SURFACE FUNCTIONALIZING METHOD FOR USE IN HIGH-THROUGHPUT IN SITU SYNTHESIS OF NUCLEIC ACIDS BY 3D INKJET PRINTING

Abstract

A surface functionalizing method for use in high-throughput in situ synthesis of nucleic acids by 3D inkjet printing. The method includes subjecting a surface of a substrate to hydroxyl enrichment treatment; adding hydrophobic molecules to the surface of the substrate, the hydrophobic molecules being not reactive with phosphoramidite monomers; spraying, by a multi-channel piezoelectric inkjet head assembly, an etching ink to a predetermined area on the surface of the substrate for micro-etching, the etching ink being prepared with a fluoride compound reactive with the hydrophobic molecules; and adding hydrophilic molecules to the surface of the substrate. By using the method, a functionalized surface with given areas being patterned can be formed on the surface of the substrate, and then a same multi-channel piezoelectric inkjet head assembly can be directly used for subsequent high-resolution printing of phosphoramidite monomers and synthesis of nucleic acids.

Claims

1. A surface functionalizing method for use in high-throughput in situ synthesis of nucleic acids by 3D inkjet printing, comprising the following steps: 1) subjecting a surface of a substrate to hydroxyl enrichment treatment; 2) adding hydrophobic molecules to the surface of the substrate, wherein the hydrophobic molecules do not react with phosphoramidite monomers; 3) spraying, by a multi-channel piezoelectric inkjet head assembly, an etching ink to a predetermined area on the surface of the substrate for micro-etching, wherein the etching ink is prepared with a fluoride compound, wherein the fluoride compound reacts with the hydrophobic molecules; and 4) adding hydrophilic molecules to the surface of the substrate, by which functionalizing of the surface of the substrate is realized.

2. The method according to claim 1, wherein the substrate is a quartz glass slide or a silicon slice provided with a silicon dioxide coating.

3. The method according to claim 1, wherein in step 1), the hydroxyl enrichment treatment is carried out using a piranha solution.

4. The method according to claim 1, wherein in step 2), the hydrophobic molecules are fluorooctyltrichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane, or (tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane.

5. The method according to claim 1, wherein in step 3), the fluoride compound is at least one selected from the group consisting of ammonium fluoride, tetrabutylammonium fluoride, phenyltrimethyl ammonium fluoride, triethylamine trihydrofluoride, and pyridine hydrofluoride.

6. The method according to claim 1, wherein in step 3), a solvent is used for preparing the etching ink and the solvent has the following characteristics: 1) the solvent has certain capability of dissolving the fluoride compound; 2) the etching ink prepared with the solvent is capable of forming ink droplets with good morphology when sprayed through a nozzle of the multi-channel piezoelectric inkjet head assembly.

7. The method according to claim 6, wherein the solvent used for preparing the etching ink is one or more selected from the group consisting of polypropylene carbonate, polyethylene glycol, acetonitrile, 2-methylglutaronitrile, and 3-methoxypropionitrile.

8. The method according to claim 1, wherein in step 3), the multi-channel piezoelectric inkjet head assembly comprises six channels, each channel comprising 180-256 independent nozzles.

9. The method according to claim 1, wherein in step 4), the hydrophilic molecules are 3-glycidyloxypropyltrimethoxysilane, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, or 3-iodo-propyltrimethoxysilane.

10. The method according to claim 7, wherein in step 3), the fluoride compound is at least one selected from the group consisting of ammonium fluoride, tetrabutylammonium fluoride, phenyltrimethyl ammonium fluoride, triethylamine trihydrofluoride, and pyridine hydrofluoride.

11. The method according to claim 10, wherein in step 2), the hydrophobic molecules are fluorooctyltrichlorosilane, (tridecafluoro-1,1,2,2-tetrahydrooctyl)trichlorosilane, or (tridecafluoro-1,1,2,2-tetrahydrooctyl)trimethoxysilane.

12. The method according to claim 11, wherein in step 3), the multi-channel piezoelectric inkjet head assembly comprises six channels, each channel comprising 180-256 independent nozzles.

13. The method according to claim 12, wherein in step 4), the hydrophilic molecules are 3-glycidyloxypropyltrimethoxysilane, N-(3-triethoxysilylpropyl)-4-hydroxybutyramide, 11-acetoxyundecyltriethoxysilane, n-decyltriethoxysilane, (3-aminopropyl)trimethoxysilane, (3-aminopropyl)triethoxysilane, or 3-iodo-propyltrimethoxysilane.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] FIG. 1 is a schematic diagram of formulas of chemical reactions occurred during patterning by a fluoride etching ink printing method.

