CN114621307B

CN114621307B - A method for encoding oligonucleotide spatial coordinates and a microfluidic device thereof

Info

Publication number: CN114621307B
Application number: CN202210383346.9A
Authority: CN
Inventors: 李传宇; 周连群; 李金泽; 张威; 李莹雪; 徐绮; 张芷齐; 姚佳; 郭振; 李超
Original assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Current assignee: Suzhou Institute of Biomedical Engineering and Technology of CAS
Priority date: 2022-04-12
Filing date: 2022-04-12
Publication date: 2024-12-13
Anticipated expiration: 2042-04-12
Also published as: CN114621307A

Abstract

The present invention relates to an oligonucleotide spatial coordinate encoding method and a microfluidic device thereof, and belongs to the technical field of molecular biology. The present invention provides an oligonucleotide spatial coordinate encoding method, wherein the full oligonucleotide sequence to be encoded is preset to be formed by connecting n+i segments of oligonucleotide sequence fragments, and according to this preset, n groups of different oligonucleotide sequence fragments X and i groups of different oligonucleotide sequence fragments Y are synthesized; an oligonucleotide synthesis array formed by orthogonal arrangement of an X flow channel and a Y flow channel is set, so that the intersection of the X flow channel and the Y flow channel forms a coding region for the connection of the oligonucleotide sequence fragments; oligonucleotide sequence fragments X and oligonucleotide sequence fragments Y are applied to each X flow channel and Y flow channel of the oligonucleotide synthesis array in turn, so that the oligonucleotide sequence fragments X and the oligonucleotide sequence fragments Y are gradually connected in the coding region to obtain the full oligonucleotide sequence to be encoded. The method greatly reduces the synthesis cost of oligonucleotides.

Description

Oligonucleotide space coordinate coding method and microfluidic device thereof

Technical Field

The invention relates to an oligonucleotide space coordinate coding method and a microfluidic device thereof, belonging to the technical field of molecular biology.

Background

Analysis of cellular spatial biological information in tissues is critical to oncology, immunology, neuroscience, genetic development, pathology, and other disciplines. For example, tumor heterogeneity in oncology, tumor microenvironment, tumor metastasis, embryonic development in genetic development, organ development patterns, individual cell layers of the brain in neuroscience, normal and abnormal partial anatomical features of the brain. The space biological information analysis can be realized by means of space coding, namely, a unique code is constructed on each point in a two-dimensional space, a sample to be detected is marked through the code, and then the space biological information analysis is realized through decoding.

The oligonucleotide (Oligo) is formed by connecting nucleotides carrying four bases of ATCG, can realize up to 4 ⁿ coding combinations by the arrangement sequence combination of the bases, and can be used for space coding. Currently, high throughput, low cost oligonucleotide in vitro synthesis has been achieved thanks to advances in DNA synthesis technology. However, there are still technical difficulties in constructing oligonucleotide arrays of known sequence in two dimensions. At present, the main strategy for constructing oligonucleotide arrays of known sequence in two dimensions and the drawbacks that exist are as follows:

1. Firstly synthesizing and fixing, namely firstly synthesizing a large number of oligonucleotide fragments with known sequences, then sequentially introducing the oligonucleotide fragments with known sequences into a substrate target position through sample application according to required spatial arrangement, and then connecting and fixing to form an oligonucleotide array;

2. the key technology of the method is to selectively introduce ATCG raw materials, which can be realized by technologies such as photoetching, ink-jet printing, type printing and the like, has the advantage of high spatial resolution (10-60 micrometers), but is limited by the chemical reaction efficiency of DNA synthesis, the coding length connected by the method is limited (generally not more than 20bp, the limit length is 60 bp), and the density and the purity of the oligonucleotides in the coding point are low;

3. Random fixing and sequencing, namely, synthesizing all required coding sequences, mixing to form a primer pool, adding the primer pool to be randomly connected to a two-dimensional substrate at one time, and reading coding information in an in-situ sequencing mode to determine coding sequence information connected to each spatial position.

