Preparation method of chip for synthesizing DNA and use of the chip, and method for synthesizing DNA

Abstract

The present application provides a method for preparing a chip for synthesizing DNA, the use of the chip, and a method for synthesizing DNA. The preparation method includes the following steps: inkjet printing a hydrophilic substance on the surface of a substrate covered with a silane film layer, and after the hydrophilic substance reacts with the silane film layer to form a covalent bond, forming a hydrophilic lattice on the surface of the silane film layer; using a hydrophobic substance to hydrophobically seal the remaining surface of the silane film layer except the hydrophilic lattice, so as to form a hydrophobic region outside the hydrophilic lattice, and the hydrophobic substance is covalently bonded to the silane film layer; wherein the hydrophilic substance includes a hydrophilic molecule, and of the two end groups of the hydrophilic molecule, one end group is used to form a covalent bond with the silane film layer, and the other end group is a base. Utilizing this preparation method, while forming the hydrophilic and hydrophobic regions, the chip preparation process can be simplified, thereby reducing the manufacturing cost of the chip.

Description

Technical Field

[0001] The present application relates to the field of DNA synthesis, and specifically to a method for preparing a chip for synthesizing DNA, uses of the chip, and a method for synthesizing DNA. Background Art

[0002] Deoxyribonucleic acid (DNA) synthesis technology is a fundamental capability for building the synthetic biology industry. It plays an increasingly important role in modern society, with applications in drug screening, clinical diagnostics, basic scientific research, and other fields. The development of related industries, particularly advances in DNA storage technology, has greatly expanded the DNA synthesis market.

[0003] As the market for DNA synthesis applications continues to expand, the demand for reducing DNA synthesis costs and improving synthesis efficiency is becoming increasingly urgent. DNA microarray synthesis technology has attracted attention due to its ability to massively parallelize the synthesis of tens of thousands or even more different DNA chains on a single chip. This synthesis technology can effectively reduce the consumption of synthesis reagents and synthesis costs, while high-throughput synthesis methods can indirectly significantly shorten synthesis time. However, since the entire synthesis process in DNA microarray synthesis technology is carried out on the chip, chip preparation is crucial. The type, density, uniformity, stability, and hydrophilicity of the chip's surface chemical modification directly affect the formation of microdroplets on the chip during the synthesis process. Poor formation can lead to marginal effects and contamination between different points, affecting the accuracy of DNA synthesis.

[0004] Currently, there are multiple approaches to chip preparation. For example, in one approach, two different silane materials, n-decyl trichlorosilane (NTS) and 10-undecenyl trichlorosilane (UTS), can be mixed in proportion and then plated onto a chip to increase the hydrophobicity of the chip surface, thereby reducing the diffusivity of reagent droplets during synthesis. The materials used in this method are highly toxic, and no difference in hydrophilicity or hydrophobicity is formed on the chip surface, resulting in a relatively weak fixation effect on the droplets. In another approach, photolithography and gas coating techniques can be used to form hydrophilic micropores and a hydrophobic surface of specific sizes on the chip surface. However, the process for preparing chips using this method is extremely complex, has stringent environmental requirements, and requires the use of large amounts of strong acids and highly toxic and polluting organic reagents during production. As a result, the chip cost is extremely high, which is not conducive to reducing the cost of DNA synthesis and poses pollution problems. Summary of the Invention

[0005] The present application provides a method for preparing a chip for synthesizing DNA, the use of the chip, and a method for synthesizing DNA, so as to form hydrophilic and hydrophobic regions on the chip surface while simplifying the chip preparation process and reducing the manufacturing cost of the chip.

[0006] In a first aspect, the present application provides a method for preparing a chip for synthesizing DNA, which comprises the following steps: inkjet printing a hydrophilic substance on the surface of a substrate covered with a silane film layer, and forming a hydrophilic lattice on the surface of the silane film layer after the hydrophilic substance reacts with the silane film layer to form a covalent connection; using a hydrophobic substance to hydrophobically seal the remaining surface of the silane film layer except the hydrophilic lattice to form a hydrophobic area outside the hydrophilic lattice, and the hydrophobic substance is covalently connected to the silane film layer; wherein the hydrophilic substance includes a hydrophilic molecule, and of the two end groups of the hydrophilic molecule, one end group is used to form a covalent connection with the silane film layer, and the other end group is a base.

