DNA microarray material surface preparation method based on polymer and diazonium salt modification
By forming three modification layers on the surface of the DNA microarray material and using diazotization and polymer modification methods, the problem of unstable modification layers of the biochip was solved, and stable connection and increased number of biological probes were achieved.
Patent Information
- Application Number
- CN202510678705.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-10
- Publication Date
- 2025-09-16
Smart Images
Figure CN120649157A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of gene detection technology, in particular to a method for preparing the surface of a DNA microarray material based on polymer and diazonium salt modification. Background Art
[0002] Currently, biochips are widely used in the fields of early disease diagnosis, infectious disease screening and rapid detection. Generally, the surface modification treatment of biochips uses diazonium salts to introduce specific functional groups on the surfaces of carbon, metals, insulators and semiconductors. The surface is further derivatized through functionalization to achieve various applications in analysis, biochemical sensors, microelectronics and biomedicine.
[0003] There are many methods for grafting diazotized surfaces, including electrochemical reduction, reduction with reducing agents, spontaneous reduction of modified substrates, sonication, photografting, heating, ball milling, and mechanical scratching. Hydrogel polymer membranes also play an important role in sequencing surface modification. Due to their dual properties as hydrogels and polymers, they provide a stable substrate for sequencing surfaces and can absorb and retain a certain amount of liquid, which may be crucial for maintaining surface wetness during sequencing. Furthermore, the large number of primer adapters in hydrogel polymers can increase the number of nucleic acid sequences.
[0004] At present, when the chip surface is diazotized, compounds such as fatty chain amino groups and hydroxyl groups are often preferentially diazotized in this process due to their active characteristics. However, the diazonium salts of fatty chain amino groups or hydroxyl groups after diazotization are very easy to decompose, resulting in their inability to connect to the chip and thus form a modified layer. Summary of the Invention
[0005] To address the above problems, the present invention provides a method for preparing the surface of a DNA microarray material based on polymer and diazonium salt modification. By combining diazotization and polymerization, a coating with specific groups can be firmly modified on the surface of the material, which can effectively solve the problem of easy shedding of the modified layer of the biochip.
[0006] The technical solution of the present invention is: A method for preparing a DNA microarray material surface based on polymer and diazonium salt modification comprises the following steps: S1, forming a first modification layer and introducing a first active functional group R1 containing an amino group on the surface of the DNA microarray material to be modified by diazotization reaction; S2, connecting the second active functional group R2 to the surface of the first modified layer through a covalent reaction to form a second modified layer, and introducing a carbon-carbon double bond; S3, connecting the carbon-carbon double bonds on the surface of the second modified layer to the polymer through a covalent reaction to form a third modified layer, and introducing a third active functional group R3; S4. The surface of the third modified layer is connected to the biomolecule probe through a covalent reaction.
[0007] Furthermore, the R3 is a functional acrylamide with a functional group.
[0008] Furthermore, the DNA microarray material is a TiN chip or other conductive chip.
[0009] Furthermore, the first modification layer is modified by an electrochemical method.
[0010] Furthermore, in step S1, the diazotization reaction method is as follows: 1) Aromatic primary amines and nitrous acid undergo diazotization reaction to form a diazonium salt solution; 2) Immerse the DNA microarray material to be modified in a diazonium salt solution and modify the chip surface using cyclic voltammetry; 3) After modification, clean the DNA microarray material.
[0011] The voltage of the cyclic voltammetry is 0-1.2 V, the cycle period is 20 mV / s, and the number of cycles is 2.5.
[0012] Furthermore, in step S2, the second modified layer is formed as follows: a. Prepare 1μM-1M R4-PEG n - a solution of acrylamide; b. Soak the DNA microarray material to be modified in PEG n -In acrylamide solution, shake and wash.
[0013] The oscillation frequency is 100-2000 rpm, the oscillation time is 1-24 hours, and the oscillation temperature is 25-55°C.