Therefore, there is a need to find high spatial resolution, long coding sequences, low cost oligonucleotide synthesis techniques to construct oligonucleotide arrays of known sequences in two dimensions, which in turn drive further development of spatial coding techniques.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for encoding spatial coordinates of an oligonucleotide, comprising the steps of:

step one, presetting the complete sequence of the oligonucleotide to be encoded as formed by connecting n+i oligonucleotide sequence fragments, and synthesizing n groups of different oligonucleotide sequence fragments X and i groups of different oligonucleotide sequence fragments Y according to the presetting;

Setting an oligonucleotide synthesis array formed by orthogonal arrangement of an X runner and a Y runner, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X runner and the Y runner;

Step three, applying an oligonucleotide sequence segment X and an oligonucleotide sequence segment Y to each X runner and each Y runner of the oligonucleotide synthesis array in a round-by-round manner, so that the oligonucleotide sequence segment X and the oligonucleotide sequence segment Y are gradually connected in a coding region to obtain an oligonucleotide complete sequence to be coded;

The number of X runners is n, the number of Y runners is i, when the oligonucleotide sequence fragments X and Y are applied round by round, the same oligonucleotide sequence fragments are applied to the coding region in the same runner, and different oligonucleotide sequence fragments are applied to the coding region in different runners, so that multiple rows can be coded in each round.

The invention also provides a microfluidic device for oligonucleotide synthesis, which comprises a first microfluidic chip and a second microfluidic chip, wherein the first microfluidic chip is provided with a plurality of X flow channels, the second microfluidic chip is provided with a plurality of Y flow channels, the X flow channels and the Y flow channels are orthogonally arranged, and a coding region is formed at the junction of the X flow channels and the Y flow channels.

In one embodiment of the invention, the microfluidic device comprises a first microfluidic module and a second microfluidic module, wherein the first microfluidic module is sequentially provided with a first substrate, a first coding chip, a first microfluidic chip, a first sample transfer plate and a first upper fixing plate from bottom to top, the first microfluidic chip is provided with a plurality of first sample inlets and a plurality of first sample outlets, the first sample inlets are arranged in pairs, the first sample inlets are communicated through an X flow channel, the first sample outlets are arranged in pairs, the first sample outlets are communicated through an X flow channel, the first sample inlets are provided with a plurality of first sample inlets and a plurality of first sample outlets, the first sample inlets are in one-to-one correspondence with the first sample inlets, the first upper fixing plate is provided with a plurality of first openings which are at least communicated with the first sample inlets or the first sample outlets, the first openings are provided with a second sealing cover plate, the second sample inlets are arranged in pairs, the second sample inlets are communicated with the second microfluidic chip from bottom to top, the second sample inlets are arranged in pairs, the second sample inlets are arranged in sequence, the second sample inlets are communicated with the second microfluidic chip, the first sample outlets are arranged in pairs, the second sample inlets are communicated with the second sample inlets are arranged in sequence from bottom to top by the second microfluidic chip, the second sample inlet and the second sample outlet are in one-to-one correspondence, the second upper fixing plate is provided with a plurality of second openings, the second openings are at least communicated with one second sample inlet or one second sample outlet, and the second openings are provided with second sealing cover plates.

In one embodiment of the present invention, the first encoding chip and/or the second encoding chip is provided with an encoding photographing area.

In one embodiment of the present invention, the code photographing area is disposed at a central position of the first code chip and/or the second code chip.

In one embodiment of the invention, a first sealing ring is arranged between the first sealing cover plate and the first opening.

In one embodiment of the invention, a second sealing ring is arranged between the second sealing cover plate and the second opening.

In one embodiment of the present invention, the first base and the first upper fixing plate are fixed by bolts.

In one embodiment of the present invention, the second substrate and the second upper fixing plate are fixed by bolts.

In one embodiment of the present invention, the first microfluidic chip, the first sampling adapter plate, the first upper fixing plate, the second microfluidic chip, the second sampling adapter plate and the second upper fixing plate are made of light-transmitting materials.

In one embodiment of the invention, the first microfluidic chip, the first sample introduction adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample introduction adapter plate and the second upper fixing plate are made of flexible light-transmitting materials.

The invention also provides a method for synthesizing the oligonucleotide, which uses the oligonucleotide space coordinate coding method to synthesize the oligonucleotide on the microfluidic device for oligonucleotide synthesis.

The invention also provides the application of the oligonucleotide space coordinate coding method or the microfluidic device for oligonucleotide synthesis or the method for synthesizing the oligonucleotide in oligonucleotide synthesis.