[0007] The present application first uses inkjet printing to prepare a hydrophilic lattice, and then forms a hydrophobic area, so that the prepared chip has a difference in hydrophilicity and hydrophobicity, with the hydrophilic area being hydrophilic and the area outside the hydrophilic area being hydrophobic. This can effectively prevent the diffusion of microdroplets, reduce the influence of marginal effects, and help improve synthesis efficiency. In this preparation method, the prepared hydrophilic lattice and hydrophobic area both directly react with the silane film layer on the surface of the substrate to form a covalent bond, so that the hydrophilic lattice and hydrophobic area both form a stable connection relationship with the substrate, and the preparation process will not cause damage to the hydrophilic lattice and hydrophobic area, which can effectively ensure the integrity of the prepared hydrophilic lattice and hydrophobic area. In addition, in the preparation method of the present application, the hydrophilic lattice is prepared using an inkjet printing process, which can significantly reduce the manufacturing cost of the chip compared to the photolithography process. The preparation cost of a chip of the same area can be reduced by more than 80%. In addition, in the hydrophilic lattice of the present application, since the hydrophilic end group in its molecular structure is a base, the base can serve as the starting point for subsequent DNA synthesis. Compared with traditional chips, the alignment step can be simplified during the DNA synthesis process, greatly reducing the process difficulty.

[0008] In one possible implementation of the present invention, the hydrophilic molecule further includes an intermediate hydrophilic group connecting the two end groups, wherein the intermediate hydrophilic group is an organic molecular chain with a length of 1 to 16 atoms. By introducing the intermediate hydrophilic group, the hydrophilic effect of the hydrophilic lattice can be further enhanced.

[0009] In a possible implementation of the present application, the intermediate hydrophilic group includes one of polyethylene glycol, polyoxyether or polyamide groups.

[0010] In one possible implementation of the present application, when the silane film layer is an aminosilane film layer, the end groups in the hydrophilic molecules used to form a covalent bond with the silane film layer include at least one of an NHS group, an anhydride group, an NCO ester group, or a phosphoramidite group. This allows for a stable bond with the silane film layer, enhancing the bond strength between the hydrophilic lattice and the silane film layer and preventing subsequent process operations from damaging the hydrophilic group structure.

[0011] In one possible implementation of the present application, when the silane film layer is a hydroxysilane film layer, the end groups in the hydrophilic molecules used to form a covalent bond with the silane film layer include at least one of anhydride groups, NCO ester groups, or phosphoramidite groups. This allows for a stable bond with the silane film layer, enhancing the bond strength between the hydrophilic lattice and the silane film layer and preventing subsequent process operations from damaging the hydrophilic group structure.

[0012] In one possible implementation of the present application, when the silane film layer is an epoxysilane film layer, the end groups in the hydrophilic molecules used to form a covalent bond with the silane film layer include at least one of an NH2 group, an OH group, an SH group, or a hydrazine group. This allows for a stable bond structure with the silane film layer, enhancing the bond strength between the hydrophilic lattice and the silane film layer and preventing damage to the hydrophilic group structure caused by subsequent processing operations.

[0013] In one possible implementation of the present application, a hydrophilic substance is inkjet printed on the surface of a substrate covered with a silane film layer. After the hydrophilic substance reacts with the silane film layer to form a covalent bond, a hydrophilic lattice is formed on the surface of the silane film layer, including:

[0014] First, a first hydrophilic substance is inkjet printed, and then a second hydrophilic substance is inkjet printed. After the first hydrophilic substance reacts with the silane film layer to form a covalent bond and the first hydrophilic substance is covalently bonded to the second hydrophilic substance, a hydrophilic lattice is formed on the surface of the silane film layer; wherein one end of the first hydrophilic substance is covalently bonded to the silane film layer, and the other end of the first hydrophilic substance is bonded to the second hydrophilic substance; and the other end of the second hydrophilic substance is a base.

[0015] In a possible implementation of the present application, the second hydrophilic substance is a phosphoramidite monomer (phosporamidite) with a 4,4-dimethoxytrityl (4,4-dimethoxytriphenylmethyl, DMT) protecting group, which can be recorded as dT-CE-DMT Phosporamidite.

[0016] In one possible implementation of the present application, the hydrophobic substance includes a hydrophobic molecule, wherein one of the two end groups of the hydrophobic molecule is used to form a covalent bond with the silane film layer, and the other end group is a hydrophobic group. Wherein, when the hydrophobic group is specifically selected, the hydrophobic group can be selected from a C chain group or a C chain group containing F. In one possible implementation of the present application, the hydrophobic group is an organic molecular chain with a length of 8-16 atoms. Thus, the hydrophobic effect of the hydrophobic area can be improved.

[0017] As an exemplary illustration, in one possible implementation of the present application, the hydrophobic group is selected from one of C8, C10, C12, C14, C16, or fully F-substituted C8, C10, C12, C14, and C16.

[0018] In one possible implementation of the present application, when the silane film layer is an aminosilane film layer, the end group of the hydrophobic molecule used to form a covalent bond with the silane film layer includes at least one of an NHS group, an anhydride group, an NCO ester group, or a phosphoramidite group. In another possible implementation of the present application, when the silane film layer is a hydroxysilane film layer, the end group of the hydrophobic molecule used to form a covalent bond with the silane film layer includes at least one of an anhydride group, an NCO ester group, or a phosphoramidite group. In another possible implementation of the present application, when the silane film layer is an epoxysilane film layer, the end group of the hydrophobic molecule used to form a covalent bond with the silane film layer includes at least one of an NH2 group, an OH group, an SH group, or a hydrazine group. In this way, a stable connection structure can be formed with the silane film layer, thereby improving the connection strength between the hydrophilic lattice and the silane film layer and preventing subsequent process operations from damaging the structure of the hydrophilic group.

