High-throughput synthesis chip of oligonucleotide chain and application of high-throughput synthesis chip

Abstract

The invention relates to an oligonucleotide chain high-flux synthesis chip and application thereof, the surface of the chip is integrated with a micropore synthesis unit array with a straight cylinder type microstructure, the area of an array region accounts for 37% or more of the area of the chip, the aperture ratio of the array region is 38%-46%, the aperture density is 13 / mm < 2 >-650 / mm < 2 >, the ratio of the total surface area of the synthesis unit to the area of the array region is not more than 0.90, and the specific surface area of the synthesis unit to the area of the array region is not more than 0.90. A functional reagent which can be grafted with the oligonucleotide monomer is coated in the synthesis unit, and an inert reagent which cannot be grafted with the oligonucleotide monomer is covered outside the synthesis unit. The chip-integrated straight-cylinder-shaped synthesis units are numerous, the three-dimensional structure is simple, the synthesis efficiency is high, the manufacturing period is short, the cost is low, and the chip-integrated straight-cylinder-shaped synthesis unit is suitable for industrialization of oligonucleotide chain high-throughput synthesis.

Description

Technical Field

[0001] The invention relates to the field of microfluidic solid-phase chemical synthesis of nucleotides, and in particular to a high-throughput synthesis chip for oligonucleotide chains. Background Art

[0002] With the development of biology, the importance of biomacromolecules in modern medicine and people's daily life has attracted more and more attention. Polysaccharides, specific proteins, nucleic acids, nucleoside analogs and other bioactive macromolecules are important directions and tools for modern medical and biological research, among which nucleic acids are the most critical biomacromolecules. The preparation of specific nucleic acids according to needs is the basis of gene library preparation, primer synthesis, gene modification and other projects.

[0003] Methods for obtaining nucleic acids in large quantities include biological methods such as microbial culture and reproduction, or the use of polymerase chain reaction (PCR amplification) to amplify a small amount of nucleic acid chains, and solid-phase chemical synthesis. The first two methods require the provision of existing nucleic acid chains as templates, while solid-phase chemical synthesis uses chemical methods to prepare target nucleic acids from scratch using basic reagents. Compared to the first two methods that are restricted by template acquisition methods, solid-phase synthesis can be separated from established biological materials and is currently the main means of synthesizing nucleic acids in the industry.

[0004] The solid phase phosphoramidite method is a relatively mature strategy for solid phase synthesis of nucleic acids. After nearly four decades of development, the synthesis of oligonucleotide chains based on the solid phase phosphoramidite method has moved from mature laboratory technology to industrial production. In recent years, in the research of synthesis technology using the solid phase phosphoramidite method, research based on microfluidic chips and inkjet printing technology has attracted attention: microfluidic chips integrate a large number of solid phase synthesis units, and the inkjet print head selectively inputs phosphoramidite reaction raw materials to each synthesis unit, thereby realizing high-throughput in situ customized synthesis of nucleic acids. Among them, the research on chips is currently mostly focused on the optimization and improvement of the structure of the synthesis unit, including processing the synthesis unit into a fancy hole with a specific aspect ratio and a specific internal structure (CN114100714B, US9981239B2), bonding multiple layers of substrates with fancy holes together (CN111036316B), etc., aiming to increase the surface area of ​​the microstructure to obtain a higher synthesis flux.

[0005] However, as the surface area of ​​the microstructure increases, the reagents in the reaction process will have a significant accumulation effect. Complex structures such as fancy holes and multi-layer holes make it more difficult to clean the reagents during the synthesis process, and the risk of reagent residues is greater, affecting the efficiency of the synthesis. At the same time, complex microstructures also bring high processing costs: the processing technology of fancy deep holes includes deep silicon etching, bonding, high-precision lithography and other technologies, which have high requirements for equipment performance and precision, long processing cycles, and high costs, which are not suitable for industrial advancement. Summary of the invention

[0006] In view of the above problems, the present invention provides a high-throughput oligonucleotide chain synthesis chip with high synthesis efficiency and easy processing. A two-dimensional array consisting of a large number of microporous synthesis units is integrated on the surface of the basic material of the chip, and the opening rate of the array area is represented by η, then 38%≤η≤46%.

[0007] Preferably, 38%≤η≤45%.

[0008] Furthermore, 40%≤η≤45%.

