CN114621307A - An oligonucleotide spatial coordinate encoding method and its microfluidic device - Google Patents

Abstract

The invention relates to an oligonucleotide space coordinate encoding method and a microfluidic device thereof, belonging to the technical field of molecular biology. The invention provides an oligonucleotide space coordinate coding method, presetting the oligonucleotide full sequence to be coded as being formed by connecting n + i sections of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting; setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel; and respectively applying the oligonucleotide sequence fragment X and the oligonucleotide sequence fragment Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pair so as to gradually connect the oligonucleotide sequence fragment X and the oligonucleotide sequence fragment Y in the coding region to obtain the oligonucleotide complete sequence to be coded. The method greatly reduces the synthesis cost of the oligonucleotide.

Description

An oligonucleotide spatial coordinate encoding method and its microfluidic device

technical field

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

Background technique

Spatial bioinformatics analysis of cells in tissues is crucial to disciplines such as oncology, immunology, neuroscience, genetic development, and pathology. For example, tumor heterogeneity in oncology, tumor microenvironment, tumor metastasis; embryonic development in genetic development, atlas of organ development, individual cell layers of the brain in neuroscience, anatomical features of normal and abnormal parts of the brain. Spatial biological information analysis can use spatial coding, that is, construct a unique code on each point in the two-dimensional space, mark the sample to be tested by coding, and then realize spatial biological information analysis by decoding.

Oligonucleotides (Oligo) are formed by connecting nucleotides carrying four ATCG bases. Up to 4 ⁿ coding combinations can be achieved through the sequence combination of bases, which can be used for spatial coding. At present, thanks to the advancement of DNA artificial synthesis technology, high-throughput and low-cost oligonucleotide synthesis in vitro has been achieved. However, it is still technically difficult to construct oligonucleotide arrays of known sequences in two-dimensional space. At this stage, the main strategies for constructing oligonucleotide arrays with known sequences in two-dimensional space and their defects are as follows:

1. Synthesize first and then fix: that is, first synthesize a large number of oligonucleotide fragments of known sequence, and then sequentially introduce the oligonucleotide fragments of known sequence into the target position of the substrate by spotting according to the required spatial arrangement, and then connect Fixed formation of oligonucleotide arrays; this method is limited by the size of the spotting droplets, the spatial resolution is generally greater than 50 microns, and the cost increases significantly as the number of encoded spots increases;

2. In situ synthesis: that is, in situ chemical synthesis of the required oligonucleotide array on a two-dimensional substrate; the key technology of this method is to selectively introduce ATCG raw materials, which can be achieved by photolithography, inkjet printing, movable type printing, etc. The technical realization has the advantage of high spatial resolution (10-60 microns), but limited by the chemical reaction efficiency of DNA synthesis, the coding length of this method is limited (generally no more than 20 bp, and the limit length is 60 bp). Low density and low purity of oligonucleotides in the spot;

3. Random fixation and re-sequencing: that is, first synthesizing all the required coding sequences, mixing them to form a primer pool, then adding random connections to the two-dimensional substrate at one time, and then reading the coding information by in situ sequencing to determine each spatial position Connected coding sequence information; although this method can construct Oligo arrays with high spatial resolution and long coding sequences, both the synthesis of primer pools in the early stage and the subsequent sequencing lead to high cost and difficult to prepare in batches.

Therefore, it is urgent to find a high spatial resolution, long coding sequence, and low-cost oligonucleotide synthesis technology to construct oligonucleotide arrays with known sequences in two-dimensional space, thereby promoting the further development of spatial coding technology.

SUMMARY OF THE INVENTION

In order to solve the above-mentioned problems, the present invention provides an oligonucleotide spatial coordinate encoding method, which comprises the following steps:

Step 1: The full sequence of the oligonucleotide to be encoded is preset to be composed of n+i segments of oligonucleotide sequence fragments, and according to this preset, n groups of different oligonucleotide sequence fragments X and i groups are synthesized Different oligonucleotide sequence fragment Y;

Step 2: Setting up an oligonucleotide synthesis array formed by the orthogonal arrangement of the X flow channel and the Y flow channel, 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;

Step 3: Apply oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y to each X channel and Y channel of the oligonucleotide synthesis array round by round, so that oligonucleotide sequence fragment X and oligonucleotide sequence fragment X are The nucleotide sequence fragment Y is gradually connected in the coding region to obtain the full sequence of the oligonucleotide to be encoded;

Among them, the number of X channels is n; the number of Y channels is i; when applying oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y one by one, the same oligonucleotide is applied to the coding region in the same channel For nucleotide sequence fragments, different oligonucleotide sequence fragments are applied to the coding region in different channels, so that each round can encode multiple lines.

The present invention also provides a microfluidic device for oligonucleotide synthesis, the microfluidic device includes a first microfluidic chip and a second microfluidic chip; on the first microfluidic chip There are several X flow channels; several Y flow channels are arranged on the second microfluidic chip; the X flow channel and the Y flow channel are arranged orthogonally; the intersection of the X flow channel and the Y flow channel forms a code area.