[0019] In a second aspect, the present application further provides a use of a chip obtained by the preparation method of the first aspect of the present application in DNA synthesis. Using this chip to synthesize DNA can effectively simplify the alignment process, reduce process difficulty, and lower synthesis costs.

[0020] In a third aspect, the present application further provides a method for synthesizing DNA, the method comprising the following steps:

[0021] A hydrophilic substance is inkjet printed on the surface of a substrate covered with a silane film layer. After the hydrophilic substance reacts with the silane film layer to form a covalent bond, a hydrophilic lattice is formed on the surface of the silane film layer. The hydrophilic substance includes a hydrophilic molecule, one of the two end groups of the hydrophilic molecule is used to form a covalent bond with the silane film layer, and the other end group is a base. The remaining surface of the substrate except the hydrophilic array is covered with a hydrophobic substance. After the hydrophobic substance reacts with the remaining silane film layer to form a covalent bond, a hydrophobic area is formed on the remaining surface of the substrate. After the substrate with the hydrophilic and hydrophobic areas is dried, synthetic DNA is inkjet printed starting from the base.

[0022] The present application provides a method for synthesizing DNA. Since bases are introduced when preparing the chip, the inkjet printing of hydrophilic dot arrays and the DNA synthesis process can be integrated and fused. After the chip is formed, DNA synthesis can be performed directly by inkjet printing. The preparation process is simple, does not involve additional photolithography / RF sputtering equipment, is low in cost, and takes a short time. Moreover, because it is not limited by the size of the mask plate, the flux scalability is strong. In the existing scheme, the preparation of the hydrophilic and hydrophobic regions of the chip is separated from the DNA printing process. Therefore, after the hydrophilic and hydrophobic regions are formed, it is necessary to visually mark some hydrophilic points, and then perform a two-dimensional position calibration before printing the synthetic DNA, and then perform inkjet printing to synthesize the DNA. In this process, the marked hydrophilic points can no longer be used for synthesis. In the scheme of the present application, DNA synthesis can be performed by direct printing on the formed chip without the need for secondary calibration, which greatly reduces the difficulty of the process, simplifies the process steps, and is more operable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 This is a schematic diagram of the chip preparation process according to one embodiment of the present application;

[0024] Figure 2 This is a schematic structural diagram of a substrate having a silane film layer coated on its surface according to an embodiment of the present application;

[0025] Figure 3 This is a schematic structural diagram of forming a hydrophilic lattice on a substrate surface according to an embodiment of the present application;

[0026] Figure 4 This is a schematic structural diagram of forming a hydrophilic and hydrophobic region on a substrate surface according to an embodiment of the present application;

[0027] Figure 5 This is a schematic diagram of the process flow of a chip according to an embodiment of the present application before preparing a hydrophilic lattice;

[0028] Figure 6 This is a schematic diagram of the molecular connection structure between the silane film layer and the substrate according to one embodiment of the present application;

[0029] Figure 7 A schematic flow chart of a method for synthesizing DNA according to an embodiment of the present application;

[0030] Figure 8 This is a schematic diagram of the structure of inkjet-printed synthetic DNA according to one embodiment of the present application;

[0031] Figure 9 This is a schematic diagram of the DNA synthesis process according to one embodiment of the present application.

[0032] Reference numerals:

[0033] 11-substrate; 12-silane film layer; 13-hydrophilic lattice; 14-hydrophobic region. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solutions and advantages of this application clearer, this application will be further described in detail below with reference to the accompanying drawings.

[0035] DNA microarray synthesis technology can synthesize tens of thousands or even more different types of DNA chains in parallel on a single chip on a large scale. In DNA microarray synthesis technology, since the entire synthesis process is carried out on the chip, the preparation of the chip is the key to DNA microarray synthesis technology. During the reaction process, in order to fix the reaction droplets, the current improvement idea is to form hydrophilic and hydrophobic areas on the surface of the chip to fix the reaction droplets. For example, an existing solution is to use photolithography technology to modify the hydrophilicity of the chip surface and use photolithography technology to form a difference in hydrophilicity and hydrophobicity on the chip surface. When inkjet synthesizing DNA, the droplets react in the hydrophilic micropores, and the hydrophobic surface plays a good physical and chemical isolation role, thereby fixing the droplets. However, the process of using photolithography to prepare chips is extremely complex and has stringent environmental requirements. Large amounts of strong acids and highly toxic and polluting organic reagents are used during production. Furthermore, the physical mask must be bonded to the chip substrate during photolithography, a difficult and incomplete bonding process. Consequently, oxygen plasma activation destroys the hydrophobicity of the masked areas. Furthermore, the use of photolithography to prepare chips also results in high chip costs, hindering the cost of DNA synthesis. The complex and costly process of preparing hydrophilic and hydrophobic regions by photolithography limits its industrialization and large-scale application.