The technical scheme of the invention has the following advantages:

1. The invention provides an oligonucleotide space coordinate coding method, which comprises the steps of presetting an oligonucleotide complete sequence to be coded, connecting n+i sections of oligonucleotide sequence fragments, synthesizing n groups of different oligonucleotide sequence fragments X and i groups of different oligonucleotide sequence fragments Y according to the presetting, setting an oligonucleotide synthesis array formed by orthogonal arrangement of X runners and Y runners, enabling an encoding region for connecting the oligonucleotide sequence fragments to be formed at the intersection of the X runners and the Y runners, finally applying the oligonucleotide sequence fragments X and the oligonucleotide sequence fragments Y to each X runner and each Y runner of the oligonucleotide synthesis array in turn respectively, enabling the oligonucleotide sequence fragments X and the oligonucleotide sequence fragments Y to be gradually connected in the encoding region, obtaining the oligonucleotide complete sequence to be coded, wherein the number of the X runners is n, the number of the Y runners is i, applying the oligonucleotide sequence fragments X and the oligonucleotide sequence fragments Y in turn by turn, applying the same oligonucleotide sequence fragments to the encoding region in turn, and applying the same oligonucleotide sequence fragments to different oligonucleotide sequence fragments in turn, and enabling multiple lines of encoding fragments to be applied to each oligonucleotide sequence. The method divides a section of oligonucleotide complete sequence to be encoded into a plurality of sections for synthesis and connection, and ensures that at least one section of coding sequence of each coding region is different through a plurality of microfluidic selection region connection reactions, thereby ensuring that the coding sequences of each site of the finally combined coding sequences are different. The method uses parallel linear micro-channel arrays to position the coding region, the same channel applies the same oligonucleotide sequence fragments to the flow region, different channels apply different oligonucleotide sequence fragments, and each round can code multiple rows. In order to complete lattice coding, the method performs multiple coding application and connection through the micro-channels, the direction or angle of each micro-channel is different, and at least one round of applied oligonucleotide sequence fragments is guaranteed to be different for different coding regions, so that the number of the coding rounds is assumed to be q, the number of the micro-channels is p (i.e. the number of the coding sequences applied per round is p), p ^q×4^m coding combinations can be completed only by synthesizing p×q oligonucleotide sequence fragments, and compared with the traditional coding method, the number of the oligonucleotide sequence fragments synthesized for achieving the same coding number is reduced by p ^q-1/q times, and the synthesis cost of the oligonucleotides is greatly reduced.

2. The invention provides a microfluidic device for oligonucleotide synthesis, which comprises a first microfluidic chip and a second microfluidic chip, wherein a plurality of X flow channels are arranged on the first microfluidic chip, a plurality of X flow channels are arranged on the second microfluidic chip, the X flow channels and the Y flow channels are orthogonally arranged, and a coding region is formed at the intersection of the X flow channels and the Y flow channels. The microfluidic device adopts independent parallel micro-channel design (X channel and Y channel), and combines a detachable pressure sealing assembly module (substrate, coding chip, micro-fluidic chip, sample injection adapter plate and upper fixing plate), so that parallel oligonucleotide sequence fragment addition, multiple oligonucleotide sequence fragment addition, positioning and connection can be realized. The micro-fluidic device can regulate and control the density of the oligonucleotide coding array through the micro-channel size design. The upper fixing plate, the sample injection adapter plate and the microfluidic chip of the microfluidic device are made of light-transmitting materials, so that optical photographing in a coding photographing area of the coding chip is facilitated, and optical information is acquired. The sample injection adapter plate and the microfluidic chip of the microfluidic device are made of flexible light-transmitting materials such as PDMS, so that the sealing effect of the detachable pressure sealing assembly module is improved.

3. The present invention provides a method for synthesizing an oligonucleotide using the above-described oligonucleotide space coordinate encoding method for synthesizing an oligonucleotide on the above-described microfluidic device for oligonucleotide synthesis. The method adopts a step-by-step connection method, firstly prepares the required oligonucleotide sequence fragments and modifications by a standard oligonucleotide synthesis method, then sequentially connects the required oligonucleotide sequence fragments to a target position by combining a microfluidic device by a chemical connection method, an enzyme connection method and other connection methods, so as to obtain the oligonucleotide coding array with a definite sequence, and the prepared oligonucleotide sequence is long in length and high in purity.

Drawings

FIG. 1 is a schematic diagram of the synthesis scheme of oligonucleotides.

Fig. 2 is a schematic view of a flow channel structure of a first microfluidic chip and a second microfluidic chip in a microfluidic device.

Figure 3 is a cross-sectional view of a first microfluidic module and a second microfluidic module in a microfluidic device.

Fig. 4 is a schematic diagram of the overall structure of a first microfluidic module and a second microfluidic module in a microfluidic device.

Fig. 5 is a top view of a first microfluidic chip in a microfluidic device.

