluorescent Chip, hereafter referred to as the FC chip). Both the FC chip and the SC chip use the same raw materials: BGI's independently developed silicon microarray chips. Their nanometer-level resolution results in a higher cost, accounting for 82.4% of the total material cost of the FC chip and 38% of the total cost of the SC chip. SC chip preparation involves three steps: Part 1, sequencing, and Part 2. Sequencing quality is the criterion for Part 1 testing, and this process requires high quality control (QC) with a success rate of approximately 50%. Furthermore, the SC chip preparation process requires 25 cycles of sequencing to obtain CID (coordinate identity) sequence information, and sequencing costs account for 46% of the total SC chip cost. Therefore, for SC chips, chip raw materials and sequencing costs together account for 84% of the material cost.
[0005] Summary of the Invention
[0006] The present invention provides a method for reusing a microarray chip to reduce chip raw material costs and sequencing costs.
[0007] In order to achieve the above objectives, the following technical solutions are adopted:
[0008] A method for reusing a microarray chip, comprising the following steps:
[0009] 1) Using strong alkali or protease to treat microarray chips that fail quality control;
[0010] 2) The microarray chip processed in step 1) is re-prepared as a microarray chip to achieve the reuse of the microarray chip.
[0011] The present invention has the beneficial effects of treating an SC chip that fails quality control with a strong base or protease to convert it into an FC chip; and treating an SC chip that fails quality control with a protease to reconstitute it into an SC chip. The present invention reuses microarray chips that fail quality control to reduce chip raw material costs and sequencing costs. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] 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.
[0013] FIG1 shows the SC chip preparation process (FIG. A) and the SC chip recycling process of the present invention (FIG. B).
[0014] FIG2 shows the poly T structure at the 3' end of the probe coupled to the surface of the FC chip prepared using the reuse method 1 (strong base, NaOH) in Example 1.
[0015] FIG3 is a schematic diagram of FC chip probe detection (poly T combined with poly A-cy5) prepared using reuse mode 1 (strong base, NaOH) in Example 1.
[0016] FIG4 shows the detection results of the FC chip probe prepared using the reuse method 1 (strong base, NaOH) in Example 1.
[0017] FIG5 shows the detection results of the FC chip probe prepared using the reuse method 2 (proteinase K) in Example 1.
[0018] FIG6 shows, from left to right, photographs of the conventional FC chip in Example 1, the FC chip prepared again after NaOH treatment, and the FC chip prepared again after proteinase K treatment.
[0019] FIG7 shows, from left to right, the capture test results of the conventional FC chip, the FC chip prepared after NaOH treatment, and the FC chip prepared after proteinase K treatment in Example 1.
[0020] FIG8 is a sequencing heat map of cycle 1 of the SC chip prepared by reusing the SC chip in Example 2. DETAILED DESCRIPTION
[0021] 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.
[0022] In this invention, a spatiotemporal gene expression SC chip (also referred to herein as an SC chip) refers to a probe chip with CID coordinate information; a spatiotemporal fluorescence FC chip (also referred to herein as an FC chip) refers to a probe chip without CID coordinate information. The difference between the two is the presence or absence of CID coordinate information, which is obtained through next-generation sequencing.
[0023] Preparation of a spatiotemporal gene expression SC chip involves three steps: Part 1, sequencing, and Part 2, as shown in Figure 1. Part 1 involves spreading DNA nanospheres onto the microarray chip surface, ensuring that each fixed modification site on the microarray is bound to a DNA nanosphere. Part 2 involves attaching probes to the DNA nanospheres. After sequencing, the algorithm software first performs quality control (QC) on the sequencing results. Quality control measures include sequencing quality (Q30 ≥ 80%), maximum impurity area (maximum impurity area within a 1 cm x 1 cm area ≤ 400 μm x 400 μm), and total impurity area (total area of all impurities within a 1 cm x 1 cm area ≤ 514 μm x 514 μm). All three are essential, and the success rate of this step is approximately 50%. If the sequencing QC passes, the sample proceeds to Part 2; otherwise, it is discarded. After Part 2, QC is repeated; if it passes, the sample proceeds to the subsequent process; otherwise, it is discarded. Furthermore, the SC chip preparation process requires sequencing, with a sequence length of 25 bases, which is costly. In summary, the cost of SC chip preparation is prohibitive.