[0036] To solve the above technical problems, the present application provides a method for preparing a chip for synthesizing DNA. The terms used in the following examples are only for the purpose of describing specific embodiments and are not intended to limit the present application. As used in the specification and claims of the present application, the singular expressions "a", "an", "above", "the" and "this" are intended to also include expressions such as "one or more", unless the context clearly indicates otherwise.

[0037] References to "one embodiment" or "some embodiments" in this specification mean that a particular feature, structure, or characteristic described in conjunction with that embodiment is included in one or more embodiments of the present application. Thus, phrases such as "in one embodiment," "in some embodiments," "in other embodiments," and "in yet other embodiments" appearing in various places in this specification do not necessarily refer to the same embodiment, but rather mean "one or more but not all embodiments," unless otherwise specifically emphasized. The terms "including," "comprising," "having," and variations thereof mean "including but not limited to," unless otherwise specifically emphasized.

[0038] Figure 1 This is a flow chart of a chip preparation method according to an embodiment of the present application. Figure 2 This is a schematic structural diagram of a substrate having a silane film layer on its surface according to an embodiment of the present application. Figure 3 This is a schematic structural diagram of forming a hydrophilic lattice on a substrate surface according to an embodiment of the present application; Figure 4 This is a schematic diagram of a structure for forming hydrophilic and hydrophobic regions on a substrate surface according to an embodiment of the present application. Figures 1 to 4 As shown, in one embodiment of the present application, a method for preparing a chip for synthesizing DNA includes the following steps:

[0039] S1), such as Figure 2 and Figure 3 As shown, a hydrophilic substance is inkjet printed on the surface of a substrate 11 covered with a silane film layer 12. After the hydrophilic substance reacts with the silane film layer 12 to form a covalent bond, a hydrophilic lattice 13 is formed on the surface of the silane film layer 12. The hydrophilic substance includes a hydrophilic molecule, and one of the two end groups of the hydrophilic molecule is used to form a covalent bond with the silane film layer 12, and the other end group is a base.

[0040] S2), such as Figure 2 and Figure 4 As shown, the remaining surface of the silane film layer 12 except the hydrophilic lattice is hydrophobically sealed by a hydrophobic substance to form a hydrophobic region 14 outside the hydrophilic lattice 13 , and the hydrophobic substance is covalently bonded to the silane film layer.

[0041] It is understood that the specific type of silane film layer is not limited in the embodiments of the present application. As an example, the material forming the silane film layer can be any of aminosilane, hydroxysilane, or epoxysilane. In addition, the substrate in the embodiments of the present application can be, for example, a glass substrate or a silicon substrate.

[0042] Figure 5 This is a schematic diagram of the process flow of a chip according to an embodiment of the present application before preparing a hydrophilic lattice. Figure 5As shown, in one embodiment of the present application, before forming the hydrophilic lattice, the chip preparation method further includes the following steps:

[0043] S11) Figure 2 , pre-treating the substrate 11;

[0044] S12), prepare a silane film layer 12 on the surface of the pre-treated substrate 11, and the formed structure is as follows Figure 2 shown.

[0045] In one embodiment of the present application, pre-treatment of the substrate 11 may include: ultrasonic cleaning of the substrate using NaOH solution or HCl solution, and then plasma bombardment to remove impurities and non-covalently bonded groups on the surface of the substrate 11, so as to improve the bonding strength between the silane film layer 12 and the substrate 11.

[0046] Figure 6 Schematic diagram of the molecular connection structure between the silane film layer and the substrate in one embodiment of the present application. Figure 2 and Figure 6 As shown, as an example, a silane film layer 12 can be formed on the surface of the substrate 11 by a liquid phase method or a vapor phase deposition method. In one embodiment of the present application, the preparation of the silane film layer 12 may include, for example: preparing an ethanol solution of silane and adjusting the pH value to the desired acidity value, such as 5.5±0.5, with acetic acid, immersing the pretreated substrate 11 in the solution, reacting at room temperature for 10-14 hours, cleaning and drying, and then forming a silane film layer 12 on the surface of the substrate 11. The silane used in the ethanol solution of silane may be one of modified silanes such as aminosilane, hydroxysilane or epoxysilane. Accordingly, Figure 6 In the above, the R1 group may represent an amino group, a hydroxyl group or an epoxy group.