In an embodiment of the present invention, the microfluidic device includes a first microfluidic module and a second microfluidic module; the first microfluidic module is sequentially provided with a first substrate, a second microfluidic module from bottom to top a coding chip, a first microfluidic chip, a first sample injection adapter plate and a first upper fixing plate; the first microfluidic chip is provided with several first sample inlets and several first sample outlets; The first sample inlets are arranged in pairs, and the pair of first inlets communicate with each other through the X flow channel; the first sample outlets are arranged in pairs, and the X flow between the pair of first sample outlets The first sample introduction adapter plate is provided with several first sample inlets and several first sample outlets; the first sample inlets are in one-to-one correspondence with the first sample inlets; A sample outlet is in one-to-one correspondence with the first sample outlet; the first upper fixing plate is provided with a plurality of first openings; the first openings communicate with at least one first sample inlet or first sample outlet; The first opening is provided with a first sealing cover plate; the second microfluidic module is sequentially provided with a second substrate, a second coding chip, a second microfluidic chip, and a second sample injection adapter from bottom to top plate and a second upper fixed plate; the second microfluidic chip is provided with a number of second injection ports and a number of second injection ports; the second injection ports are arranged in pairs, and a pair of second injection ports The ports are connected through the Y flow channel; the second sample outlets are arranged in pairs, and the pair of second sample outlets are connected through the Y flow channel; the second sample injection adapter plate is provided with several The second sample inlet and several second sample outlets; the second sample inlet corresponds to the second sample inlet one-to-one; the second sample outlet corresponds to the second sample outlet one-to-one; the second upper fixing plate A plurality of second openings are arranged thereon; the second openings communicate with at least a second sample inlet or a second sample outlet; and a second sealing cover plate is arranged on the second openings.

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

In an embodiment of the present invention, the encoding photographing area is set at the center of the first encoding chip and/or the second encoding chip.

In an embodiment of the present invention, a first sealing ring is provided between the first sealing cover plate and the first opening.

In an embodiment of the present invention, a second sealing ring is provided between the second sealing cover plate and the second opening.

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

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

In an embodiment of the present invention, the first microfluidic chip, the first sample injection adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample injection adapter plate and the second The upper fixing plate is made of light-transmitting material.

In an embodiment of the present invention, the first microfluidic chip, the first sample injection adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample injection adapter plate and the second The upper fixing plate is made of flexible light-transmitting material.

The present invention also provides a method for synthesizing oligonucleotides, which uses the above-mentioned oligonucleotide spatial coordinate encoding method to perform oligonucleotide synthesis on the above-mentioned microfluidic device for oligonucleotide synthesis .

The present invention also provides the application of the above-mentioned oligonucleotide spatial coordinate encoding method or the above-mentioned microfluidic device for oligonucleotide synthesis or the above-mentioned method for synthesizing oligonucleotides in oligonucleotide synthesis.

The technical scheme of the present invention has the following advantages:

1. The present invention provides a method for encoding oligonucleotide spatial coordinates. The method is as follows: first, the full sequence of the oligonucleotide to be encoded is preliminarily formed by connecting n+i segments of oligonucleotide sequence fragments, And according to this preset, synthesize n groups of different oligonucleotide sequence fragments X and i groups of different oligonucleotide sequence fragments Y, and then set up the oligonucleotide synthesis formed by the X flow channel and the Y flow channel orthogonally set. Array, so that the intersection of X flow channel and Y flow channel form a coding region for oligonucleotide sequence fragments to connect, and finally apply oligonucleotides to each X flow channel and Y flow channel of the oligonucleotide synthesis array round by round. The nucleotide sequence fragment X and the oligonucleotide sequence fragment Y, so that the oligonucleotide sequence fragment X and the oligonucleotide sequence fragment Y are gradually connected in the coding region to obtain the full sequence of the oligonucleotide to be encoded, wherein X The number of flow channels is n, and the number of Y channels is i. When applying oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y one by one, the same oligonucleotide sequence is applied to the coding region in the same channel Fragments, different oligonucleotide sequence fragments are applied to the coding region in different channels, so that each round can encode multiple lines. The method divides a whole sequence of oligonucleotides to be encoded into multiple segments for synthesis and connection, and through multiple microfluidic selection region ligation reactions, it is ensured that at least one segment of the coding sequence in each coding region is different, thereby ensuring that The coding sequence of each site of the final combined coding sequence is different. The method uses parallel linear microfluidic arrays for the positioning of coding regions. The same oligonucleotide sequence fragment is applied to the flow-through region in the same channel, and different oligonucleotide sequence fragments are applied to different channels. Encode multiple lines. In order to complete the dot matrix coding in the method, multiple rounds of coding application and connection are performed through the microchannel, and the direction or angle of each round of the microchannel is different to ensure that for different coding regions, at least one oligonucleotide sequence fragment is applied. The rounds are different. Therefore, assuming that the number of coding rounds is q and the number of microchannels in each round is p (that is, the number of coding sequences applied in each round is p), it is only necessary to synthesize p×q kinds of oligonucleotide sequence fragments to complete p ^q ×4 ^m encoding combinations, compared with the traditional encoding method, the number of oligonucleotide sequence fragments required to be synthesized to achieve the same encoding number is reduced by p ^q-1 /q times, which greatly reduces the synthesis of oligonucleotides cost.

