Full-printing synthesis method and application thereof in DNA (deoxyribonucleic acid) preparation

Abstract

The invention belongs to the field of chemistry and biology, and particularly relates to a full-printing synthesis method and application of the full-printing synthesis method in DNA preparation. The method has the advantages of being simple, efficient, low in cost, high in precision and high in flux, and low-deletion-rate synthesis of DNA can be achieved by conducting super-hydrophobic and oleophobic treatment on the surface of the base material and combining regulation and control of the printing mode, the composition of the printing material and the variety and the adding amount of reactants.

Description

Technical Field

[0001] This invention belongs to the fields of chemistry and biology, specifically relating to a simple, efficient, low-cost, high-throughput fully printed synthesis method and its application in DNA preparation. Background Technology

[0002] High-throughput DNA synthesis methods have evolved through three generations. The first generation was column-based synthesis, including phosphodiester, phosphotriester, phosphite, phosphite amide, and solid-phase phosphite amide methods. These methods used synthesis columns filled with porous glass or polystyrene sieves as solid-phase carriers, with each DNA molecule synthesized in a separate column. Yields were high, typically in the nmol range, but the large amount of reagents consumed resulted in high production costs and limited throughput. The second generation was microarray synthesis. Microarrays served as solid-phase carriers for DNA synthesis, allowing for high-density, integrated synthesis reactions at specific sites on their surface. This saved reagents while achieving high-throughput DNA synthesis. Microarray DNA synthesis technology is based on the four-step cyclic reaction in solid-phase phosphite amide synthesis. By employing different site-specific deprotection control methods, photochemical, electrochemical, microfluidic, and inkjet printing DNA synthesis methods have been developed. Photochemical synthesis is characterized by high selectivity and controllability, but its application is limited to a very small number of scenarios, such as gene chips, due to the high cost of photosensitive deprotection monomers and short synthesis lengths. Electrochemical synthesis can achieve localized high-precision DNA synthesis, but it is gradually being phased out because it requires specific electrode surfaces and has a high error rate. Microfluidic-based synthesis methods have achieved automation and high efficiency in DNA synthesis, but the need to introduce micropumps and microvalves makes their structures relatively complex, cumbersome to operate, and reduces efficiency, thus preventing them from becoming mainstream. Inkjet printing DNA synthesis offers high throughput, low cost, and longer synthesis lengths, and is gradually becoming the mainstream high-throughput oligonucleotide synthesis method. However, when using inkjet printing alone, the selected synthesis materials are limited by printing parameters such as viscosity and surface tension, thus restricting its application range.

[0003] Shanghai Diwin Co., Ltd. has achieved the fabrication of functionalized surfaces with hydrophilic and hydrophobic regions by enriching the surface of a substrate with hydroxyl groups, modifying the surface with hydrophobic molecules, and then using a multi-channel piezoelectric inkjet head assembly to achieve in-situ droplet etching and subsequent addition of functional groups to the substrate surface. This functionalized surface can stabilize reaction droplets in specific locations, solving the problem of how to achieve independent partitioning of chemical reactions for each droplet on the surface of glass or silicon substrates used as DNA synthesis carriers without physical separation (CN114106588A). However, the precision of in-situ droplet etching is difficult to control in this method.

[0004] To address the limitations of inkjet printing alone in terms of materials used for DNA synthesis, and to achieve the construction of non-interfering micro-reaction chambers on different substrate surfaces, this invention is the first to combine 3D printing with inkjet printing. By controlling the composition of printing materials, the types and amounts of reactants, it achieves DNA synthesis that is simple, efficient, low-cost, low-deletion-rate, high-precision, and high-throughput. Summary of the Invention

[0005] One of the objectives of this invention is to provide a simple, universally applicable, cost-effective, high-precision, and high-throughput fully printed synthesis method.

[0006] Another objective of this invention is to achieve low-cost, low-deletion-rate DNA synthesis by combining powder bed fusion 3D printing with inkjet printing.