[0047] Continue to refer to Figure 2 and Figure 6In one embodiment of the present application, when the silane film layer 12 is an aminosilane film layer, the end groups in the hydrophilic molecules of the hydrophilic substance used to form a covalent bond with the silane film layer 12 include at least one of an NHS group, an anhydride group, an NCO ester group, or a phosphoramidite group. In another embodiment of the present application, when the silane film layer 12 is a hydroxysilane film layer, the end groups in the hydrophilic molecules used to form a covalent bond with the silane film layer 12 include at least one of an anhydride group, an NCO ester group, or a phosphoramidite group. In yet another embodiment of the present application, when the silane film layer 12 is an epoxysilane film layer, the end groups in the hydrophilic molecules used to form a covalent bond with the silane film layer 12 include at least one of an NH2 group, an OH group, an SH group, or a hydrazine group. When the substance of the silane film layer 12 changes, by optimizing the end groups in the hydrophilic molecules, the hydrophilic substance and the silane film layer 12 can more easily react to form a covalent bond, thereby effectively improving the binding force between the hydrophilic lattice and the silane film layer.

[0048] In one embodiment of the present application, the hydrophilic molecule has an intermediate hydrophilic group, which is connected between the two end groups, and the intermediate hydrophilic group is an organic molecular chain with a length of 1-16 atoms. As an example, the intermediate hydrophilic group can be, for example, a polyethylene glycol, a polyoxyether, or a polyamide group. The length of the molecular chain in the polyethylene glycol, polyoxyether, or polyamide group can be, for example, 8-16 atoms.

[0049] In the preparation method of the present invention, inkjet printing can be used to form a hydrophilic lattice. This can be used in conjunction with the subsequent DNA synthesis process. After the chip is prepared in the same device, the same inkjet printing device can be used to directly print and synthesize DNA, avoiding the need to move the chip. This solves the chip alignment problem during DNA synthesis, saves process steps, and improves work efficiency.

[0050] In one embodiment of the present application, the hydrophilic substance may be dT-CE-DMT Phosporamidite. During the inkjet printing process, dT-CE-DMT Phosporamidite may be directly inkjet printed, thereby directly forming a hydrophilic substance with a base on the surface of the silane film layer.

[0051] In order to improve the hydrophilic properties of the hydrophilic dot matrix and improve the connection strength between the hydrophilic dot matrix and the silane film layer, in another embodiment of the present application, in the process of forming the hydrophilic dot matrix, the first hydrophilic substance can be inkjet printed first, and then the second hydrophilic substance can be inkjet printed; wherein, one end of the molecule of the first hydrophilic substance is covalently connected to the silane film layer, and the other end of the molecule of the first hydrophilic substance is covalently connected to the molecule of the second hydrophilic substance; the other end of the molecule of the second hydrophilic substance is a base.

[0052] In one embodiment of the present application, the hydrophobic substance includes a hydrophobic molecule. Of the two end groups of the hydrophobic molecule, one end group is used to form a covalent connection with the silane film layer, and the other end group is a hydrophobic group.

[0053] Continue to refer to Figure 2 and Figure 6 In one embodiment of the present application, when the silane film layer 12 is an aminosilane film layer, the end groups in the hydrophobic molecules used to form a covalent bond with the silane film layer 12 include at least one of an NHS group, an anhydride group, an NCO ester group, or a phosphoramidite group. In another embodiment of the present application, when the silane film layer 12 is a hydroxysilane film layer, the end groups in the hydrophobic molecules used to form a covalent bond with the silane film layer 12 include at least one of an anhydride group, an NCO ester group, or a phosphoramidite group. In yet another embodiment of the present application, when the silane film layer 12 is an epoxysilane film layer, the end groups in the hydrophobic molecules used to form a covalent bond with the silane film layer 12 include at least one of an NH2 group, an OH group, an SH group, or a hydrazine group. When the substance of the silane film layer 12 changes, by optimizing the end groups in the hydrophobic molecules, the hydrophobic substance and the silane film layer 12 can more easily react to form a covalent bond, thereby effectively improving the binding force between the hydrophobic region and the silane film layer.

[0054] In one embodiment of the present application, the hydrophobic group is a C chain group or a C chain group containing F. In one embodiment of the present application, the hydrophobic group is an organic molecular chain with a length of 8-16 atoms. As an example, the hydrophobic group is selected from one of C8, C10, C12, C14, C16, polyethylene glycol, polyoxyether, or polyamide groups.

[0055] In one embodiment of the present application, when preparing the hydrophobic region, a hydrophobic material can be used to hydrophobically seal the entire substrate surface to form a hydrophobic region outside the hydrophilic lattice. The remaining surface of the substrate excluding the hydrophilic array is covered with the hydrophobic material. After the hydrophobic material reacts with the remaining silane film before the hydrophilic lattice to form a covalent bond, the hydrophobic region can be formed on the remaining surface of the substrate. For example, when hydrophobic sealing, the hydrophobic material can be applied to the area of ​​the substrate excluding the hydrophilic lattice by immersion, spin coating, spray coating, pulling, microfluidic flow, or the like.

[0056] The following is a detailed description of the chip preparation method of the present application in conjunction with specific embodiments.