[0009] The distribution of the synthesis units in the array area includes every mm 2 Lay out 13~650 synthesis units.

[0010] Preferably, per mm 2 The number of deployed synthesis units ranges from 70 to 200.

[0011] The micropore synthesis unit includes a straight cylindrical microstructure extending downward perpendicularly to the chip surface.

[0012] The ratio W of the total surface area of ​​the straight-tube microstructure to the area of ​​the array region is between 0.40 and 0.90.

[0013] Preferably, 0.42≤W≤0.51.

[0014] Preferably, the value of W is controlled by controlling the depth h of the microstructure extending downward.

[0015] Preferably, 1 µm≤h≤30 µm.

[0016] Furthermore, 1 µm≤h≤10 µm.

[0017] The microporous synthesis units are arranged on the chip surface in the shape of a regular triangle, a regular quadrilateral or an isosceles triangle.

[0018] Preferably, the microporous synthesis units are arranged in an isosceles triangle on the chip surface.

[0019] The array area of ​​the chip accounts for more than 37% of the surface area of ​​the chip base material.

[0020] The basic material of the chip includes one of a silicon wafer, a silicon-doped solid solution slice, a ceramic wafer or a quartz glass wafer.

[0021] Preferably, the base material comprises one of a silicon wafer or a quartz glass wafer.

[0022] The processing method of the straight-tube microstructure includes conventional microelectronic processing technology, including photolithography and dry etching and / or photolithography and wet etching.

[0023] Preferably, the processing method of the microporous structure includes ultraviolet lithography and dry etching.

[0024] The synthesis of the oligonucleotide chain is based on the phosphoramidite triester method.

[0025] The inner surface of the straight-tube microstructure is covered with a functionalized reagent for grafting phosphoramidite monomers.

[0026] The chip is covered with an inert reagent that cannot be grafted with the phosphoramidite monomer outside the straight-tube microstructure.

[0027] The application of the chip includes application in a high-throughput synthesis process of oligonucleotide chains.

[0028] The application of the chip includes integrating the chip into other chips or devices for high-throughput synthesis of oligonucleotide chains.

[0029] The application of the chip includes application in the high-throughput synthesis process of macromolecules with biological activity.

[0030] The beneficial effects of the present invention are:

[0031] 1. The present invention provides a high-throughput synthesis chip for oligonucleotide chains with high synthesis efficiency: a reaction array composed of a large number of straight-tube microporous synthesis units is arranged on the surface of the chip, providing a carrier for the high-throughput synthesis of oligonucleotide chains; the appropriate microstructure surface area eliminates the accumulation effect of reagents during the synthesis process and improves the accuracy of the synthesis.

[0032] 2. The chip provided by the present invention can be obtained by conventional microelectronic processing methods, has simple process requirements, short processing cycle, is easy to produce in a standardized manner, and has the potential for industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 It is a chip mask on a 4-inch substrate.

[0034] Figure 2 It is a chip mask on a 6-inch substrate.

[0035] Figure 3 Schematic diagram of the horizontal arrangement of straight-cylindrical microstructures on the chip surface (a is a regular quadrilateral arrangement; b is a regular triangle arrangement; c is an isosceles triangle arrangement).

[0036] Figure 4 Schematic diagram of a straight-tube microstructure (a: chip localization; b: enlargement).

[0037] Figure 5 Schematic diagram of the functionalized reagent processing chip structure.

[0038] Figure 6 This is a standard curve of nucleotide concentration.

[0039] Meaning of the symbols in the figure:

[0040] 101: chip cutting lines on a 4-inch substrate; 102: array area mask; 201: chip cutting lines on a 6-inch substrate; 202: array area mask; 501: non-reactive area covered with inert reagents; 502: functional reagents that can be grafted with phosphoramidite monomers; 503: chip base materials; 504: inkjet microdroplets containing phosphoramidite monomers during the reaction process; 601: the position of the sample in Example 3 corresponding to the standard curve; 602: the position of the sample in Example 4 corresponding to the standard curve. DETAILED DESCRIPTION

[0041] The present invention provides a chip for high-throughput synthesis of oligonucleotide chains. The basic material of the chip includes a silicon wafer, a ceramic wafer or a glass wafer, and the surface has a two-dimensional array composed of microporous synthesis units. The array area accounts for more than 37% of the surface area of ​​the chip. The synthesis units are arranged in a regular quadrilateral, a regular triangle or an isosceles triangle. The number of units arranged per square millimeter is represented by a, 13≤a≤650; the opening rate of the array area is represented by η, 38%≤η≤46%. The synthesis unit includes a straight cylindrical microstructure extending perpendicular to the chip surface to the bottom of the surface. The ratio of the total surface area of ​​the microstructure to the area of ​​the array area is represented by W, 0.4≤W≤0.9. The microstructure can be obtained by conventional microelectronic processing technology. The inner wall of the microstructure is covered with a functionalized reagent that can be grafted with a phosphoramidite monomer, and the part outside the microstructure is covered with an inert reagent that cannot be grafted with the monomer.