[0007] To fabricate rigid three-dimensional microstructures using 3D printing, the substrate surface needs to be treated with superhydrophobic and oleophobic agents. A synthesis method combining 3D printing and inkjet printing requires controlling the reaction conditions during DNA preparation by adjusting the composition of the printing materials, printing conditions, and the types and amounts of reactants, resulting in a simple and efficient DNA synthesis process.

[0008] The fully 3D-printed DNA synthesis method includes the following steps: First, a rigid three-dimensional microstructure is prepared on the surface of a superhydrophobic and oleophobic substrate using 3D printing. Then, a universal solid-phase carrier (DMT-protected phosphorylated amide solid powder and an insoluble polymer selected from silica gel, cross-linked polystyrene, or porous glass beads) is filled into the microstructure. After filling, sintering is performed, and after cooling to room temperature, a solution of deprotecting agent, ATCG monomer, activator, capping agent, and oxidant is sequentially printed into the micropores using inkjet printing to prepare DNA. Each reaction step is followed by rinsing with acetonitrile. A flowchart of the fully 3D-printed DNA synthesis process is attached. Figure 1 .

[0009] Superhydrophobic and oleophobic treatment: First, the substrate is ultrasonically cleaned sequentially with acetone-ethanol-isopropanol. After drying, a 2-10 μm thick layer of soot is deposited on its surface. Then, it is treated with ammonia vapor under vacuum conditions in a vacuum dryer with either tetramethoxysilane or tetraethoxysilane for 24-48 hours. Next, it is sintered in a muffle furnace at 500-800℃ for 2-6 hours. After cooling to room temperature, it is treated with oxygen plasma under the following conditions: 150-250W, 200-400s. Then soak in a 0.1-0.5 wt% fluorosilane-alkane solution for 1-5 hours, remove and then heat at 50-80℃ for 2-5 hours; the fluorosilane is selected from one of triethylfluorosilane, dodecafluoroheptylpropyltrimethoxysilane, dodecafluoroheptylpropylmethyldimethoxysilane, tridecafluorooctyltrimethoxysilane, heptadecafluorodecyltrimethoxysilane, and heptadecafluorodecyltriethoxysilane; the alkane is selected from one of n-hexane, isopentane, cyclohexane, n-pentane, n-heptane, and isooctane.

[0010] The substrate used for 3D printing is selected from one of silicon substrate, silica substrate, quartz substrate or glass substrate, preferably silicon substrate.

[0011] The 3D printing method of this invention employs powder bed fusion technology, and the printing material consists of alloy powder and binder. The designed three-dimensional microstructure is one of the following: a bottom-through inverted opal structure, a porous structure, or a polygonal 3D structure, wherein the polygonal 3D microstructure is selected from one of a pyramid, a cylinder, or a hemisphere; the specific design of the polygonal 3D microstructure is shown in the appendix. Figure 2 .

[0012] The alloy in the 3D printing material is selected from one of titanium alloy, aluminum alloy and stainless steel; the binder is a photosensitive resin, selected from one of furan resin, phenolic resin, alkyd resin and silicone resin.

[0013] The sintering temperature used in powder bed melting technology in 3D printing is 500-1000℃.

[0014] The deprotectant solution used in the inkjet printing of this invention comprises 1-5 wt% trichloroacetic acid and the balance dichloromethane; the activator solution comprises 1-5 wt% 5-ethimercaptotetrazole and the balance acetonitrile; the capping agent solution comprises 5-15 wt% acetic anhydride, 10-20 wt% N-methylimidazolium and the balance tetrahydrofuran; and the oxidant solution comprises 1-5 wt% iodine, 15-25 wt% pyridine, 50-80 wt% tetrahydrofuran and the balance water.

[0015] The specific process for preparing DNA by inkjet printing is as follows: 1) Print a deprotecting agent solution into a microstructure filled with a universal solid-phase carrier, reacting for 0.5-3 min; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Sequentially print 1-20 wt% acetonitrile solutions of A, T, C, and G base monomers and activator solutions, reacting for 2-10 min; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print a capping agent solution, reacting for 0.5-3 min; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print an oxidizing agent solution, reacting for 0.5-5 min; 8) Rinse with sufficient acetonitrile to remove unreacted reactants. To ensure sufficient reaction of reactants in each step, each step is inkjet printed at least 5 times. The number of times the deprotecting agent solution is printed depends on the amount of solid-phase carrier.