[0028] FIG. 2A is an overall schematic diagram of a multi-channel piezoelectric inkjet head assembly.

[0029] FIG. 2B is a side perspective schematic diagram of the multi-channel piezoelectric inkjet head assembly.

[0030] FIG. 2C is a bottom perspective schematic diagram of the multi-channel piezoelectric inkjet head assembly.

[0031] FIGS. 3A-3E are a schematic process flow showing functionalization of a surface with two different coating materials (a hydrophobic molecular layer and a hydrophilic molecular layer) by the fluoride etching ink printing method.

[0032] FIG. 4 is a schematic diagram of reaction active areas (white) and reaction inactive areas (gray) patterned on the surface treated and functionalized by the fluoride etching ink printing method, with oligonucleotides layers extending from active functionalized surfaces.

[0033] FIG. 5A is a schematic diagram showing piezoelectric inkjet printing by first to sixth channels onto the surface of the substrate.

[0034] FIG. 5B is a side perspective schematic diagram showing the piezoelectric inkjet printing by the first to sixth channels onto the surface of the substrate.

[0035] FIGS. 6A-6D are a schematic flow chart showing forming of a patterned surface including different active and inactive functionalized areas by the fluoride etching ink printing method.

[0036] FIG. 7A is a photograph of distribution of droplets spontaneously formed by direct treatment with a large amount of water vapor on the surface of the substrate differentially functionalized by the fluoride etching ink printing method.

[0037] FIG. 7B is a photograph is a photograph of distribution of droplets formed after printing with a phosphoramidite monomer ink used for synthesis of oligonucleotides on the surface of the substrate differentially functionalized by the fluoride etching ink printing method.

[0038] FIG. 8 is a diagram showing effect of microarray-based synthesis of oligonucleotides with 150 bases on the surface of the substrate treated and functionalized by the fluoride etching ink printing method of the present disclosure.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] Experimental methods used in the following embodiments are conventional methods unless otherwise specified.

[0040] Materials, reagents and so on used in the following embodiments are commercially available unless otherwise specified.

[0041] 1. Pretreatment of a Surface to be Functionalized

[0042] 1.1 High-purity quartz glass, with its main component being silicon dioxide and hereinafter referred to as glass slide, was used as a substrate.

[0043] 1.2 The glass slide was ultrasonically rinsed with deionized water for 30 minutes and then dried at 80° C.

[0044] 1.3 The glass slide was immersed in a freshly prepared piranha solution (H.sub.2SO.sub.4/30% H.sub.2O.sub.2, volume ratio being 3:1), and incubated for 1 hour at room temperature, in which case a large number of air bubbles were generated. It should be noted that the process of preparing the piranha solution will be accompanied by a violent exothermic phenomenon, and therefore hydrogen peroxide should be carefully poured into concentrated sulfuric acid along an inner wall of a beaker with constant stirring to speed up heat dissipation. The solution is very corrosive and should be handled with care.

[0045] 1.4 The above glass slide was transferred to a new staining tank, rinsed with a large amount of deionized water for several times until resultant wastewater was nearly neutral, then soaked in acetonitrile for 20 seconds, taken out and quickly dried with nitrogen.

[0046] 2. Treatment for a First Layer of Hydrophobic Molecules 2.1 The dried glass slide was immersed in a freshly prepared 0.02 M FOTS solution in dichloromethane, and incubated at room temperature for 30 minutes in a dry atmosphere.

[0047] 2.2 The glass slide was taken out, rinsed successively with dichloromethane and acetonitrile, and then dried for later use.

[0048] 3. Preparation of a Fluoride Compound Etching Ink

[0049] 3.1 The fluoride compound etching ink was prepared as follows. Trifluoroacetic acid was dropwise added to 2M TBAF solution in polypropylene carbonate to adjust the pH to near neutral (pH=6-7) to obtain the fluoride compound etching ink.

[0050] 4. Etching by Printing and Post-Treatment

[0051] 4.1 The ink was rinsed off with a large amount of absolute ethanol to terminate an etching reaction. After being dried, the glass slide was fully coated with GOPS (i.e., a second layer of hydrophilic molecules) or a general linker solution in DMF for a reaction overnight at room temperature in a dry atmosphere, and then rinsed with a large amount of acetonitrile solution and dried for later use. The treatment of the glass slide in this step can effectively prevent re-hydrophobicization of etched areas, thus playing the role of immobilizing hydrophilic groups. Meanwhile, the addition of the general linker can make it easier to cut final synthesized oligonucleotides from the glass slide.