Fig. 6 is a top view of a second microfluidic chip in a microfluidic device.

Fig. 7 is a top view of a first encoding chip and a second encoding chip in a microfluidic device.

Fig. 8 is a top view of a first sample injection adapter plate and a second sample injection adapter plate in a microfluidic device.

Figure 9 is a top view of a first microfluidic module and a second microfluidic module in a microfluidic device.

Fig. 10 is a schematic view showing the overall structure of a first upper fixing plate and a second upper fixing plate in a microfluidic device.

Fig. 11 is a schematic diagram of the overall structure of a first seal ring and a second seal ring in a microfluidic device.

FIG. 12 is a schematic view of the overall structure of a first seal cover plate and a second seal cover plate in a microfluidic device.

FIG. 13 shows the synthesis of oligonucleotides.

FIG. 14 results of oligonucleotide synthesis of a localized coding region.

In fig. 2 to 12, a first microfluidic module 1, a second microfluidic module 2, a first substrate 3, a first coding chip 4, a first microfluidic chip 5, a first sample inlet adapter plate 6, a first upper fixing plate 7, a first sample inlet 8, a first sample outlet 9, an X-channel 10, a first sample inlet 11, a first sample outlet 12, a first opening 13, a first sealing cover plate 14, a second substrate 15, a second coding chip 16, a second microfluidic chip 17, a second sample inlet adapter plate 18, a second upper fixing plate 19, a second sample inlet 20, a second sample outlet 21, a Y-channel 22, a coding region 23, a second sample inlet 24, a second sample outlet 25, a second opening 26, a second sealing cover plate 27, a coding photographing region 28, a first sealing ring 29 and a second sealing ring 30.

Detailed Description

The following examples are provided for a better understanding of the present invention and are not limited to the preferred embodiments described herein, but are not intended to limit the scope of the invention, any product which is the same or similar to the present invention, whether in light of the present teachings or in combination with other prior art features, falls within the scope of the present invention.

The following examples do not identify specific experimental procedures or conditions, which may be followed by procedures or conditions of conventional experimental procedures described in the literature in this field. The reagents or apparatus used were conventional reagent products commercially available without the manufacturer's knowledge.

Example 1 spatial coordinate encoding method of oligonucleotide

This example provides a method for encoding the spatial coordinates of an oligonucleotide (see FIG. 1 for a scheme for synthesizing an oligonucleotide using this method), comprising the steps of:

Example 2: microfluidic device

As shown in fig. 2-12, the present embodiment provides a microfluidic device (the flow of synthesizing oligonucleotides using the device is shown in fig. 1) for implementing the spatial coordinate encoding method of the oligonucleotide of embodiment 1, the microfluidic device is composed of a first microfluidic module 1 and a second microfluidic module 2, the first microfluidic module 1 is sequentially provided with a first substrate 3, a first encoding chip 4, a first microfluidic chip 5, a first sample injection adapter plate 6 and a first upper fixing plate 7 from bottom to top, the first microfluidic chip 5 is provided with a plurality of first sample injection ports 8, The first sample injection adapter plate 6 is provided with a plurality of first sample inlets 11 and a plurality of first sample outlets 12, the first sample inlets 11 are in one-to-one correspondence with the first sample inlets 8, the first upper fixing plate 7 is provided with a plurality of first openings 13, the first openings 13 are at least communicated with one first sample inlet 11 or one first sample outlet 12, the first openings 13 are provided with a first sealing cover plate 14, the second microfluidic module 2 is sequentially provided with a second substrate 15 from bottom to top, The second coding chip 16, the second micro-fluidic chip 17, the second sample introduction adapter plate 18 and the second upper fixing plate 19, wherein the second micro-fluidic chip 17 is provided with a plurality of second sample introduction ports 20, A plurality of second sample outlets 21 and a plurality of Y flow channels 22; the second sample inlets 20 are arranged in pairs, the second sample inlets 20 are communicated with each other through Y flow channels 22, the second sample outlets 21 are arranged in pairs, the second sample outlets 21 are communicated with each other through Y flow channels 22, the X flow channels 10 and the Y flow channels 22 are orthogonally arranged, a coding region 23 is formed at the junction of the X flow channels 10 and the Y flow channels 22, a plurality of second sample inlets 24 and a plurality of second sample outlets 25 are arranged on the second sample adapter plate 18, the second sample inlets 24 are in one-to-one correspondence with the second sample inlets 20, the second sample outlets 25 are in one-to-one correspondence with the second sample outlets 21, a plurality of second openings 26 are arranged on the second upper fixing plate 19, the second openings 26 are at least communicated with one second sample inlet 24 or second sample outlet 25, a second sealing cover plate 27 is arranged on the second opening 26, a coding photographing region 28 is arranged on the junction of the first coding chip 4 and the second coding chip 16, the coding region 28 is arranged on the center position of the first coding chip 4 and the second coding chip 16, the second sample outlets 25 are in one-to-one correspondence with the second sample outlets 21, a plurality of second openings 26 are communicated with the second sample inlets 24 or the second sample outlets 25 through a plurality of second sealing plates 27, a plurality of sealing rings 3 and a sealing ring 30 are arranged between the first sealing plate and the first sealing plate 7 and the second sealing plate 13 and the second sealing plate 30 and the second sealing plate, m4, etc.), the second substrate 15 and the second upper fixing plate 19 are fixed by bolts, and the first microfluidic chip 5, the first sample injection adapter plate 6, the first upper fixing plate 7, the second microfluidic chip 17, the second sample injection adapter plate 18 and the second upper fixing plate 19 are made of flexible light-transmitting materials (such as fluororubber, neoprene, PDMS, etc.).

