Method for preparing a carboxylated DNA chip

Abstract

The application provides a preparation method of a carboxylated DNA chip. The carboxylated DNA chip is prepared by using a common glass sheet as a carrier, modifying the surface of the glass sheet, introducing an amino oligonucleotide chain through a covalent bond, and achieving the purpose of subsequent DNA capture. Compared with a traditional synthesis process, the carboxylated chip substrate prepared by the application has the advantages of low fluorescent background, high signal intensity, high fixing efficiency, low manufacturing cost, simple and easy-to-operate steps, and the like. The application is easy to be industrialized and processed, and can be widely applied to various DNA chips.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biochip processing, in particular to a preparation method of a carboxylated DNA chip. BACKGROUND

[0002] Biochip technology is a new technology developed in the 80s. Biochip usually uses silicon wafer, glass or polymer as substrate material, integrates various biological active substances (DNA, protein, tissue cells, etc.), uses fluorescence or isotope probe to hybridize with biological active substances on the substrate, and uses advanced imaging equipment to obtain image information, thereby obtaining micro information. Like semiconductor chips, biochips can quickly perform complex and accurate operations. Biochips mainly include three categories: gene chips (DNA chips), protein chips and tissue chips. Among them, DNA chip is the fastest developing in biochip. Specifically, it detects the signal strength after the substrate material hybridizes with the probe molecule with fluorescent label to obtain the quantity and sequence information of the sample molecule.

[0003] Compared with traditional gene diagnosis technology, DNA chip technology has high diagnosis efficiency. It can complete detection within 30 minutes by using hybridization detection method. DNA chip can detect multiple sequences at the same time because it can modify multiple sequence structures on the substrate.

[0004] The preparation process of DNA chip is complex and requires many process flows. Therefore, various factors can cause low yield of the chip, thereby affecting the later use. The conventional glass chip substrate preparation method usually uses aldehyde group or amino group modification. The prepared aldehyde substrate or amino substrate has various disadvantages. Due to different modification methods or efficiency problems of passivation reaction, the hybridization probe or fluorescent monomer with fluorescent label often has deep background and low signal-to-noise ratio during detection. In the application process of gene chip, sensitivity and selectivity are two very important parameters. Therefore, it is the key of gene chip technology to make an ideal chip substrate with high immobilization efficiency and without non-specific reaction with other substances in the reaction system. SUMMARY

[0005] The purpose of the present application is to provide a preparation method of a carboxylated DNA chip, which is particularly suitable for spatial transcriptome tissue permeabilization time optimization chip.

[0006] In order to achieve the purpose of the present application, in the first aspect, the present application provides a preparation method of a carboxylated DNA chip, comprising the following steps:

[0007] (1) cleaning the glass slide;

[0008] (2) silanization modification of the surface of the glass slide;

[0009] (3) Carboxyl modification of the surface of the slide;

[0010] (4) Activating the carboxyl groups of the carboxylated slide obtained in step (3) by placing it in a mixed solution of EDC and NHS;

[0011] (5) Condensing the activated slide obtained in step (4) with the amino-modified oligonucleotide chain solution to obtain a carboxylated DNA chip.

[0012] In step (2), the slide is treated with the silane reagent 3-aminopropyl triethoxysilane to obtain a silanized slide.

[0013] In step (3), the silanized slide is washed and then placed in a mixture of 30-60 ml of deionized water and 250-500 μl of polyacrylic acid (preferably 60 ml of deionized water and 500 μl of polyacrylic acid), and the pH is adjusted to 8.0 with hydrochloric acid to perform a carboxyl reaction, thereby obtaining a carboxylated slide.