[0052] Formulas of chemical reactions occurred during the above treatment are shown in FIG. 1. From the right column to the left column, the first reaction is adding the first layer of hydrophobic molecules to the surface of the silicon dioxide; the second reaction is the fluoride compound selectively removing the hydrophobic molecules from a given area; the third reaction is selectively adding a layer of hydrophilic molecules to the given area; the fourth reaction is adding a layer of spacer molecules to the layer of hydrophilic molecules; and the fifth reaction is adding the general linker molecules for use in synthesis and subsequent cutting of oligonucleotides.

[0053] The present embodiment adopts a multi-channel piezoelectric inkjet head assembly with six channels, each channel including 180-256 independent nozzles. The first channel is used for inkjet printing of an activator Tetrazole; the second, third, fourth and fifth channels are used for inkjet printing of four types of phosphoramidite monomers, including 2′-F-Ac-dC phosphoramidite monomer, DMF-dG phosphoramidite monomer, def-dA phosphoramidite monomer, and TBB-dT phosphoramidite monomer; and the sixth channel is used for printing of the fluoride etching ink. An overall schematic diagram of the multi-channel piezoelectric inkjet head assembly is shown in FIG. 2A, a side perspective schematic diagram thereof is shown in FIG. 2B, and a bottom perspective schematic diagram thereof is shown in FIG. 2C. A schematic diagram showing printing by the six channels onto the surface of the substrate is shown in FIG. 5A, and a side perspective schematic diagram thereof is shown in FIG. 5B.

[0054] FIGS. 3A-3E are a schematic process flow showing functionalization of a surface with two different coating materials by the fluoride etching ink printing method (it is different from FIG. 1 in that it is viewed from the perspective of a given area of the surface). The first step represents treatment for a layer of hydrophobic molecules (FIG. A-FIG. B); the second step represents selective etching with a fluoride compound (FIG. B-FIG. C); the third step represents addition of a layer of hydrophilic molecules (FIG. C-FIG. D); and the fourth step represents addition of a layer of a linker used for synthesis of oligonucleotides on the layer of hydrophilic molecules (FIG. D-FIG. E).

[0055] FIG. 4 is a schematic diagram of reaction active areas (areas inside the white circles represent the reaction active areas) and reaction inactive areas (gray) patterned on the surface treated and functionalized by the fluoride etching ink printing method of the present embodiment, with oligonucleotide layers extending from active functionalized surfaces.

[0056] FIGS. 6A-6D are a schematic flow chart showing forming of a patterned surface including different active and inactive functionalized areas by the fluoride etching ink printing method of the present embodiment. The first step represents treatment for a layer of hydrophobic molecules (FIG. A-FIG. B); the second step represents selective etching treatment with a fluoride compound (FIG. B-FIG. C); the third step represents addition of a layer of hydrophilic molecules and a layer of a linker used for synthesis of oligonucleotides (FIG. C-FIG. D), oligonucleotides being to be synthesized on the active functionalized areas on the surface (the areas filled with the black crosses in the figure).

[0057] FIG. 7A is a photograph of distribution of droplets spontaneously formed by direct treatment with a large amount of water vapor on the surface of the substrate differentially functionalized by the fluoride etching ink printing method of the present embodiment. As can be seen, patterned areas exhibit high hydrophilicity, while other areas exhibit hydrophobicity. The patterned areas can be used for subsequent synthesis of oligonucleotides.

[0058] FIG. 7B is a photograph of distribution of droplets formed after printing with a phosphoramidite monomer ink used for synthesis of oligonucleotides on the surface of the substrate differentially functionalized by the fluoride etching ink printing method of the present embodiment. It can be seen that patterned areas used for the synthesis exhibit good droplet morphology.

[0059] FIG. 8 is a diagram showing effect of microarray-based synthesis of oligonucleotides with 150 bases on the surface of the substrate treated and functionalized by the fluoride etching ink printing method of the present embodiment, with the last base indicated by a Cy3 fluorescently labeled monomer. It can be seen from this figure that after 150 rounds of synthesis, synthetic features of the oligonucleotides still maintain good morphology, and the synthesized last base being fluorescently labeled indicates that the surface treated and functionalized according to the present disclosure remains normal after repeated rinsing. The method can be successfully used for high-throughput synthesis of long-chain oligonucleotides.

INDUSTRIAL APPLICATION

[0060] By using the method of the present disclosure, a functionalized surface with given areas being patterned can be formed on a surface of a substrate, and then a same multi-channel piezoelectric inkjet head assembly can be directly used for subsequent high-resolution printing of phosphoramidite monomers and synthesis of nucleic acids. Compared with current methods of the same kind, the method of the present disclosure does not require expensive photolithography devices and designs for repeated position calibration, thereby reducing synthesis cost. The method can also increase density of available active sites on functionalized sites, and improve yield of a single oligonucleotide.