[0042] Where η = A a / A s ; W = A0 × n / A s ; a=n / A s (A a is the total opening area on the chip surface, A s is the array area, A0 is the micropore inner surface area, and n is the number of micropores).

[0043] 1. Chip Preparation

[0044] Taking silicon wafer as an example, the chip is prepared as follows:

[0045] Step 1: Forming microstructures. First, spin-coat photoresist on a silicon wafer, dry it, and then perform UV lithography and cleaning on the photoresist surface to obtain a mask with a reaction array pattern. Then, etch the substrate with the mask to form a straight-tube microstructure array. After etching, cut it according to the predetermined size.

[0046] Step 2: Surface functionalization: After the surface micropore structure is formed, functional reagents are applied to the inner wall of the micropores, and inert reagents are used to cover the non-reactive areas outside the micropores.

[0047] 2. Application of the chip

[0048] Based on the inkjet biosynthesizer, the chip is applied as follows:

[0049] The first step: placing the chip in the reaction chamber of a synthesizer.

[0050] Step 2: Fill the reaction chamber with protective gas until the water-oxygen content reaches a predetermined value.

[0051] Step 3: Treat the synthesis unit on the chip surface with a deprotecting agent and drain it after a period of reaction.

[0052] Step 4: Introduce cleaning solution to rinse the chip surface.

[0053] Step 5: Fill the reaction chamber with protective gas, drain the cleaning liquid, and then use the inkjet print head to deliver the required monomer reagents to each reaction unit on the surface of the synthesis chip, and then deliver the activation reagents for reaction.

[0054] Step 6: Clean the chip surface again.

[0055] Step 7: Refill the reaction chamber with protective gas, drain the cleaning solution, deliver the capping reagent (a 1:1 mixture of CapA and CapB) to the surface of the synthesis chip, and drain the reagent after a period of reaction.

[0056] Step 8: Introduce cleaning solution to clean excess CapA and CapB reagents from the chip surface.

[0057] Step 9: Fill the reaction chamber with protective gas, drain the cleaning liquid, and then deliver the oxidant to the surface of the synthesis chip, and drain it after a period of reaction.

[0058] Step 10: After introducing the cleaning liquid to clean the oxidant, fill in the protective gas to exhaust the cleaning reagent.

[0059] The third to tenth steps are cycled to graft monomer reagents in sequence according to the target sequence to complete the synthesis of the oligonucleotide chain.

[0060] 3. Determination of synthesis results

[0061] a. Sequencing of synthesis results

[0062] The oligonucleotide chains synthesized on the chip are amino-deprotected, purified and recovered to obtain free oligonucleotide sequence products, which are then subjected to PCR cycle amplification to obtain a double-stranded DNA library suitable for second-generation sequencing to complete high-throughput sequencing.

[0063] b. Determination of synthesis amount

[0064] The obtained free oligonucleotide sequence was subjected to real-time fluorescence quantitative polymerase chain reaction (qPCR) using SYBR Green fluorescent dye method, and the total amount of synthesized oligonucleotide chain was accurately determined using standard curve method.

[0065] The content of the present invention is explained below by using specific examples. The examples used only represent one implementation method of the present invention and do not represent any limitation to the present invention.

[0066] Example 1 Chip Preparation

[0067] A positive photoresist was spin-coated at 2000 r / min on a 4-inch diameter circular silicon wafer. After slow drying, the photoresist surface was subjected to UV lithography and cleaning to form a mask with a microporous synthetic unit array area pattern ( Figure 1 The array area is 53.68 mm long and 12.36 mm wide, and contains 46,700 straight cylindrical holes with a diameter of 90 µm, arranged in an isosceles triangle ( Figure 3 c). The masked silicon wafer is then dry-etched to a depth of 3 µm.