[0014] Further, step (2) comprises:

[0015] 1) Mix 49 ml of 90%-98% (preferably 95%) ethanol and 1 ml of 3-aminopropyl triethoxysilane in a beaker, pour it into a culture dish containing the slide, seal the culture dish with plastic wrap, avoid light, and place it on a decolorizing shaker at 50-150 rpm (preferably 80 rpm) at room temperature for 30 minutes; remove the slide and rinse it with deionized water for 6-8 times, and dry it with nitrogen;

[0016] 2) Place the slide in a culture dish containing 90%-98% (preferably 95%) ethanol, and shake it at 50-150 rpm (preferably 80 rpm) for 10 minutes; then remove the slide and place it in a culture dish containing deionized water, and shake it at 50-150 rpm (preferably 80 rpm) for 5-15 minutes (preferably 5 minutes); remove the slide, rinse it with deionized water for 6-8 times, dry it with nitrogen, and then place the slide in an oven at 120°C for 50-90 minutes (preferably 50 minutes); finally, remove the slide from the oven, avoid light, and cool it to room temperature to obtain a silanized slide.

[0017] Further, step (3) comprises:

[0018] 1) Place the silanized slide in a culture dish, mix 60 ml of deionized water and 500 μl of polyacrylic acid, adjust the pH to 8.0 with 2M hydrochloric acid, and then pour it into the culture dish containing the slide, seal the culture dish with plastic wrap, avoid light, and place it on a decolorizing shaker at 50-150 rpm (preferably 80 rpm) at room temperature for 20-40 minutes (preferably 20 minutes);

[0019] 2) Take out the glass slide, rinse it with deionized water for 6-8 times, and then dry it with nitrogen to obtain the carboxylated glass slide.

[0020] 4. The method of claim 1, wherein step (4) comprises: preparing a mixed solution of EDC and NHS, placing the carboxylated glass slide into the mixed solution, oscillating at room temperature at 1000-2000 rpm (preferably 2000 rpm) for 30 min to complete the activation of the glass slide.

[0021] The mixed solution of EDC and NHS is prepared as follows: dissolve 1.09 mg of EDC and 0.65 mg of NHS in 0.1 M MES 100 ul to obtain the mixed solution.

[0022] Further, step (5) comprises: placing the activated glass slide into a solution of amino-modified oligonucleotide chain with a final concentration of 50 μM prepared with 0.1 M MES, oscillating at room temperature at 2000 rpm overnight; after the reaction is completed, washing the glass slide with deionized water for 3 times, and then soaking the glass slide in 0.5% BSA for 15-30 min to obtain the carboxylated DNA chip.

[0023] Further, step (1) comprises:

[0024] 1) Pour deionized water with washing solution into a culture dish, place the glass slide into the culture dish, wipe the surface of the glass slide gently, then rinse the glass slide with deionized water for 6-8 times, dry it with nitrogen, and then place it into a clean culture dish;

[0025] 2) Mix 25 ml of methanol and 25 ml of 37% concentrated hydrochloric acid, pour them into the culture dish containing the glass slide, seal the culture dish with plastic wrap, and then place it on a decolorizing shaker to oscillate at 80 rpm for 30 minutes;

[0026] 3) Take out the glass slide, rinse it with deionized water for 6-8 times, and then dry it with nitrogen, and then place it into a clean culture dish;

[0027] 4) Pour 50 ml of 98% concentrated sulfuric acid into the culture dish containing the glass slide, seal the culture dish with plastic wrap, and then place it on a decolorizing shaker to oscillate at 50-150 rpm (preferably 80 rpm) for 30 minutes, then take out the glass slide, rinse it with deionized water for 6-8 times, dry it with nitrogen, and then place it into a clean staining jar;

[0028] 5) Pour 100 ml of 95% ethanol into the staining jar containing the glass slide, place the staining jar into an ultrasonic cleaner to ultrasonically clean it for 10 minutes, then pour out the ethanol, add 100 ml of deionized water, and then place the staining jar into the ultrasonic cleaner to ultrasonically clean it for 5 minutes, take out the glass slide, rinse it with deionized water for 6-8 times, and finally dry it with nitrogen.

[0029] Preferably, the size of the glass slide is 75 mm x 25 mm x 1 mm.

[0030] In one embodiment of the present application, the sequence of the amino-modified oligonucleotide chain is: 5'-Amino-C6CTACACGACGCTCTTCCGATCTCGTAAGGGCATGGTTGATTTTTTTTTTTTTTTTTTTTTTTVN-3'.