[0024] Therefore, in order to reuse the SC chip that fails the QC of Part 1 sequencing, the present invention provides a method for reusing a microarray chip, which comprises the following steps:
[0025] 1) Using strong alkali or protease to treat microarray chips that fail quality control;
[0026] 2) The microarray chip processed in step 1) is re-prepared as a microarray chip to achieve the reuse of the microarray chip.
[0027] In one embodiment, the microarray chip that fails quality control is an SC chip that fails the first quality control during the preparation process (ie, the QC process after Part 1 in FIG. 1 ).
[0028] In one embodiment, the SC chip is treated with a strong base to prepare the SC chip into an FC chip. The strong base may be NaOH.
[0029] In one embodiment, the SC chip is treated with a protease to prepare the SC chip into an FC chip. The protease may be selected from pepsin, proteinase K, trypsin, or papain.
[0030] In a further embodiment, preparing the SC chip into a FC chip comprises the following steps:
[0031] 1) Soaking the treated chip in an amino compound-containing reagent so that the chip surface carries amino groups;
[0032] 2) washing off the amino-containing compound on the chip, and immersing the chip in a first coupling reagent having an azide group, so that the first coupling reagent reacts with the amino groups on the chip surface to form an amide bond;
[0033] 3) washing off the first coupling reagent on the chip and immersing the chip in a second coupling reagent, wherein the second coupling reagent is a probe solution with a 5'-end modification, and the second coupling reagent reacts with the azide group of the first coupling reagent through a click chemistry reaction, so that the probe is bound to the chip surface.
[0034] The amino-containing compound is arginine or lysine; the first coupling reagent is selected from: N-hydroxysuccinimide active ester, imide ester, fluorophenyl ester, STP ester, maleimide or SMCC; the 5' end modification of the probe is selected from: DBCO dibenzocyclooctyne or thiol; the 3' end of the probe is a poly-T structure.
[0035] In the present invention, the amino-containing compound can generate strong adhesion through the interaction of its cations with the anions on the chip. While the amino-containing compound adheres to the chip, the surface amino groups are also successfully bound to the chip. The first coupling reagent is an important "bridge" used to tightly bind the probe in step 3) and the amino groups in step 1). The ester-activated cross-linker and the labeled compound in the first coupling reagent react with the amino groups on the chip surface under physiological to weakly alkaline conditions (i.e., pH 7.2-9) to form a stable amide bond. Therefore, the first coupling reagent is also successfully bound to the chip, and the azide group on the first coupling reagent is also exposed. 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 first coupling reagent through a click chemistry reaction, ultimately allowing the probe with a poly T tail to be successfully bound to the chip surface.
[0036] In one embodiment, the SC chip is treated with a protease to reconstitute the SC chip into a SC chip. The protease is selected from pepsin, proteinase K, trypsin, or papain.
[0037] In a further embodiment, the steps of re-preparing the SC chip into a SC chip are as described above.
[0038] The present invention specifically describes the reuse of SC chips that fail quality control to prepare SC chips or FC chips. In addition, the method of the present invention is also applicable to other conventional microarray chips, that is, using protease to treat unqualified microarray chips to achieve the reuse of the microarray chip.
[0039] Example
[0040] Example 1: Reusing SC chips that failed sequencing QC to prepare FC chips
[0041] This example demonstrates a method for reusing an SC chip that fails sequencing QC to prepare an FC chip.