Preferably, the width of the X flow channel and the Y flow channel is 2-200 mu m, the center distance of the X flow channel and the Y flow channel is 4-500 mu m, the height of the X flow channel and the Y flow channel is 2-200 mu m, the number of the first sample inlet, the first sample outlet, the second sample inlet, the second sample outlet, the second sample inlet and the second sample outlet is 12-240, the aperture is 0.5-5.0 mm, and the length of the bolt is 3-12 mm.

Example 3A method of synthesizing an oligonucleotide

The embodiment provides a method for synthesizing an oligonucleotide, the method uses the oligonucleotide space coordinate coding method of embodiment 1 to synthesize the oligonucleotide on the microfluidic device for oligonucleotide synthesis of embodiment 2, the synthesis target is an oligonucleotide with a nucleotide sequence shown as SEQ ID NO:1(AAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACACACGACGCTCTTCCGATCTNNNNNNNNNNGAGTGATTGCTTGTGACGCCTTNNNNNNNNNNNNNNNNNNNNNNT30VN), the oligonucleotide with a nucleotide sequence shown as SEQ ID NO:1 is preset according to the oligonucleotide space coordinate coding method of embodiment 1, the oligonucleotide is formed by connecting an oligonucleotide 1, an oligonucleotide 2 and an oligonucleotide 3, wherein the oligonucleotide 1 is a universal head sequence with a nucleotide sequence shown as SEQ ID NO:2 (TTAAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACAC), the oligonucleotide 2 and the oligonucleotide 3 are coding sequences, the method respectively comprises 4 groups (namely n=4) of different oligonucleotide sequence fragments X and 4 groups (namely i=4) of different oligonucleotide sequence fragments Y, according to the preset method, the oligonucleotide with a nucleotide sequence shown as SEQ ID NO: 3-35 NO 6 and the oligonucleotide sequence shown as SEQ ID NO:6 are synthesized, and then the oligonucleotide with a specific sequence shown as SEQ ID NO: 35 is carried out by connecting the oligonucleotide 3 to the oligonucleotide with the microfluidic device shown as follows (see fig. 35:35):

1. preparing a substrate, performing surface treatment on the substrate, and then connecting a universal head sequence Oligo1;

2. Constructing an X-direction micro-channel on a substrate;

3. Applying an Oligo2 coding sequence by sucking an X-direction micro-channel through a positive pressure pump, and connecting an oligonucleotide sequence fragment X1 to a head sequence through a connection reaction;

4. Disassembling the X-direction micro-channel, and reconstructing a new Y-direction micro-channel, wherein the new micro-channel and the fluid direction of the front micro-channel are distributed in an orthogonal direction (the first micro-fluidic module and the second micro-fluidic module share one substrate, and the X-direction micro-channel and the Y-direction micro-channel are disassembled and reconstructed on the same substrate through bolts);

5. Applying an Oligo3 coding sequence through a microchannel, and connecting the oligonucleotide sequence fragment Y1 to the oligonucleotide sequence fragment X1 through a connection reaction;

6. repeating the steps 4 and 5 until the number of coding rounds is reached, and disassembling the micro-flow channel to obtain an Oligo coding array on the substrate;

wherein, oligo1, oligo2 and Oligo3 were synthesized by the division of the biological engineering (Shanghai) and the reagent used for the ligation of the oligonucleotides was T4 DNALIGASE (Nuo Wei Zan, cat. N103-01) and the ligation of the oligonucleotides was room temperature (25 ℃);