[0016] The number of times the reaction solution was printed by inkjet printing was related to the amount of general-purpose solid carrier used to fill the microstructure. In order to ensure that the reactants in the microstructure reacted fully, the number of printing times in the preliminary test was more than 5 times.

[0017] This invention provides a fully printed synthesis method and its application in DNA preparation.

[0018] Compared with existing inventions, the present invention has the following advantages:

[0019] (1) This invention is the first to achieve simple and efficient DNA synthesis by combining powder bed fusion 3D printing with inkjet printing.

[0020] (2) The full-printing synthesis method of the present invention has the characteristics of low cost, high precision and high throughput.

[0021] (3) In order to achieve the preparation of high-precision rigid three-dimensional microstructures by 3D printing, the present invention uses a superhydrophobic and oleophobic substrate surface.

[0022] (4) The fully printed synthesis method of the present invention can be further extended to other biosynthetic fields that require rigid microstructures and micro-reactions. Attached Figure Description

[0023] Figure 1 This is a flowchart illustrating the DNA preparation process using the fully printed synthesis method of this invention.

[0024] Figure 2 Scanning electron microscope image of the inverse opal structure 3D printed according to the present invention.

[0025] Figure 3 Design drawings for polygonal 3D microstructures Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the present invention clearer, the following description is provided in conjunction with embodiments and appendices. Figures 1-2 To further illustrate this invention, it should be understood that the following embodiments are merely illustrative and explanatory of the invention and should not be construed as limiting the scope of protection of the invention. All technologies implemented based on the above description of this invention are covered within the scope of protection intended by this invention.

[0027] Unless otherwise stated, the raw materials and reagents used in the following examples are commercially available products or can be prepared by known methods.

[0028] Example 1

[0029] Preparation of superhydrophobic and oleophobic substrate surface: The silicon substrate was ultrasonically cleaned sequentially with acetone-ethanol-isopropanol. After drying, soot was deposited on the substrate surface. Then, it was treated with tetraethoxysilane and ammonia water under vacuum conditions in a vacuum dryer for 24 hours. Then, it was sintered in a muffle furnace at 500℃ for 6 hours. After cooling to room temperature, it was treated with oxygen plasma (treatment conditions: 150W, treatment time: 400s). Then, it was soaked in a 0.5wt% heptadecafluorodecyltriethoxysilane-hexane solution for 1 hour. After removal, the substrate was heated at 70℃ for 2 hours.

[0030] 3D printing for rigid three-dimensional microstructures: Rigid three-dimensional microstructures are prepared using powder bed fusion 3D printing technology and filled with a general-purpose solid carrier. The microstructures are prepared using powder composed of titanium alloy and phenolic resin. The relative content of the printing components is adjusted according to the requirements of DNA molecule synthesis.

[0031] DNA preparation by inkjet printing: 1) Print 5 wt% trichloroacetic acid-dichloromethane solution 5 times into a microstructure filled with a universal solid-phase carrier, reacting for 0.5 min each time; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Print 5 times sequentially, alternating between 1 wt% acetonitrile solution of A, T, C, and G base monomers and 5 wt% 5-ethimercaptotetrazole-acetonitrile solution, reacting for 10 min each time; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print 5 times with 10 wt% acetic anhydride-tetrahydrofuran solution and 10 wt% N-methylimidazolium-tetrahydrofuran solution, reacting for 0.5 min each time; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print 5 times with an oxidant solution containing 5 wt% iodine, 15 wt% pyridine, 60 wt% tetrahydrofuran, and the remainder water, reacting for 0.5 min each time; 8) Rinse with sufficient acetonitrile to remove unreacted reactants.