[0014] In step S3, the method for forming the third modified layer is as follows: 1) Prepare 0.1%-15% acrylamide aqueous solution, 0.1%-15% ammonium persulfate aqueous solution, 0.1%-15% functional acrylamide monomer solution, and 0.01%-5% N,N-diisopropylethylamine; 2) adding ammonium persulfate solution, functional acrylamide monomer solution, and N,N-diisopropylethylamine to the acrylamide aqueous solution to obtain a polymer solution; 3) Add the polymer solution obtained in step 2) dropwise to the surface of the DNA microarray material to be modified and react at 25-55°C for 0.5-6 hours; 4) After the reaction is completed, the surface of the DNA microarray material to be modified is cleaned.
[0015] Furthermore, the acrylamide aqueous solution needs to be subjected to argon exhaust treatment.
[0016] The beneficial effects of the present invention are: This technology uses polymer modification to form a three-dimensional structure on the chip surface, which can increase the sites for biomolecule connection. Compared with traditional biochips, it can connect a large number of biological probes. In addition, due to the functional groups in the polymer, the biological probe molecules can be connected through chemical covalent bonds rather than non-specific adsorption on the polymer. Using the polymer as a modification layer on the chip surface can solve the current problem of the small number of biomolecule probes connected to the chip; at the same time, the amino groups of the fatty chain can be protected during the diazotization process, which can retain the active sites required for subsequent experiments, thereby increasing the number of target active sites and the stability of the modification layer. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] Figure 1 This is a flow chart of the overall method for preparing the surface of a DNA microarray material based on polymer and diazonium salt modification according to an embodiment of the present invention; Figure 2a This is a photograph of a substrate chip before modification in the method for preparing a DNA microarray material surface based on polymer and diazonium salt modification described in an embodiment of the present invention; Figure 2b is the molecular structure diagram of functional monomer 2; Figure 2c is the molecular structure diagram of functional monomer 3; Figure 2d is the molecular structure diagram of functional monomer 4; Figure 3 This is a photo of a modified chip according to the method for preparing a DNA microarray material surface based on polymer and diazonium salt modification described in an embodiment of the present invention; Figure 4 This is a fluorescence comparison photograph after the first hybridization, melting, and second hybridization of the method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to an embodiment of the present invention; Figure 5 This is a fluorescence comparison diagram of thiol primers and non-thiol primers connected to the method for preparing the surface of a DNA microarray material based on polymer and diazonium salt modification described in an embodiment of the present invention. DETAILED DESCRIPTION
[0018] like Figure 1 As shown, the method for preparing the surface of a DNA microarray material based on polymer and diazonium salt modification includes the following steps: S1, forming a first modification layer and introducing a first active functional group R1 containing an amino group on the surface of the DNA microarray material to be modified by diazotization reaction; S2, connecting the second active functional group R2 to the surface of the first modified layer through a covalent reaction to form a second modified layer, and introducing a carbon-carbon double bond; S3, connecting the carbon-carbon double bonds on the surface of the second modified layer to the polymer through a covalent reaction to form a third modified layer, and introducing a third active functional group R3; S4. The surface of the third modified layer is connected to the biomolecule probe through a covalent reaction. Example 1
[0019] S1, the first modification layer: 10 mM 4-(2-amino)phenylethylamine and 10 mM sodium nitrite were weighed and dissolved in 0.5 M HCl solution pre-cooled at 4°C. The mixture was mixed to obtain a diazonium salt solution. A three-electrode electrochemical workstation was used to modify specific areas of the chip. The chip served as the working electrode, the Pt wire as the counter electrode, and the Ag / AgCl as the reference electrode. The electrolyte was the 10 mM diazonium salt solution. Modification was performed using a potentiostatic method at -1.2 V for 5 minutes. After modification, the cells were rinsed with deionized water by vortexing for 30 seconds. The chemical formula of p-aminophenylethylamine is as follows: ; S2, second modification layer: Prepare a 10 mM NHS-PEGn-acrylic acid solution in DMSO; place the chip in the solution and shake it at 37°C for 2 h (oscillation frequency: 1500 rpm); then rinse with deionized water by vortexing for 30 s (vortexing).
[0020] S3, the third modification layer: Prepare a 5% acrylamide aqueous solution and evacuate it with argon for 30 minutes. Then, add 10% functional monomer 1 solution, 5% ammonium persulfate aqueous solution, and 100 μL of tetramethylethylenediamine to the evacuated acrylamide aqueous solution in sequence and vortex mix for 30 seconds. Each time a liquid is added, vortex mix for 30 seconds. Add the solution dropwise to the chip surface and react at 25°C for 2 hours. After the reaction, wash with deionized water and then DMSO, respectively.