[0031] wherein C6 represents the amino modification site on the 5' sugar ring of the primer, 5'Aminolinker(C6) is added to the 5' sugar ring of the primer in the form of phosphor amine in the last step of the synthesis cycle through B-cyanoethyl chemical reaction, 5'C6 amino modification can be used to prepare functionalized oligonucleotides, which are widely used in DNA microarrays and multiple marker diagnostic systems; V is A, G or C; and N is A, T, G or C.

[0032] In a second aspect, the present application provides the use of the carboxylated DNA chip prepared according to the method in spatial transcriptome research.

[0033] By means of the above technical solution, the present application has at least the following advantages and beneficial effects:

[0034] (I) In the present application, ordinary glass slides are used as carriers, and after carboxyl modification on the surface of the glass slides, amino oligonucleotide chains can be introduced through covalent bonds, so as to achieve the purpose of capturing DNA on the subsequent DNA chip. Compared with the traditional synthesis process, the carboxyl-modified chip substrate prepared by the present application has the advantages of low fluorescent background, high signal intensity, high fixation efficiency, low manufacturing cost, simple and easy-to-operate steps, etc. It is easy to be industrialized and streamlined, and can be widely used in various DNA chips.

[0035] (II) The connection process is simple: the condensation reaction conditions are mild, and the reaction process is fast;

[0036] (III) The connection efficiency is high: the glass slides are treated by carboxylation to cover the surface with carboxyl active sites, which is convenient for subsequent connection;

[0037] (IV) The detection sensitivity is high: the number of DNA chains connected is large, which can greatly increase the detection of the tested substances;

[0038] (V) The operation success rate is high: the reagents required in the whole chip manufacturing process are few, which can effectively improve the success rate. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 The figure shows the parameters of the chip optimized for spatial transcriptome permeabilization time and the schematic diagram of the capture probe. As can be seen from the figure, the target area of the chip is densely covered with capture probes.

[0040] Figure 2The chip capture probe structure diagram for optimizing the spatial transcriptome permeabilization time in the preferred embodiment of the present application.

[0041] The oligonucleotide chain is composed of three parts, and the connection sequence region, the quality inspection probe binding region and the mRNA capture region are sequentially arranged from the surface of the slide.

[0042] Figure 3 The chip permeabilization fluorescence result diagram for optimizing the spatial transcriptome permeabilization time in the preferred embodiment of the present application.

[0043] Figure 4 The preparation process diagram of the carboxylated DNA chip. DETAILED DESCRIPTION

[0044] The present application provides a preparation method of a spatial transcriptome tissue permeabilization time optimization chip, and the specific method is as follows:

[0045] 1. Slide surface cleaning: the slide surface is cleaned thoroughly.

[0046] 2. Slide surface silanization modification: the clean slide surface is subjected to silanization modification process through a silanization reagent.

[0047] 3. Carboxylation modification process: the slide of step 2 is treated by a polyacrylic acid solution to obtain a carboxyl modified slide.

[0048] 4. Condensation reaction: the amino modified oligonucleotide chain is connected to the slide through condensation reaction to prepare a DNA chip.

[0049] The terms involved in the present application are as follows:

[0050] EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

[0051] NHS: N-hydroxysuccinimide.

[0052] MES: 2-morpholinoethanesulfonic acid.