The microfluidic device for oligonucleotide synthesis of example 2 has the specific parameters that the width of the X channel and the Y channel is 50 μm, the center distance of the X channel and the Y channel is 100 μm, the height of the X channel and the Y channel is 50 μm, the number of the first sample inlet, the first sample outlet, the second sample inlet, the second sample outlet, the second sample inlet and the second sample outlet is 48, the aperture is 1.0mm, the first microfluidic chip, the first sample introduction adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample introduction adapter plate and the second upper fixing plate are made of PDMS, the bolts are made of M4, and the length is 8mm.

In the oligonucleotide synthesis process, the X flow channel and the Y flow channel are respectively and continuously connected with 4 oligonucleotide sequence fragments X and 4 oligonucleotide sequence fragments Y, and 16 hetero-intersections with clear boundaries theoretically exist, so that after the Y flow channel is connected with a fluorescent hybridization probe, the local coding photographing region (namely one half coding photographing region) after oligonucleotide synthesis is photographed by a confocal microscope, and the observation result is shown in fig. 14. As can be seen from FIG. 14, the confocal microscope imaged 8 clear-bordered hetero-intersections on the locally encoded photographed region after oligonucleotide synthesis, resulting in a satisfactory result.

It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations or modifications of the above teachings will be apparent to those of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. While still being apparent from variations or modifications that may be made by those skilled in the art are within the scope of the invention.

Sequence listing

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Claims

1. A microfluidic device for synthesizing oligonucleotides is characterized by comprising a first microfluidic module and a second microfluidic module, wherein the first microfluidic module is sequentially provided with a first substrate, a first coding chip, a first microfluidic chip, a first sample adapter plate and a first upper fixing plate from bottom to top, the first microfluidic chip is provided with a plurality of X-channels, a plurality of first sample inlets and a plurality of first sample outlets, the first sample inlets are arranged in pairs, the first sample inlets are communicated through the X-channels, the first sample outlets are arranged in pairs, the first sample outlets are communicated through the X-channels, the first sample adapter plate is provided with a plurality of first sample inlets and a plurality of first sample outlets, the first sample inlets are in one-to-one correspondence with the first sample inlets, the first upper fixing plate is provided with a plurality of first openings, the first openings are at least communicated with one first sample inlet or a plurality of first sample outlet, the first sample inlets are arranged in pairs, the first sample inlets are communicated with a plurality of second sample inlets, the second sample inlets are arranged in pairs, the second sample inlets are communicated with a plurality of second sample outlet through the second sample inlet, the second sample inlet is arranged in pairs, the second sample inlet is communicated with the first sample outlet, the first sample inlet is arranged in one-to the first upper fixing plate, the sample inlet is communicated with the second sample outlet through the second sample inlet, the first sample inlet is arranged in sequence, the second sample inlet and the second sample outlet are in one-to-one correspondence, the second upper fixing plate is provided with a plurality of second openings which are at least communicated with one second sample inlet or one second sample outlet, the second openings are provided with second sealing cover plates, the X flow channels and the Y flow channels are arranged in an orthogonal mode, and the junction of the X flow channels and the Y flow channels forms a coding region.

2. The microfluidic device for oligonucleotide synthesis according to claim 1, wherein the first encoding chip and/or the second encoding chip is provided with an encoding photographing region.

3. The microfluidic device for oligonucleotide synthesis according to claim 2 wherein the encoded photographing region is provided at a central position of the first encoding chip and/or the second encoding chip.

4. A microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 3 wherein a first sealing ring is provided between the first sealing cover plate and the first opening and a second sealing ring is provided between the second sealing cover plate and the second opening.

5. A microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 3 wherein the first substrate and the first upper fixing plate are fixed by bolts and the second substrate and the second upper fixing plate are fixed by bolts.

6. The microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 3, wherein the first microfluidic chip, the first sample introduction adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample introduction adapter plate and the second upper fixing plate are made of light-transmitting materials.

7. A method of synthesizing an oligonucleotide, wherein the method uses an oligonucleotide space coordinate coding method to perform oligonucleotide synthesis on the microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 6;

the oligonucleotide space coordinate coding method comprises the following steps:

8. Use of a microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 6 or a method of synthesizing an oligonucleotide according to claim 7 in oligonucleotide synthesis.