[0021] S4. Connect the probe: When using thiol-modified primers, the ligation method is as follows: 1. Primer reduction: Weigh dithiothreitol and dissolve it in DMSO to obtain a dithiothreitol solution. For a 100 μM primer, take 20 μL of the solution, 100 μL of DMSO, and 20 μL of a 1 mM dithiothreitol solution. Mix and let it stand at room temperature for 30 minutes for reduction to obtain a primer reduction solution. 2. Add 2g of sodium iodide to 20ml of DMSO, then add 10μL of N,N-diisopropylethylamine. Measure 500μL of primer reduction solution and 500μL of sodium iodide solution to prepare primer connection solution. Immerse the chip in the primer connection solution and react at 37℃ for 2h. After completion, rinse with deionized water.
[0022] Verification method: 1. XPS characterization of element content (%): The element content (%) of the substrate before modification and the chip surface after the third layer modification was tested respectively. The results are as follows:
[0023] Experimental conclusion: XPS elemental analysis shows that the Ti and O content on the chip surface decreased significantly after the third modification layer, while the C, N, and Br elements increased significantly. Among them, Br is a characteristic element of the functional modification group of the third modification layer primer, which indicates that the third modification layer is successfully modified.
[0024] 2. SEM test of the thickness of the third modified layer: Chip 1 is the base chip before modification; Chip 2 is the chip after the third layer modification. Figure 2a 、 3 shown.
[0025] Experimental conclusion: Chip 1 can see the conductive layer of the base chip with a thickness of about 57.5nm, and the conductive layer thickness of chip 2 is 43.1nm. Above the conductive layer, a modification layer with clear boundaries and a thickness of about 34.0nm can be seen, indicating that the modification of the third modification layer was successful.
[0026] 3. DNA ligation efficiency and stability test: The test method is as follows: 1. After modifying the TiN chip surface using this method, the modified chip undergoes a first hybridization using a DNA oligonucleotide attached to the chip surface and a complementary paired nucleotide with a fluorescent-modified group. Multiple sets of hybridized chip surface images are taken using a fluorescence microscope, recorded as Chip 1, Chip 2, and Chip 3. The images are analyzed using image processing software such as ImageJ, and the fluorescence extraction value is calculated. 2. After the first hybridization, melt the chip with strong alkali. After melting, perform a second hybridization using the same method as the first hybridization. Use a fluorescence microscope to take a picture of the chip surface after the second hybridization. Use image processing software such as ImageJ to analyze the picture and calculate the fluorescence extraction value. 3. Compare the fluorescence retention rate after the first hybridization and the second hybridization. The fluorescence photos are as follows: Figure 4 As shown: According to the fluorescence photos, the fluorescence value data are extracted as shown in Table 1 below:
[0027] Test conclusion: This method was used to successfully connect DNA oligonucleotides to the TiN chip surface; strong alkali depolymerization was successful, and the surface modification layer was stable under depolymerization conditions.
[0028] 4. Specificity Verification: Experimental method: Replace the thiol primers in the DNA oligonucleotide linker with non-thiol modified primers, use the same method to modify the chip and hybridize, and compare with the chip using non-thiol primers. Use fluorescence microscope to photograph the two groups of chips (two chips each), record the thiol primers as chips 4 and 5, and the non-thiol primers as chips 6 and 7, and compare the fluorescence intensity. The fluorescence photos are as follows: Figure 5 shown.
[0029] Based on the fluorescence photos, the fluorescence value data were extracted and the results are shown in Table 2 below.
[0030]
[0031] Experimental conclusion: The polymer and biomolecular probe are specifically chemically linked rather than nonspecifically adsorbed.