[0053] The following examples are used to illustrate the present application, but not to limit the scope of the present application. If not specifically indicated, the technical means used in the examples are conventional means familiar to those skilled in the art, and the raw materials used are commercially available. Example 1 Preparation method of carboxylated DNA chip

[0054] The present embodiment provides a preparation method of a spatial transcriptome permeabilization time optimization chip (carboxylated DNA chip), and the specific method is as follows:

[0055] 1. Preparation of carboxylated slide

[0056] Colorless transparent glass sheet (size of 75 mm x 25 mm x 1 mm) was used as carrier. Its preparation process was divided into three processes: washing of glass sheet surface, silanization modification of glass sheet surface, and carboxyl modification. The specific steps are as follows:

[0057] (1) Washing of glass sheet surface

[0058] A. Pour deionized water with washing liquid into a 15 cm diameter culture dish, put the glass sheet into it, wipe the surface of the glass sheet gently, rinse both sides of the glass sheet 6 times with deionized water, dry with nitrogen, and put it into a clean culture dish (the glass sheets cannot be stacked);

[0059] B. Respectively, 25 ml of methanol and concentrated hydrochloric acid were measured in a 100 ml clean beaker, stirred uniformly with a magnetic stirrer, then poured into the culture dish containing the glass sheet (at this time, the glass sheet is upward, and the upward side is defined as the front side), sealed with plastic wrap, and placed on a decolorization shaker at 80 rpm for 30 minutes;

[0060] C. The glass sheet was taken out from the methanol-hydrochloric acid solution with tweezers, rinsed with deionized water for 6-8 times, dried with nitrogen, and placed in a clean culture dish with the front side upward;

[0061] D. Pour 50 ml of concentrated sulfuric acid into the culture dish containing the glass sheet, seal it with plastic wrap, and place it on a decolorization shaker at 80 rpm for 30 minutes. Then take out the glass sheet with tweezers, rinse both sides with deionized water for 6-8 times, dry with nitrogen, and put it into a clean staining cylinder;

[0062] E. Pour 100 ml of 95% ethanol into the staining cylinder containing the glass sheet, and place it in an ultrasonic cleaner for 10 minutes. Then pour out the ethanol, add 100 ml of deionized water, and place it in an ultrasonic cleaner for 5 minutes. Take out the glass sheet with tweezers, rinse both sides with deionized water for 6 times, dry with nitrogen, and place it in a clean culture dish with the front side upward;

[0063] (2) Silanization modification of glass sheet surface

[0064] A. Respectively, 49 ml of 95% ethanol and 1 ml of silane reagent APTES (3-aminopropyl triethoxysilane, A3648, Sigma-Aldrich) were measured in a 100 ml clean beaker, stirred uniformly with a magnetic stirrer, then poured into the culture dish containing the glass sheet, sealed with plastic wrap, and placed in the dark on a decolorization shaker at 80 rpm for 30 minutes. Take out the glass sheet with tweezers, rinse both sides with deionized water for 6 times, and dry with nitrogen;

[0065] B. Put the glass slide into a petri dish containing 50 ml 95% ethanol, and wash for 10 minutes at 80 rpm. Take out the glass slide and put it into a petri dish containing deionized water, and wash for 5 minutes at 80 rpm. Take out the glass slide, wash it with deionized water for 6-8 times, and dry it with nitrogen. Put the glass slide into a clean chip rack, and bake it at 120°C for 50 minutes. Take out the glass slide from the oven, and place it in the dark. Cool it to room temperature;

[0066] (3) Carboxyl modification of the surface of the glass slide

[0067] A. Put the glass slide into a clean petri dish. Then, respectively take 60 ml deionized water and 500 μl polyacrylic acid, pour them into a 100 ml clean beaker, stir them, adjust the pH to 8.0 with 2M hydrochloric acid, and pour them into the petri dish containing the glass slide. Seal the petri dish with plastic wrap, and place it in the dark on a decolorizing shaker at 80 rpm for 20 minutes;

[0068] B. Take out the glass slide with a pair of tweezers, wash both sides with deionized water for 6 times, dry it with nitrogen, and put it into a clean chip box for vacuum storage at 4°C.

[0069] 2. Linking of the amino-modified oligonucleotide chain

[0070] Accurately weigh 1.09 mg EDC and 0.65 mg NHS, and prepare 0.1M MES at the same time. Dissolve the weighed EDC and NHS in the prepared MES. Add 100 μL of the EDC and NHS solution to the carboxylated glass slide fixed in the clamp. Then, place the glass slide in the room temperature, and shake it in a metal bath (2000 rpm) for 30 minutes.