[0057] Example 1

[0058] This embodiment is a method for preparing a chip for synthesizing DNA, which comprises the following steps:

[0059] S101), take a 2.5 cm × 7.5 cm glass substrate, ultrasonically clean it with 0.1 M NaOH solution, 0.1 M HCl, and double distilled water for 10 minutes, blow dry it with nitrogen, and immediately bombard it with plasma for 1 minute at a power of 300 W;

[0060] S102), prepare a 2% aminosilane ethanol solution, adjust the pH to 5.5 with acetic acid, and immediately immerse the substrate after the plasma treatment in the solution, react at room temperature for 12 hours, take it out and wash it with ethanol and water once, and then blow dry with nitrogen;

[0061] S103), placing the substrate in a vacuum drying oven, vacuum drying at 110°C for 15 minutes, cooling to room temperature, and then washing with ethanol and water twice each for standby use;

[0062] S104), preparation of hydrophilic dot array: placing the substrate in the printing area of ​​the inkjet printing device, printing a PBS solution of hydrophilic molecules NHS-PEG4-OH according to the preset array, and then printing an acetonitrile solution containing dT-CE-DMT Phosporamidite, and reacting for 1 minute;

[0063] S105), hydrophobic sealing: placing the substrate in a reaction tank, flowing an acetonitrile solution containing a hydrophobic molecule NHS-C8, and reacting for 1 minute;

[0064] S106), washing with acetonitrile solution for three times, and drying to complete the preparation of the hydrophilic and hydrophobic regions on the surface of the amino chip.

[0065] Example 2

[0066] This embodiment is a method for preparing a chip for synthesizing DNA, which comprises the following steps:

[0067] S201), taking a 2.5 cm × 7.5 cm glass substrate, ultrasonically cleaning it with 0.1 M NaOH solution, 0.1 M HCl, and double-distilled water for 10 min, blowing it dry with nitrogen, and immediately bombarding it with plasma for 1 min at a power of 300 W;

[0068] S202), prepare a 2% hydroxysilane ethanol solution, adjust the pH to 5.5 with acetic acid, and immediately immerse the substrate after the plasma treatment in the solution, react at room temperature for 12 hours, take it out and wash it with ethanol and water once, and then blow dry with nitrogen;

[0069] S203), placing the substrate in a vacuum drying oven, vacuum drying at 110°C for 15 minutes, cooling to room temperature, and then washing with ethanol and water twice each for standby use;

[0070] S204), preparation of hydrophilic dot array: placing the substrate in the printing area of ​​the inkjet printing device, and printing an acetonitrile solution containing dT-CE-DMT Phosporamidite directly on the surface of the substrate treated in S203) according to a preset array, and reacting for 1 minute;

[0071] S205), hydrophobic sealing: placing the substrate in a reaction tank, flowing a solution containing hydrophobic acid anhydride, and reacting for 1 minute;

[0072] S206), acetonitrile solution is circulated for washing three times, and after drying, the hydrophilic and hydrophobic regions on the surface of the hydroxyl chip are prepared.

[0073] Example 3

[0074] This embodiment is a method for preparing a chip for synthesizing DNA, which comprises the following steps:

[0075] S301), take a 2.5 cm × 7.5 cm glass substrate, ultrasonically clean it with 0.1 M NaOH solution, 0.1 M HCl, and double distilled water for 10 minutes, blow dry it with nitrogen, and immediately perform plasma bombardment for 1 minute at a power of 300 W;

[0076] S302), prepare a 2% ethanol solution of epoxy silane, adjust the pH to 5.5 with acetic acid, and immediately immerse the substrate after the plasma treatment in the solution, react at room temperature for 12 hours, take it out and wash it with ethanol and water once, and then blow dry with nitrogen;

[0077] S303), placing the substrate in a vacuum drying oven, vacuum drying at 110°C for 15 minutes, cooling to room temperature, and then washing with ethanol and water twice each, for standby use;

[0078] S304), preparation of hydrophilic dot array: placing the substrate in the printing area of ​​the inkjet printing device, printing a hydrophilic aminoethanol PBS solution (pH=9.0) according to the preset array, and then printing an acetonitrile solution containing dT-CE-DMT Phosporamidite, and reacting for 30 minutes;

[0079] S305), hydrophobic blocking: placing the substrate in a reaction tank, flowing a DMSO solution containing hydrophobic dodecylamine (pH=9.0), and reacting for 30 minutes;

[0080] S306), circulate acetonitrile solution for three times of cleaning, and after drying, complete the preparation of the hydrophilic and hydrophobic regions on the surface of the epoxy chip.