[0032] 5. Verification of DNA oligonucleotide ligation quantity: Five DNA microarray chips were prepared. Fluorescent primer probes that could be paired with DNA oligonucleotides were added and paired by hybridization. After hybridization, the oligonucleotides were melted with a strong base. The melted solution was collected and quantified by qPCR. The number of connected DNA oligonucleotides was calculated. The results are shown in the following table:
[0033] Example 2: The difference between Example 2 and Example 1 lies in the method for preparing the first modification layer. The specific differences are as follows: 2 mM N-p-aminobenzyl-trifluoroacetamide and 2 mM sodium nitrite were weighed and dissolved in 0.5 M HCl solution precooled at 4°C and mixed thoroughly to obtain a diazonium salt solution. A specific area of the chip was modified using an electrochemical workstation and a three-electrode system, with the chip serving as the working electrode, a Pt wire as the counter electrode, and Ag / AgCl as the reference electrode, using a 2 mM diazonium salt solution as the electrolyte. Cyclic voltammetry was used for modification, with a voltage of 0 V to 0.8 V, 20 mV / s, and two cycles. After modification, the sample was vortexed and washed with deionized water for 30 seconds. A 0.1 M potassium carbonate solution (solvent: water / methanol in a 1 / 2 volume ratio) was prepared and reacted at 60°C for 12 hours. After the reaction, the sample was vortexed and washed with deionized water for 30 seconds. N-p-Aminobenzyl-trifluoroacetamide.
[0034] The chemical formula of N-p-aminobenzyl-trifluoroacetamide is as follows: .
[0035] Example 3: The difference between Example 3 and Example 1 lies in the method for preparing the first modification layer. The specific differences are as follows: 2 mM 4-[2-(Boc-amino)ethyl]aniline, 2 mM tert-butyl nitrite or nitrosotetrafluoroborate, and 0.1 M tetraethylammonium tetrafluoroborate were weighed and dissolved in 5 ml of acetonitrile and mixed evenly to obtain a diazonium salt solution. Specific areas of the chip were modified using an electrochemical workstation and a three-electrode system, with the chip serving as the working electrode, a Pt wire as the counter electrode, and an Ag / AgCl as the reference electrode, using a 2 mM diazonium salt solution as the electrolyte. Modification was performed by cyclic voltammetry at a voltage of 0 V to 0.8 V, 20 mV / s, and 10 cycles. After modification, the chip was vortex-washed in anhydrous ethanol for 30 seconds, then immersed in 4.0 M hydrochloric acid solution at room temperature for 15 minutes, and then vortex-washed in DMF, deionized water, and anhydrous ethanol for 30 seconds.
[0036] The chemical formula of 4-[2-(Boc-amino)ethyl]aniline is as follows: .
[0037] Example 4: The first difference between Example 4 and Example 1 lies in the method for preparing the third modification layer. The specific differences are as follows: The active energy group R3 introduced into the third modified layer is azide, and the preparation method is as follows: Prepare 5% acrylamide aqueous solution and exhaust it with argon for 30 min; 10% functional monomer 2 (such as Figure 2b ) solution, 5% ammonium persulfate aqueous solution, and 100 μL of tetramethylethylenediamine were sequentially added to the degassed acrylamide aqueous solution and vortex-mixed for 30 seconds. Each time a liquid was added, vortex-mixed for 30 seconds. The solution was then added dropwise to the chip surface and reacted at 25°C for 2 hours. After the reaction, the chip was washed with deionized water and then DMSO.
[0038] The second difference between Example 4 and Example 1 lies in the biological probe connection method, and the specific differences are as follows: Prepare DNA oligonucleotide ligation solution: 2 μM alkynyl-modified DNA oligonucleotide, 0.1 mM tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine, 0.1 mM cuprous bromide, and 0.1 mM sodium ascorbate dissolved in dimethyl sulfoxide and mixed thoroughly. Soak the chip in the ligation solution and react at room temperature overnight. Rinse with dimethyl sulfoxide and purified water in this order.
[0039] Embodiment 5: The first difference between Example 5 and Example 1 lies in the method for preparing the third modification layer. The specific differences are as follows: The active energy group R3 introduced into the third modified layer is a carboxyl group, and its preparation method is as follows: Prepare 5% acrylamide aqueous solution and exhaust it with argon for 30 min; 10% functional monomer 3 (such as Figure 2c ) solution, 5% ammonium persulfate aqueous solution, and 100 μL of tetramethylethylenediamine were added to the degassed acrylamide aqueous solution in sequence and vortexed for 30 seconds. Each time a liquid was added, vortexed for 30 seconds. The solution was then added dropwise to the chip surface and allowed to react at 25°C for 2 hours. After the reaction, the chip was washed with deionized water and then DMSO. The second difference between Example 5 and Example 1 lies in the biological probe connection method, and the specific differences are as follows: Prepare DNA oligonucleotide ligation solution: 2 μM NH2-modified DNA oligonucleotide and 20 mg / ml EDC dissolved in 0.1 M MES solution (pH 4.5). Immerse the chip in the ligation solution and react at 37°C overnight. Wash with purified water after the reaction.