[0032] Example 2

[0033] Preparation of superhydrophobic and oleophobic substrate surface: The silica substrate was ultrasonically cleaned sequentially with acetone-ethanol-isopropanol. After drying, soot was deposited on the substrate surface. Then, it was treated with tetraethoxysilane and ammonia water under vacuum conditions in a vacuum dryer for 48 hours. After that, it was sintered in a muffle furnace at 600℃ for 4 hours. After cooling to room temperature, it was treated with oxygen plasma (treatment conditions: 250W, treatment time: 200s). Then, it was soaked in a 0.1wt% heptadecafluorodecyltriethoxysilane-hexane solution for 2 hours. After taking it out, the substrate was heated at 50℃ for 5 hours.

[0034] Preparation of three-dimensional microstructures: Rigid three-dimensional microstructures were prepared using powder bed fusion 3D printing technology and filled with a general-purpose solid carrier. The microstructures were prepared using powder composed of aluminum alloy and furan resin. The relative content of the printing components was adjusted according to the requirements of DNA molecule synthesis.

[0035] DNA preparation by inkjet printing: 1) Print 8 times with a 1 wt% trichloroacetic acid-dichloromethane solution into a microstructure filled with a universal solid-phase carrier, reacting for 2 min each time; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Print 10 times alternately with 10 wt% acetonitrile solution of A, T, C, and G base monomers and 1 wt% 5-ethimercaptotetrazole-acetonitrile solution, reacting for 5 min each time; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print 10 times with a 15 wt% acetic anhydride-tetrahydrofuran solution and a 20 wt% N-methylimidazolium-tetrahydrofuran solution, reacting for 2 min each time; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print 10 times with an oxidant solution containing 1 wt% iodine, 20 wt% pyridine, 80 wt% tetrahydrofuran, and the remainder water, reacting for 5 min each time; 8) Rinse with sufficient acetonitrile to remove unreacted reactants.

[0036] Example 3

[0037] Preparation of superhydrophobic and oleophobic substrate surface: The quartz substrate was ultrasonically cleaned sequentially with acetone-ethanol-isopropanol. After drying, soot was deposited on the substrate surface. Then, it was treated with tetramethoxysilane and ammonia water under vacuum conditions in a vacuum dryer for 36 hours. Then, it was sintered in a muffle furnace at 800℃ for 2 hours. After cooling to room temperature, it was treated with oxygen plasma (treatment conditions: 200W, treatment time: 300s). Then, it was soaked in a 0.3wt% triethylfluorosilane-hexane solution for 5 hours. After removal, the substrate was heated at 60℃ for 3 hours.

[0038] Fabrication of three-dimensional microstructures: Rigid three-dimensional microstructures were fabricated using powder bed fusion 3D printing technology and filled with a general-purpose solid carrier. The microstructures were fabricated using powder composed of stainless steel and silicone resin.

[0039] DNA preparation by inkjet printing: 1) Print 3 wt% trichloroacetic acid-dichloromethane solution 10 times into a microstructure filled with a universal solid-phase carrier, reacting for 3 min; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Print alternately 20 wt% acetonitrile solution of A, T, C, and G base monomers and 2 wt% 5-ethimercaptotetrazole-acetonitrile solution 10 times, reacting for 10 min; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print 10 wt% acetic anhydride-tetrahydrofuran solution and 25 wt% N-methylimidazolium-tetrahydrofuran solution 8 times, reacting for 3 min; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print 10 times with an oxidant solution containing 3 wt% iodine, 15 wt% pyridine, 70 wt% tetrahydrofuran, and the remainder water, reacting for 1 min; 8) Rinse with sufficient acetonitrile to remove unreacted reactants.

[0040] Example 4

[0041] Preparation of superhydrophobic and oleophobic substrate surface: The glass substrate was ultrasonically cleaned sequentially with acetone-ethanol-isopropanol. After drying, soot was deposited on the substrate surface. Then, it was treated with tetraethoxysilane and ammonia water under vacuum conditions in a vacuum dryer for 36 hours. Then, it was sintered in a muffle furnace at 580℃ for 4.5 hours. After cooling to room temperature, it was treated with air plasma (treatment conditions: 180W, treatment time: 250s). Then, it was soaked in a 0.2wt% tridecafluorooctyltrimethoxysilane-hexane solution for 3 hours. After removal, the substrate was heated at 65℃ for 2.5 hours.