[0068] After etching is completed, the reaction area is cut from the silicon wafer according to 75 mm × 24 mm. The distance between the two long boundaries of the array area and the two long boundaries of the chip is 6 mm, and the distance between the two short boundaries and the two short boundaries of the chip is 11 mm. The schematic diagram of the obtained chip microstructure is shown in the figure. Figure 4 .

[0069] The inner wall of the micropores of the cut silicon wafer is coated with a functional reagent that can be grafted with the phosphoramidite monomer, and the other parts of the silicon wafer surface are coated with an inert reagent (such as Figure 5 As shown), a high-throughput synthesis chip was obtained.

[0070] Calculate the chip parameters, the array area opening rate η = 45%, the synthesis unit layout density a = 70 / mm 2 , hole area / array area W=0.51.

[0071] Comparative Example 1 Chip Preparation

[0072] A positive photoresist was spin-coated at 3000 r / min on a 6-inch diameter circular silicon wafer. After slow drying, the photoresist surface was subjected to UV lithography and cleaning. A mask with three sets of parallel patterns of synthetic cell arrays was formed in the center of the silicon wafer ( Figure 2 Each array is 12 mm apart, with an array area of ​​53.68 mm long and 12.36 mm wide. Each array consists of 46,700 cylindrical holes with a diameter of 90 µm, arranged in an isosceles triangle ( Figure 3c). The masked silicon wafer is then etched to a depth of 32 µm.

[0073] After etching is completed, the reaction area is cut from the silicon wafer into 75 mm × 24 mm pieces. The distance between the two long boundaries of the array area and the two long boundaries of the chip is 6 mm, and the distance between the two short boundaries and the two short boundaries of the chip is 11 mm.

[0074] The inner wall of the micropores of the cut silicon wafer is coated with a functional reagent that can be grafted with the phosphoramidite monomer, and the other parts of the silicon wafer surface are coated with an inert reagent (such as Figure 5 As shown), a high-throughput synthesis chip was obtained.

[0075] Calculate the chip parameters, the array area opening rate η = 45%, the synthesis unit layout density a = 70 / mm 2 , hole area / array area W=0.93.

[0076] Example 2 Using the Chip to Synthesize Oligonucleotide Sequences

[0077] The chip obtained in Example 1 is placed on the chip carrier in the reaction chamber of the synthesizer, and the target oligonucleotide chain is synthesized using the obtained chip according to the following steps:

[0078] Step 1: Use high-purity argon as the protective gas to fill the reaction chamber until the water and oxygen contents reach 0.1 ppm respectively.

[0079] Step 2: Treat the synthesis unit on the chip surface with dichloroacetic acid to carry out a deprotection reaction, and then empty it after a period of time.

[0080] Step 3: Introduce acetonitrile to rinse the chip surface and then drain it.

[0081] Step 4: Fill the reaction chamber with argon gas again to exhaust the acetonitrile, and deliver the phosphoramidite monomer solution to each reaction unit on the surface of the synthesis chip through the inkjet print head, and then deliver dicyanoimidazole to each reaction unit for activation.

[0082] Step 5: Clean the chip surface with acetonitrile.

[0083] Step 6: Refill the reaction chamber with argon gas until the acetonitrile is emptied, deliver a mixed reagent of CapA and CapB in a volume ratio of 1:1 to the surface of the synthesis chip, and empty the reagent after a period of reaction.

[0084] Step 7: Use acetonitrile to clean the excess CapA and CapB reagents on the chip surface.

[0085] Step 8: After the acetonitrile is emptied by filling with argon gas, iodine solution is transported to the surface of the synthesis chip for oxidation and emptied after a period of reaction.

[0086] Step 9: After cleaning the oxidant with acetonitrile, evacuate with argon.

[0087] The second step to the ninth step are cycled, and phosphoramidite monomers are grafted onto each synthetic unit in sequence according to the target sequence of synthesis (see Table 1) to complete the synthesis of the oligonucleotide chain.

[0088] The chip with the completed nucleotide chain was placed in an aminolysis instrument. After 4 hours of aminolysis, the oligonucleotide sequence was deprotected and purified to obtain free oligonucleotide sequence. The elution volume was 400 µL.