[0040] Example 6: The first difference between Example 6 and Example 1 lies in the method for preparing the third modification layer. The specific differences are as follows: The active energy group R3 introduced into the third modification layer is streptavidin, and its preparation method is as follows: Prepare 5% acrylamide aqueous solution and exhaust it with argon for 30 min; 10% functional monomer 4 (such as Figure 2d ) solution, 5% ammonium persulfate aqueous solution, and 100 μL of tetramethylethylenediamine were sequentially added to the degassed acrylamide aqueous solution and vortex-mixed for 30 seconds. Each time a liquid was added, vortex-mixed for 30 seconds. The solution was then added dropwise to the chip surface and reacted at 25°C for 2 hours. After the reaction, the chip was washed with deionized water and then DMSO.
[0041] The second difference between Example 6 and Example 1 lies in the biological probe connection method, and the specific differences are as follows: Prepare primer linking solution: 2 μM biotin-modified DNA oligonucleotide, 20 mM HEPES pH 7.0; soak the chip in primer linking solution and react at 25°C for 2 h; rinse the chip with purified water.
[0042] The above-described embodiments merely represent specific implementations of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, and all such variations and improvements fall within the scope of protection of the present invention.
Claims
1. A method for preparing a DNA microarray material surface based on polymer and diazonium salt modification, characterized in that: The steps include: S1, forming a first modification layer and introducing a first active functional group R1 containing an amino group on the surface of the DNA microarray material to be modified by diazotization reaction; S2, connecting the second active functional group R2 to the surface of the first modified layer through a covalent reaction to form a second modified layer, and introducing a carbon-carbon double bond; S3, connecting the carbon-carbon double bonds on the surface of the second modified layer to the polymer through a covalent reaction to form a third modified layer, and introducing a third active functional group R3; S4. The surface of the third modified layer is connected to the biomolecule probe through a covalent reaction.
2. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: The R3 is a functional acrylamide with a functional group.
3. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: The DNA microarray material is a TiN chip or other conductive chip.
4. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: The first modification layer is modified by an electrochemical method.
5. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: In step S1, the diazotization reaction method is as follows: 1) Aromatic primary amines and nitrous acid undergo diazotization reaction to form a diazonium salt solution; 2) Immerse the DNA microarray material to be modified in a diazonium salt solution and modify the chip surface using cyclic voltammetry; 3) After modification, clean the DNA microarray material.
6. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 5, characterized in that: The voltage of the cyclic voltammetry is 0-1.2 V, the cycle period is 20 mV / s, and the number of cycles is 2.
5.
7. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: In step S2, the second modified layer is formed as follows: a. Prepare 1μM-1M R4-PEG n - a solution of acrylamide; b. Soak the DNA microarray material to be modified in PEG n -In acrylamide solution, shake and wash.
8. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 7, characterized in that: The oscillation frequency is 100-2000 rpm, the oscillation time is 1-24 hours, and the oscillation temperature is 25-55°C.
9. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 1, characterized in that: In step S3, the method for forming the third modified layer is as follows: 1) Prepare 0.1%-15% acrylamide aqueous solution, 0.1%-15% ammonium persulfate aqueous solution, 0.1%-15% functional acrylamide monomer solution, and 0.01%-5% N,N-diisopropylethylamine; 2) adding ammonium persulfate solution, functional acrylamide monomer solution, and N,N-diisopropylethylamine to the acrylamide aqueous solution to obtain a polymer solution; 3) Add the polymer solution obtained in step 2) dropwise to the surface of the DNA microarray material to be modified and react at 25-55°C for 0.5-6 hours; 4) After the reaction is completed, the surface of the DNA microarray material to be modified is cleaned.
10. The method for preparing a DNA microarray material surface based on polymer and diazonium salt modification according to claim 8, characterized in that: The acrylamide aqueous solution needs to be exhausted with argon gas.