[0042] Fabrication of three-dimensional microstructures: Rigid three-dimensional microstructures were fabricated using powder bed fusion 3D printing technology and filled with a general-purpose solid carrier. The microstructures were fabricated using powder composed of aluminum alloy and alkyd resin.

[0043] DNA preparation by inkjet printing: 1) Print 8 times with a 4 wt% trichloroacetic acid-dichloromethane solution into a microstructure filled with a universal solid-phase carrier, reacting for 1 min; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Print 10 times alternately with 15 wt% acetonitrile solution of A, T, C, and G base monomers and 3 wt% 5-ethimercaptotetrazole-acetonitrile solution, reacting for 8 min; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print 10 times with 8 wt% acetic anhydride-tetrahydrofuran solution and 18 wt% N-methylimidazolium-tetrahydrofuran solution, reacting for 2.5 min; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print 10 times with an oxidant solution containing 4 wt% iodine, 20 wt% pyridine, 65 wt% tetrahydrofuran, and the remainder water, reacting for 3 min; 8) Rinse with sufficient acetonitrile to remove unreacted reactants.

[0044] Comparative Example 1

[0045] Preparation of three-dimensional microstructures: Rigid three-dimensional microstructures were prepared on the surface of a silica substrate after sequential ultrasonic cleaning with acetone-ethanol-isopropanol using powder bed fusion 3D printing technology. A general-purpose solid carrier was filled into the microstructures. The microstructures were prepared using powder composed of aluminum alloy and furan resin. The relative content of the printing components was adjusted according to the requirements of DNA molecule synthesis.

[0046] Inkjet printing for DNA preparation: 1) Print 8 times with a 1 wt% trichloroacetic acid-dichloromethane solution into a microstructure filled with a universal solid-phase carrier, reacting for 2 min each time. 2) Rinse with sufficient acetonitrile to remove unreacted reactants. 3) Print 10 times sequentially, alternating between a 10 wt% acetonitrile solution of A, T, C, and G base monomers and a 1 wt% 5-ethimercaptotetrazole-acetonitrile solution, reacting for 5 min each time. 5) Print 10 times with a 15 wt% acetic anhydride-tetrahydrofuran solution and a 20 wt% N-methylimidazolium-tetrahydrofuran solution, reacting for 2 min each time. 6) Rinse with sufficient acetonitrile to remove unreacted reactants. 7) Print 10 times with an oxidant solution containing 1 wt% iodine, 20 wt% pyridine, 80 wt% tetrahydrofuran, and the remainder water, reacting for 5 min each time. 8) Rinse with sufficient acetonitrile to remove unreacted reactants.

[0047] The above examples and comparative examples only illustrate the preparation process. DNA cutting and purification use general methods, and this invention is not limited thereto. The DNA deletion rates prepared in the examples and comparative examples of this invention are shown in Table 1.

[0048] Table 1 Comparison of DNA loss rate results between the examples and the comparative examples

[0049] Example Missing rate (%) 1 4 2 3 3 2 4 4 Comparative Example 1 8

[0050] The foregoing embodiments are merely illustrative, used to explain some features of the method described in this invention. The appended claims are intended to claim the broadest possible scope, and the embodiments presented herein are merely illustrative of a selection of combinations of all possible embodiments. Therefore, the applicant intends that the appended claims are not limited by the selection of feature examples illustrating the invention. Some numerical ranges used in the claims also include sub-ranges within them, and variations within these ranges should also be interpreted as being covered by the appended claims where possible.

Claims

1. A fully printed synthesis method and its application in DNA preparation, characterized in that, By treating the substrate surface with superhydrophobic and oleophobic agents, and by controlling the printing method, printing material composition, and the types and amounts of reactants, DNA can be synthesized simply and efficiently.