[0089] Example 3 Synthesizing oligonucleotide sequences using the obtained chip

[0090] The chip obtained in Comparative Example 1 was placed on the chip carrier in the reaction chamber of the synthesizer, and the target oligonucleotide chain was synthesized using the obtained chip according to the following steps.

[0091] Step 1: Use high-purity argon as the protective gas to fill the reaction chamber until the water and oxygen contents reach 0.1 ppm respectively.

[0092] Step 2: Treat the synthesis unit on the chip surface with dichloroacetic acid to carry out a deprotection reaction, and then empty it after a period of time.

[0093] Step 3: Introduce acetonitrile to rinse the chip surface and then drain it.

[0094] Step 4: Fill the reaction chamber with argon gas again to exhaust the acetonitrile, and deliver the phosphoramidite monomer solution to each reaction unit on the surface of the synthesis chip through the inkjet print head, and then deliver dicyanoimidazole to each reaction unit for activation.

[0095] Step 5: Clean the chip surface with acetonitrile.

[0096] Step 6: Refill the reaction chamber with argon gas until the acetonitrile is emptied, deliver a mixed reagent of CapA and CapB in a volume ratio of 1:1 to the surface of the synthesis chip, and empty the reagent after a period of reaction.

[0097] Step 7: Use acetonitrile to clean the excess CapA and CapB reagents on the chip surface.

[0098] Step 8: After the acetonitrile is emptied by filling with argon gas, iodine solution is transported to the surface of the synthesis chip for oxidation and emptied after a period of reaction.

[0099] Step 9: After cleaning the oxidant with acetonitrile, evacuate with argon.

[0100] The second step to the ninth step are cycled, and phosphoramidite monomers are grafted onto each synthesis unit in sequence according to the target sequence of synthesis (see Table 1) to complete the synthesis of the oligonucleotide chain.

[0101] The chip with the completed nucleotide chain was placed in an aminolysis instrument. After 4 hours of aminolysis, the oligonucleotide sequence was deprotected and purified to obtain free oligonucleotide sequence. The elution volume was 400 µL.

[0102] Example 4 Determination of synthetic sequence

[0103] 1. Design sequencing primers for the target sequence. The primer sequences are shown in Table 1.

[0104] 2. The oligonucleotide sequence obtained in Example 2 was subjected to 25 PCR cycles to obtain a double-stranded DNA library with a length of 212 bp connected with a second-generation sequencing adapter. The obtained sequence was subjected to high-throughput sequencing using an Illumina novaseq sequencer, and the synthesis accuracy was 99.6%.

[0105] Example 5 Determination of synthetic sequence

[0106] 1. Design sequencing primers for the target sequence. The primer sequences are shown in Table 1.

[0107] 2. The oligonucleotide sequence obtained in Example 3 was subjected to 25 PCR cycles to obtain a double-stranded DNA library with a length of 212 bp connected with a second-generation sequencing adapter. The obtained sequence was subjected to high-throughput sequencing using an Illumina novaseq sequencer, and the synthesis accuracy was 98.2%.

[0108] Example 6 Synthesis Amount Determination

[0109] 1. According to the sequence determination in Example 4, qPCR primers were designed for the flanking sequences at both ends of the synthetic sequence. The sequences are shown in Table 1.

[0110] 2. According to the sequence determined in Example 4, nucleotide standards with the same sequence were synthesized using the column synthesis method.

[0111] 3. The free oligonucleotide sequences obtained in Example 2 and Example 3 were subjected to qPCR reaction using the Sybr Green fluorescent dye method. At the same time, the same method was used to amplify nucleotide standards of different concentrations (concentrations were 1 pM, 10 pM, 100 pM, and 1000 pM, respectively), and the CT values ​​of the standards and the samples to be tested at different amplification stages were determined, and a standard curve was drawn ( Figure 6 ), the CT value measured for the target synthetic sequence was matched to the standard curve (601, 602), and it was deduced that the total amount of oligonucleotide chains synthesized on the chip of Example 3 was 40.0 pmol, and the total amount of oligonucleotide chains synthesized on the chip of Example 4 was 20.7 pmol.