[0081] Example 4

[0082] This embodiment is a method for preparing a chip for synthesizing DNA, which comprises the following steps:

[0083] S401), take a 2.5 cm × 7.5 cm glass substrate, ultrasonically clean it with 0.1 M NaOH solution, 0.1 M HCl, and double distilled water for 10 minutes, blow dry it with nitrogen, and immediately bombard it with plasma for 1 minute at a power of 300 W;

[0084] S402), prepare a 2% aminosilane ethanol solution, adjust the pH to 5.5 with acetic acid, and immediately immerse the substrate after the plasma treatment in the solution, react at room temperature for 12 hours, take it out and wash it with ethanol and water once, and then blow dry with nitrogen;

[0085] S403), placing the substrate in a vacuum drying oven, vacuum drying at 110°C for 15 minutes, cooling to room temperature, and then washing with ethanol and water twice each for standby use;

[0086] S404), preparation of hydrophilic dot array: placing the substrate in the printing area of ​​the inkjet printing device, printing the acetonitrile solution of dT-CE-DMT Phosporamidite according to the preset array, and reacting for 1 minute;

[0087] S405), hydrophobic sealing: placing the substrate in a reaction tank, flowing an acetonitrile solution containing a hydrophobic molecule NHS-C8, and reacting for 1 minute;

[0088] S406), rinsing with acetonitrile solution for 3 times, and drying to complete the preparation of the hydrophilic and hydrophobic regions on the surface of the amino chip.

[0089] The chip of the above embodiment of the present application is first prepared by inkjet printing on the surface of the substrate to form a hydrophilic lattice, and then the entire substrate is hydrophobically sealed, thereby forming a hydrophilic and hydrophobic region on the surface of the substrate to obtain a hydrophilic and hydrophobic chip. In the embodiment of the present application, by changing the types of hydrophilic and hydrophobic substances, covalent connection with various silane film layers can be achieved, thereby forming hydrophilic and hydrophobic regions on the surface of the substrate. Compared with the preparation of chips using existing photolithography technology, the hydrophilic and hydrophobic surface manufacturing process of the embodiment of the present application does not rely on high-end equipment, and the hydrophilic and hydrophobic regions are not achieved by zoning or step-by-step coating, but by first inkjet printing a hydrophilic lattice and then sealing the entire solid-phase substrate surface in one step. Therefore, this method has the characteristics of simple process and low cost.

[0090] Based on the same technical concept, the present application also provides a method for synthesizing DNA, such as Figure 7 As shown, the method may include the following steps:

[0091] S31), inkjet printing a hydrophilic substance on the surface of the substrate covered with the silane film layer, and after the hydrophilic substance reacts with the silane film layer to form a covalent bond, forming a hydrophilic lattice on the surface of the silane film layer; wherein the hydrophilic substance includes a hydrophilic molecule, and of the two end groups of the hydrophilic molecule, one end group is used to form a covalent bond with the silane film layer, and the other end group is a base;

[0092] S32), using a hydrophobic substance to perform hydrophobic sealing on the remaining surface of the silane film layer except the hydrophilic lattice to form a hydrophobic region outside the hydrophilic lattice, and the hydrophobic substance is covalently bonded to the silane film layer;

[0093] S33), after drying the substrate with the hydrophilic and hydrophobic regions, inkjet printing is performed to synthesize DNA starting from the bases.

[0094] Figure 8 This is a schematic diagram of inkjet printing of synthetic DNA according to an embodiment of the present application, as shown in FIG. Figure 8 As shown, in one embodiment of the present application, inkjet printing can be used to synthesize DNA during the DNA synthesis process. In this process, the chip can remain in place during both the chip preparation process and the DNA synthesis process. The same inkjet printing equipment can be used to complete the inkjet synthesis of the hydrophilic dot array 13 and DNA. This effectively reduces chip costs during the DNA synthesis process and eliminates the need for equipment alignment during DNA synthesis, reducing process complexity and improving synthesis efficiency.

[0095] Figure 9 Schematic diagram of the DNA synthesis process in one embodiment of the present application. Figure 9 As shown, in one embodiment of the present application, the synthesis of DNA can adopt the phosphoramidite synthesis method, which generally includes four processes: DMT deprotection, coupling, capping, and oxidation. The method of the embodiment of the present application can be used for high-throughput synthesis of DNA. During the synthesis process, a chip is used as a carrier and the bases of the hydrophilic lattice are used as the starting point for synthesis. After synthesis by the phosphoramidite method, a chip with specific DNA can be obtained. In addition, the DNA synthesized using the method of the present application can also be eluted, purified, or amplified from the chip to obtain an oligonucleotide pool containing tens of thousands of DNA chains. In addition, the DNA product synthesized using the method of the embodiment of the present application can be sheared and dissociated from the chip.

[0096] The above are only specific embodiments of the present application, but the scope of protection of this application is not limited thereto. Any changes or substitutions that can be easily conceived by a person skilled in the art within the technical scope disclosed in this application should be included in the scope of protection of this application. Therefore, the scope of protection of this application should be based on the scope of protection of the claims.