2. The fully printed synthesis method according to claim 1 and its application in DNA preparation, characterized in that, The described full-print synthesis method includes the following steps: First, a rigid three-dimensional microstructure is prepared on the surface of a superhydrophobic and oleophobic substrate using 3D printing. Then, a general-purpose solid carrier is filled into the microstructure, and the filling is completed and sintered. After cooling to room temperature, a deprotecting agent, ATCG base monomer, activator, capping agent, and oxidant are sequentially printed into the micropores using inkjet printing to prepare DNA. The deletion rate of the obtained DNA is less than 5%.

3. The fully printed synthesis method according to claim 1 or 2 and its application in DNA preparation, characterized in that, The superhydrophobic and oleophobic treatment involves first ultrasonically cleaning the substrate sequentially with acetone, ethanol, and isopropanol. After drying, a layer of soot with a thickness of 2-10 μm is deposited on the surface. Then, the substrate is treated with a mixture of tetramethoxysilane or tetraethoxysilane and ammonia vapor under vacuum conditions in a vacuum dryer for 24-48 hours. Next, the substrate is sintered in a muffle furnace at 500-800°C for 2-6 hours. After cooling to room temperature, the substrate is treated with plasma in an air or oxygen atmosphere at 150-250W for 200-400 seconds. Then, the substrate is immersed in a 0.1-0.5wt% fluorosilane-alkane solution for 1-5 hours. Finally, the substrate is heated at 50-80°C for 2-5 hours.

4. The fully printed synthesis method according to claim 1 or 2 and its application in DNA preparation, characterized in that, The substrate is selected from one of silicon substrate, silicon dioxide substrate, quartz substrate or glass substrate, preferably silicon substrate.

5. The fully printed synthesis method according to claim 1 or 2 and its application in DNA preparation, characterized in that, The 3D printing uses powder bed fusion technology, and the printing material is composed of alloy powder and binder; the three-dimensional microstructure is one of the following: a bottom-through inverted opal structure, a porous structure, or a polygonal 3D microstructure, wherein the polygonal 3D microstructure is selected from one of pyramidal, cylindrical, or hemispherical shapes.

6. The fully printed synthesis method according to claim 5 and its application in DNA preparation, characterized in that, The alloy powder in the 3D printing material is selected from one of titanium alloy, aluminum alloy and stainless steel, and the binder is a photosensitive resin selected from one of furan resin, phenolic resin, alkyd resin and silicone resin.

7. The fully printed synthesis method according to claim 5 and its application in DNA preparation, characterized in that, The sintering temperature of the powder bed melting technology is 500-1000℃.

8. The fully printed synthesis method according to claim 1 or 2 and its application in DNA preparation, characterized in that, The deprotectant solution used in inkjet printing consists of 1-5 wt% trichloroacetic acid and the balance dichloromethane; the activator solution consists of 1-5 wt% 5-ethimercaptotetrazole and the balance acetonitrile; the capping agent solution consists of 5-15 wt% acetic anhydride, 10-20 wt% N-methylimidazolium and the balance tetrahydrofuran; and the oxidant solution consists of 1-5 wt% iodine, 15-25 wt% pyridine, 50-80 wt% tetrahydrofuran and the balance water.

9. The fully printed synthesis method according to claim 1 or 2 and its application in DNA preparation, characterized in that, The specific process for preparing DNA by inkjet printing is as follows: 1) Print a deprotecting agent solution into a microstructure filled with a universal solid-phase carrier, reacting for 0.5-3 min; 2) Rinse with sufficient acetonitrile to remove unreacted reactants; 3) Sequentially print 1-20 wt% acetonitrile solutions of A, T, C, and G base monomers and activator solutions, reacting for 2-10 min; 4) Rinse with sufficient acetonitrile to remove unreacted reactants; 5) Print a capping agent solution, reacting for 0.5-3 min; 6) Rinse with sufficient acetonitrile to remove unreacted reactants; 7) Print an oxidizing agent solution, reacting for 0.5-5 min; 8) Rinse with sufficient acetonitrile to remove unreacted reactants. To ensure sufficient reaction of reactants in each step, each step is inkjet printed at least 5 times. The number of times the deprotecting agent solution is printed depends on the amount of solid-phase carrier.

10. A fully printed synthesis method using any one of claims 1-9 and its application in DNA preparation.

Images