[0042] During the SC chip preparation process, a silicon chip is used to complete Part 1 and then perform 25 cycles of sequencing. If the chip QC is found to be unqualified in cycle 1, sequencing is immediately stopped and the chip is reused to prepare an FC chip.
[0043] Reuse method 1
[0044] The SC chip that failed the sequencing QC was treated with strong alkali, that is, soaked in 0.5M NaOH at 55℃ for 20 minutes, and then rinsed with PBS, and repeated three times. TMThe sequencer platform took photos and collected signals. The results are shown in Table 1. The signals of the four channels, A, T, C, and G, were all very low, only around 200, which is about 1% of the level before treatment, indicating that the DNBs (DNA Nano Balls) on the chip were all cleaned.
[0045] Table 1: Signals of four channels after SC chip was treated with strong alkali
[0046] The above SC chip is re-prepared into an FC chip, and the preparation process is as follows:
[0047] 1) Soaking the surface of the SC chip in arginine, an amino compound, so that the surface of the chip carries amino groups;
[0048] 2) washing off the amino compound, and then immersing in a first coupling reagent, imidoester, which reacts with the amino groups on the chip surface to form an amide bond;
[0049] 3) The first coupling reagent is washed off, and the chip surface is then immersed in a solution of a probe modified at the 5' end. The 5' end of the probe is modified with DBCO dibenzocyclooctyne. The probe solution reacts with the azide group of the first coupling reagent via click chemistry to bind the probe to the chip surface. The 3' end of the probe has a poly T structure, as shown in Figure 2;
[0050] 4) The chip is hybridized with polyA-cy5. If the chip is successfully seeded with the probe, polyA-cy5 will bind to the poly T on the probe, emitting cy5 fluorescence. Under the laser imaging of the sequencer, the G channel can collect the probe signal. The schematic diagram of the principle is shown in Figure 3.
[0051] After the FC chip was prepared, probe testing was performed. The results are shown in Figure 4. The probe signal was 1.6W, which is relatively high. The signal was also very uniform across the entire 6cm×6cm chip, indicating that the chip was successfully probed after the strong alkali treatment.
[0052] Reuse method 2
[0053] For SC chips that failed the sequencing QC test, the chip was treated with proteinase K at 55°C for 20 minutes, and then the chip surface was rinsed with PBS, and the process was repeated three times. TM The sequencer platform took photos and collected signals. The results are shown in Table 2. The signals of the four channels A, T, C, and G were all very low, only around 1000, which was about 10% of the level before treatment, indicating that the DNBs on the chip were all cleaned.
[0054] Table 2: Signals of four channels after protease treatment on SC chip
[0055] The SC chip was re-prepared into an FC chip according to the preparation process described in Reuse Method 1, and probe detection was performed. The results are shown in FIG5 . The probe signal was 4w, indicating that the chip successfully generated probes after protease treatment.
[0056] Chip final quality control (FQC)
[0057] Capture tests were performed on FC chips prepared using the two treatment methods described above and a conventional FC chip (STOmics Fluorescent Reagent Set-F1). All reagents and operations followed the STOmics Fluorescent Reagent Set-F1 and instructions. The experimental procedures are 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 any excess OCT embedding medium surrounding the block. Use OCT to secure the tissue block to the sample holder. Use a microtome to slice the tissue into 10 μm thick slices, then mount them on the FC chip. Figure 6 shows, from left to right, a conventional FC chip, an FC chip prepared using repeat method 1, and an FC chip prepared using repeat method 2.
[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 580 ms.
[0067] The results are shown in FIG7 , and the FC chips prepared by the two treatment methods can be used normally.
[0068] Example 2: Reusing SC chips that failed sequencing QC to prepare SC chips
[0069] This example demonstrates a method for reusing SC chips that failed sequencing QC to prepare SC chips. This method is also applicable to conventional sequencing chips because Part 1 of the SC chip preparation process involves spreading DNA nanospheres onto the microarray chip surface, allowing each fixed modification site on the microarray chip to bind to a DNA nanosphere. This is a standard sequencing preparation step.