[0071] After the activation is completed, add 2.5 μL of the amino-modified oligonucleotide chain (dissolved in 0.1M MES, with a final concentration of 50 μM) to the glass slide, mix well, and then shake it in a metal bath (2000 rpm) at room temperature overnight. After the reaction is completed, wash the glass slide with enzyme-free water for 3 times, and soak it in 0.5% BSA for 20 minutes.

[0072] The sequence of the amino-modified oligonucleotide chain is as follows:

[0073] 5'-Amino-C6 CTACACGACGCTCTTCCGATCTCGTAAGGGCATGGTTGATTTTTTTTTTTTTTTTTTTTTTTVN-3'

[0074] Wherein, C6 represents the amino-modified site on the 5' sugar ring of the primer, V is A, G or C, and N is A, T, G or C.

[0075] 3. Scanning detection

[0076] The prepared DNA chip is inserted into a scanner, background scanning of the whole slide is carried out using cy3 and cy5 channels, and then the collected image is analyzed and processed by using the software provided by the instrument; after the scanning is completed, DNA probe hybridization solution is prepared, the chip is treated by spotting, hybridization reaction is carried out at 50 DEG C for 15 min, after the hybridization is completed, the spotting area is cleaned by using cleaning buffer, and after being dried, the spotting area is scanned by using the scanner; after the scanning is completed, the image is analyzed and processed under the same parameters.

[0077] The quality inspection probe sequence is:

[0078] 5'-cy5-TCAACCATGCCCTTACG-3'

[0079] The parameter of the spatial transcriptome permeabilization time optimization chip prepared in the embodiment and the capture probe schematic diagram are shown in Figure 1 .

[0080] The capture probe structure schematic diagram of the spatial transcriptome permeabilization time optimization chip prepared in the embodiment is shown in Figure 2 .

[0081] The permeabilization fluorescence result schematic diagram of the spatial transcriptome permeabilization time optimization chip prepared in the embodiment is shown in Figure 3 .

[0082] The preparation process schematic diagram of the carboxylated DNA chip is shown in Figure 4 .

[0083] By using the chip preparation method provided in the application, the carboxyl group can be effectively modified on the surface of the glass slide, and the chip can be used for the production of various DNA chips; in addition, the application also provides a method for capturing DNA condensation, which can be used for capturing DNA, and can generally be used as a test chip for spatial transcriptome tissue permeabilization.

[0084] The chip is subjected to a relatively rigorous pre-cleaning process before carboxylation, which can greatly reduce the fluorescence background of the chip and effectively ensure the use of the subsequent chip.

[0085] The application fully removes the possible background fluorescence through the pre-treatment of the slide, and connects the required oligonucleotide chain to the slide through condensation reaction, which can effectively ensure the number of required oligonucleotide sequences, so as to ensure the stable detection of the experimental fluorescence signal.