[0112] Table 1

[0113] Target sequence Acgtgccgaacttagattagcaaggcacccacttgtaaaacgcgctggtatcgagaacggtatgctgtaaccccgcgggaactacactcgggccttttattgggt Sequencing primer sequences Primer1:5'AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTAcgtgccgaacttagPrimer2:5'CAAGCAGAAGACGGCATACGAGATNNNNNNNNGTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTAcccaataaaaggcc Flanking sequence Upstream: 5'Acgtgccgaacttag; Downstream: 5'Acccaataaaaggcc

[0114] In the above experiments, two chips were obtained through Example 1 and Comparative Example 1. The chip array area opening rate was 45%, and the density of synthetic unit layout was 70 / mm 2 , the ratio of the hole surface area to the array area is 0.51 and 0.93 respectively; a certain amount of oligonucleotide chains were synthesized by using these two chips through Examples 2 and 3 respectively, and sequencing (Examples 4 and 5) showed that the synthesis accuracy of the chip obtained in Comparative Example 1 was 98.2%, which was lower than 99.6% of the chip in Example 1; through quantitative detection of the obtained oligonucleotide chains, the nucleotide chain synthesis amount of the chip in Example 1 can reach 40.0 pmol, and the nucleotide chain synthesis amount of the chip in Comparative Example 1 is 20.7 pmol.

[0115] The synthesis flux of the two chips is consistent, the synthesis experiment process and the elution volume are the same, and only the surface area of ​​the synthesis unit of the chip is different. The surface area of ​​the synthesis unit of the chip in Example 1 is moderate. While providing more than 40,000 stable synthesis carriers, it maintains a low reagent accumulation rate and reduces the influence of residual reagents on the synthesis process, thereby achieving a higher synthesis efficiency and obtaining more oligonucleotide chains; the synthesis unit of Comparative Example 1 is deeper. Although the material exchange space of the reaction process is expanded, too many precursor reagents are retained, which reduces the coupling efficiency and obtains less nucleotide chain synthesis.

[0116] In summary, the present invention provides a high-throughput oligonucleotide chain synthesis chip with high synthesis efficiency and low cost. The chip integrates a large number of solid-phase synthesis units, has high synthesis flux, low retention rate of precursor reagents, high synthesis accuracy, low processing cost, and is conducive to industrial application.

Claims

1. A high-throughput synthesis chip for oligonucleotide chains, characterized in that: A two-dimensional array composed of microporous synthetic units is integrated on the surface of the chip's base material. The opening ratio of the array area is represented by η, 38%≤η≤46%.

2. The chip according to claim 1, characterized in that: The opening ratio of the array area is represented by η, 38%≤η≤45%, preferably, 40%≤η≤45%.

3. The chip according to claim 1 or 2, wherein the synthesis unit has a width of 1 mm 2 The number of array areas is represented by a, 13≤a≤650.

4. The chip according to any one of claims 1 to 3, wherein the synthesis unit has a width of 1 mm 2 The number of array areas is represented by a, 70≤a≤200. 5 . The chip according to claim 1 , wherein the microporous synthesis unit comprises a straight cylindrical microstructure perpendicular to the chip surface. 6 . The chip according to claim 1 , wherein the microporous synthesis units are arranged on the chip surface in the form of an equilateral triangle, a regular quadrilateral or an isosceles triangle, preferably in the form of an isosceles triangle.

7. The chip according to any one of claims 1 to 6, wherein the ratio of the total surface area of ​​the straight cylindrical microstructure to the area of ​​the array region is represented by W, 0.40≤W≤0.90, preferably, 0.42≤W≤0.

51.

8. The chip according to any one of claims 1 to 7, wherein the array area accounts for more than 37% of the total area of ​​the chip.

9. The chip according to any one of claims 1 to 8, wherein the base material comprises one of single crystal silicon or silicon-doped solid solution slices, ceramic slices or glass slices, and preferably, the base material comprises a silicon slice.

10. The chip according to any one of claims 1 to 9, wherein the high-throughput synthesis of the oligonucleotide chains is based on the phosphoramidite triester method. 11 . The chip according to claim 1 , wherein the inner surface of the straight-tube microstructure is covered with a functionalizing agent for grafting phosphoramidite monomers. 12 . The chip according to claim 1 , wherein the chip is partially covered with an inert reagent that cannot be grafted with the phosphoramidite monomer outside the straight-tube microstructure.

13. The chip according to any one of claims 1 to 12, used in a high-throughput synthesis process of oligonucleotide chains.

14. The chip according to any one of claims 1 to 12, integrated into other chips or devices for high-throughput synthesis of oligonucleotide chains.

15. The chip according to any one of claims 1 to 12, used in a high-throughput synthesis process of biologically active substances.

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