[0070] During the SC chip preparation process, sequencing is performed after completing Part 1, and the sequencing length is 25 bases (i.e., 25 cycles). If the chip QC is found to be unqualified in cycle 1, sequencing is stopped immediately and the chip is reused to prepare the SC chip.
[0071] For SC chips that failed the sequencing QC test, the chip was treated with proteinase K at 55°C for 20 minutes, and then the chip surface was rinsed with PBS, and the process was repeated three times. TM The sequencer platform captured signals using a camera. The results, shown in Table 3, show very low signals for all four channels, A, T, C, and G, below 1000, approximately 10% of the pre-processing level, indicating that the DNBs on the chip were completely removed. The treated chip was reloaded with DNBs and then subjected to conventional sequencing. The sequencing heatmap for cycle 1 is shown in Figure 8. The Q30 was approximately 80%, indicating normal sequencing quality after chip reuse. After sequencing was complete, Part 2 and subsequent processes were performed to produce the final SC chip.
[0072] Table 3: Signals of four channels after protease treatment on SC chip
[0073] In summary, SC chips that fail sequencing QC can be reused as FC or SC chips after strong alkali or protease treatment. This treatment method, in addition to its application in spatiotemporal chips, can also be used to reuse other common sequencing chips (non-spatiotemporal chips). For example, if the initial sequencing quality of a common sequencing chip is poor, or if an unexpected error occurs during sequencing, the chip surface can be treated with protease, and DNA nanospheres can be re-applied to the chip surface for resequencing.
[0074] 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 reusing a microarray chip, characterized in that: The method comprises the following steps: 1) Using strong alkali or protease to treat microarray chips that fail quality control; 2) The microarray chip treated in step 1) is re-prepared as a microarray chip to achieve the reuse of the microarray chip.
2. The method according to claim 1, characterized in that The microarray chip that fails the quality control is a SC chip that fails the first quality control during the preparation process.
3. The method according to claim 2, characterized in that The SC chip is treated with a strong base to prepare the SC chip into a FC chip.
4. The method according to claim 3, characterized in that The strong base is NaOH.
5. The method according to claim 2, characterized in that: The SC chip was treated with protease to prepare the SC chip into a FC chip.
6. The method according to claim 5, characterized in that The protease is selected from: pepsin, proteinase K, trypsin or papain.
7. The method according to any one of claims 3 to 6, characterized in that: The preparation of the SC chip into a FC chip comprises the following steps: 1) Soaking the treated chip in an amino compound-containing reagent so that the chip surface carries amino groups; 2) washing away the amino-containing compound on the chip, and immersing the chip in a first coupling reagent having an azide group, so that the first coupling reagent reacts with the amino groups on the surface of the chip to form an amide bond; 3) Washing off the first coupling reagent on the chip, soaking the chip in a second coupling reagent, wherein the second coupling reagent is a probe solution with a 5'-end modification, and the second coupling reagent reacts with the azide group of the first coupling reagent through a click chemistry reaction, so that the probe is bound to the chip surface.
8. The method according to claim 7, characterized in that The amino group-containing compound is arginine or lysine.
9. The method according to claim 7 or 8, characterized in that: The first coupling reagent is selected from: N-hydroxysuccinimide active ester, imide ester, fluorophenyl ester, STP ester, maleimide or SMCC.
10. The method according to any one of claims 7 to 9, characterized in that: The 5' end modification of the probe is selected from: DBCO dibenzocyclooctyne or thiol.
11. The method according to any one of claims 7 to 10, characterized in that: The 3' end of the probe is a poly-T structure.
12. The method according to claim 2, characterized in that: The SC chip was treated with protease to re-prepare the SC chip into a SC chip.
13. The method according to claim 12, characterized in that The protease is selected from: pepsin, proteinase K, trypsin or papain.