[0086] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. A method for preparing a carboxylated DNA chip, characterized by, The method comprises the following steps: (1) washing the slide; (2) silanization modification of the surface of the slide; (3) carboxyl modification of the surface of the slide; (4) placing the carboxylated slide obtained in step (3) in a mixed solution of EDC and NHS to activate the carboxyl group; (5) placing the activated slide obtained in step (4) in a solution of amino-modified oligonucleotide chains to perform condensation reaction, thereby obtaining the carboxylated DNA chip; In step (2), the slide is treated with silanization reagent 3-aminopropyl triethoxysilane to obtain a silanized slide; In step (3), after the silanized slide is washed, it is placed in a mixture of 30-60 ml deionized water and 250-500 μl polyacrylic acid, and hydrochloric acid is used to adjust the pH to 8.0 to perform carboxyl reaction, thereby obtaining a carboxylated slide; Step (2) comprises: 1) mixing 49 ml of 90%-98% ethanol and 1-3 ml of 3-aminopropyl triethoxysilane in a beaker, pouring into a culture dish containing the slide, sealing the culture dish with plastic wrap, avoiding light, and placing on a decolorizing shaker at 50-150 rpm for 30 minutes at room temperature; removing the slide, washing the slide with deionized water for 6-8 times, and blowing dry with nitrogen; 2) placing the slide in a culture dish containing 90%-98% ethanol, and oscillating at 50-150 rpm for 10-30 minutes; then removing the slide and placing it in a culture dish containing deionized water, oscillating at 50-150 rpm for 5-15 minutes; removing the slide, washing it with deionized water for 6-8 times, and blowing dry with nitrogen, and then placing the slide in an oven at 120°C for 50-90 minutes; finally, removing the slide from the oven, avoiding light, and cooling to room temperature, thereby obtaining the silanized slide; Step (3) comprises: 1) placing the silanized slide in a culture dish, mixing 60 ml of deionized water and 500 μl of polyacrylic acid, adjusting the pH to 8.0 with 2M hydrochloric acid, and then pouring into the culture dish containing the slide, sealing the culture dish with plastic wrap, avoiding light, and placing on a decolorizing shaker at 50-150 rpm for 20-40 minutes at room temperature; 2) removing the slide, washing it with deionized water for 6-8 times, and then blowing dry with nitrogen, thereby obtaining the carboxylated slide; Step (4) comprises: preparing a mixed solution of EDC and NHS, placing the carboxylated slide in the mixed solution, oscillating at 1000-2000 rpm at room temperature for 30 minutes to complete the activation of the slide; The preparation of the mixed solution of EDC and NHS is as follows: dissolving 1.09 mg of EDC and 0.65 mg of NHS in 0.1M MES 100 ul, thereby obtaining the mixed solution; Step (5) comprises: placing the activated slide in a solution of amino-modified oligonucleotide chains with a final concentration of 50 μM prepared with 0.1M MES, oscillating at 2000 rpm at room temperature for overnight reaction; after the reaction is completed, washing with enzyme-free water for 3 times, and soaking in 0.5% BSA for 15-30 minutes, thereby obtaining the carboxylated DNA chip.

2. The method of claim 1, wherein, The size of the slide is 75 mm x 25 mm x 1 mm.

3. The method of claim 1, wherein, The sequence of the amino-modified oligonucleotide chain is: 5'-Amino-C6CTACACGACGCTCTTCCGATCTCGTAAGGGCATGGTTGATTTTTTTTTTTTTTTTTTTTTTTVN-3'; Wherein, C6 represents the amino modification site on the 5' sugar ring of the primer, V is A, G or C, and N is A, T, G or C.

4. The method according to any one of claims 1 to 3, characterized in that, Step (1) comprises: 1) Pour deionized water with washing liquid into a culture dish, put the glass slide into it, wipe the surface of the glass slide gently, then rinse the glass slide with deionized water for 6-8 times, dry with nitrogen, and put it into a clean culture dish; 2) Mix 25ml of methanol and 25ml of 37% concentrated hydrochloric acid, pour into the culture dish containing the glass slide, seal the culture dish with plastic wrap, and put it on the decolorization shaker at 80rpm for 30 minutes; 3) Take out the glass slide, rinse it with deionized water for 6-8 times, dry it with nitrogen, and put it into a clean culture dish; 4) Pour 50ml of 98% concentrated sulfuric acid into the culture dish containing the glass slide, seal the culture dish with plastic wrap, and put it on the decolorization shaker at 50-150rpm for 30 minutes, then take out the glass slide, rinse it with deionized water for 6-8 times, dry it with nitrogen, and put it into a clean staining jar; 5) Pour 100ml of 95% ethanol into the staining jar containing the glass slide, put the staining jar into the ultrasonic cleaner for ultrasonic cleaning for 10 minutes, then pour out the ethanol, add 100ml of deionized water, put the staining jar into the ultrasonic cleaner for ultrasonic cleaning for 5 minutes, take out the glass slide, rinse it with deionized water for 6-8 times, and finally dry it with nitrogen.

5. The application of the carboxylated DNA chip prepared by the method according to any one of claims 1-4 in spatial transcriptome research.

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