Claims

1. A method for preparing a chip for synthesizing DNA, characterized in that: include: inkjet printing a hydrophilic substance on the surface of the substrate covered with the silane film layer, and forming a hydrophilic lattice on the surface of the silane film layer after the hydrophilic substance reacts with the silane film layer to form a covalent bond; Using a hydrophobic substance to hydrophobically seal the remaining surface of the silane film layer except the hydrophilic lattice to form a hydrophobic area outside the hydrophilic lattice, wherein the hydrophobic substance is covalently bonded to the silane film layer; The hydrophilic substance includes a hydrophilic molecule. Among the two end groups of the hydrophilic molecule, one end group is used to form a covalent connection with the silane film layer, and the other end group is a base.

2. The preparation method according to claim 1, characterized in that The hydrophilic molecule further comprises an intermediate hydrophilic group connecting the two end groups, and the intermediate hydrophilic group is an organic molecular chain with a length of 1 to 16 atoms.

3. The preparation method according to claim 2, characterized in that The intermediate hydrophilic group includes one of polyethylene glycol, polyoxyether or polyamide groups.

4. The preparation method according to any one of claims 1 to 3, characterized in that When the silane film layer is an aminosilane film layer, the end groups of the hydrophilic molecules for forming a covalent connection with the silane film layer include at least one of an NHS group, an anhydride group, an NCO ester group or a phosphoramidite group.

5. The preparation method according to any one of claims 1 to 3, characterized in that When the silane film layer is a hydroxysilane film layer, the end groups of the hydrophilic molecules for forming a covalent connection with the silane film layer include at least one of anhydride group, NCO ester group or phosphoramidite group.

6. The preparation method according to any one of claims 1 to 3, characterized in that When the silane film layer is an epoxy silane film layer, the end groups of the hydrophilic molecules for forming a covalent connection with the silane film layer include at least one of an NH2 group, an OH group, an SH group or a hydrazine group.

7. The preparation method according to any one of claims 1 to 3, characterized in that The method comprises: inkjet printing a hydrophilic substance on the surface of the substrate covered with the silane film layer, and forming a hydrophilic lattice on the surface of the silane film layer after the hydrophilic substance reacts with the silane film layer to form a covalent bond. First, inkjet printing a first hydrophilic substance, and then inkjet printing a second hydrophilic substance, after the first hydrophilic substance reacts with the silane film layer to form a covalent bond and the first hydrophilic substance and the second hydrophilic substance are covalently bonded, a hydrophilic lattice is formed on the surface of the silane film layer; Wherein, one end of the first hydrophilic substance is covalently connected to the silane film layer, and the other end of the first hydrophilic substance is connected to the second hydrophilic substance; The other end of the second hydrophilic substance is a base.

8. The preparation method according to claim 7, characterized in that The second hydrophilic substance is a phosphoramidite monomer with a DMT protecting group.

9. The preparation method according to any one of claims 1 to 3, characterized in that The hydrophobic substance includes a hydrophobic molecule. Among the two end groups of the hydrophobic molecule, one end group is used to form a covalent connection with the silane film layer, and the other end group is a hydrophobic group.

10. The preparation method according to claim 9, characterized in that The hydrophobic group is a C chain group or a C chain group containing F.

11. The preparation method according to claim 10, characterized in that: The hydrophobic group is an organic molecular chain with a length of 8 to 16 atoms.

12. The preparation method according to claim 11, characterized in that The hydrophobic group is selected from one of C8, C10, C12, C14, C16, or all-F substituted C8, C10, C12, C14, C16.

13. The preparation method according to claim 9, characterized in that When the silane film layer is an aminosilane film layer, the end groups of the hydrophobic molecules for forming a covalent connection with the silane film layer include at least one of an NHS group, an anhydride group, an NCO ester group or a phosphoramidite group.

14. The preparation method according to claim 9, characterized in that When the silane film layer is a hydroxysilane film layer, the end groups of the hydrophobic molecules for forming a covalent connection with the silane film layer include at least one of anhydride group, NCO ester group or phosphoramidite group.

15. The preparation method according to claim 9, characterized in that When the silane film layer is an epoxy silane film layer, the end groups of the hydrophobic molecules for forming a covalent connection with the silane film layer include at least one of an NH2 group, an OH group, an SH group or a hydrazine group.

16. Use of a chip obtained by the preparation method according to any one of claims 1 to 15 in synthesizing DNA.

17. A method for synthesizing DNA, characterized in that: The following steps are involved: A hydrophilic substance is inkjet-printed on the surface of a substrate covered with a silane film layer, and after the hydrophilic substance reacts with the silane film layer to form a covalent bond, a hydrophilic lattice is formed on the surface of the silane film layer; wherein the hydrophilic substance comprises a hydrophilic molecule, and one of the two end groups of the hydrophilic molecule is used to form a covalent bond with the silane film layer, and the other end group is a base; Using a hydrophobic substance to hydrophobically seal the remaining surface of the silane film layer except the hydrophilic lattice to form a hydrophobic area outside the hydrophilic lattice, wherein the hydrophobic substance is covalently bonded to the silane film layer; After the substrate with the hydrophilic and hydrophobic regions formed thereon is dried, synthetic DNA is inkjet printed starting from the base.

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