2. The present invention provides a microfluidic device for oligonucleotide synthesis, the microfluidic device comprises a first microfluidic chip and a second microfluidic chip, the first microfluidic chip There are several X flow channels on the second microfluidic chip, and several X flow channels are arranged on the second microfluidic chip. The X flow channel and the Y flow channel are arranged orthogonally, and the intersection of the X flow channel and the Y flow channel is formed. coding area. The microfluidic device adopts an independent parallel microfluidic channel design (X channel + Y channel), combined with a detachable pressure-sealed assembly module (substrate + coding chip + microfluidic chip + sample injection adapter plate + upper fixed plate). ), can realize parallel oligonucleotide sequence fragment addition, and multiple oligonucleotide sequence fragment addition, positioning and connection. The microfluidic device can control the oligonucleotide encoding array density through the design of the microfluidic channel size. The upper fixing plate, the sample injection adapter plate, and the microfluidic chip of the microfluidic device all need to be made of light-transmitting materials, so that it is convenient to take an optical photograph in the coded photographing area of the coded chip to obtain optical information. The sample injection adapter plate and the microfluidic chip of the microfluidic device are all selected from flexible light-transmitting materials such as PDMS, which is beneficial to improve the sealing effect of the detachable pressure sealing assembly module.

3. The present invention provides a method for synthesizing oligonucleotides, which uses the above-mentioned oligonucleotide spatial coordinate encoding method to perform oligonucleotide synthesis on the above-mentioned microfluidic device for oligonucleotide synthesis. synthesis. The method adopts a step-by-step ligation method. First, the required oligonucleotide sequence fragments and modifications are prepared by standard oligonucleotide synthesis methods, and then the required oligonucleotides are sequentially combined with a microfluidic device by chemical ligation and enzymatic ligation. The nucleotide sequence fragments are ligated to the target position to obtain a sequence-determined oligonucleotide coding array, and the prepared oligonucleotide sequence has a long length and high purity.

Description of drawings

Figure 1: Schematic flow chart of oligonucleotide synthesis.

Figure 2: Schematic diagram of the flow channel structure of the first microfluidic chip and the second microfluidic chip in the microfluidic device.

Figure 3: Cross-sectional views of the first microfluidic module and the second microfluidic module in the microfluidic device.

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

Figure 5: Top view of the first microfluidic chip in the microfluidic device.

Figure 6: Top view of the second microfluidic chip in the microfluidic device.

Figure 7: Top view of the first encoding chip and the second encoding chip in the microfluidic device.

Figure 8: Top view of the first sampling adapter plate and the second sampling adapter plate in the microfluidic device.

Figure 9: Top view of the first microfluidic module and the second microfluidic module in the microfluidic device.

Figure 10: Schematic diagram of the overall structure of the first upper fixing plate and the second upper fixing plate in the microfluidic device.

Figure 11: Schematic diagram of the overall structure of the first sealing ring and the second sealing ring in the microfluidic device.

Figure 12: Schematic diagram of the overall structure of the first sealing cover plate and the second sealing cover plate in the microfluidic device.

Figure 13: Synthesis process of oligonucleotides.

Figure 14: Results of oligonucleotide synthesis of local coding regions.

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

Detailed ways

The following examples are provided for a better understanding of the present invention, and are not limited to the best embodiments, and do not limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by combining with the features of other prior art shall fall within the protection scope of the present invention.

If the specific experimental steps or conditions are not indicated in the following examples, it can be carried out according to the operations or conditions of the conventional experimental steps described in the literature in this field. The reagents or instruments used without the manufacturer's indication are all conventional reagent products that can be obtained from the market.

Embodiment 1: A kind of oligonucleotide spatial coordinate encoding method

This embodiment provides a method for encoding oligonucleotide spatial coordinates (see Figure 1 for the process of synthesizing oligonucleotides using this method), and the method includes the following steps:

Step 1: The full sequence of the oligonucleotide to be encoded is preset to be composed of n+i segments of oligonucleotide sequence fragments, and according to this preset, n groups of different oligonucleotide sequence fragments X and i groups are synthesized Different oligonucleotide sequence fragment Y;

Step 2: Setting up an oligonucleotide synthesis array formed by the orthogonal arrangement of the X flow channel and the Y flow channel, 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;

Step 3: Apply oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y to each X channel and Y channel of the oligonucleotide synthesis array round by round, so that oligonucleotide sequence fragment X and oligonucleotide sequence fragment X are The nucleotide sequence fragment Y is gradually connected in the coding region to obtain the full sequence of the oligonucleotide to be encoded;

Among them, the number of X channels is n; the number of Y channels is i; when applying oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y one by one, the same oligonucleotide is applied to the coding region in the same channel For nucleotide sequence fragments, different oligonucleotide sequence fragments are applied to the coding region in different channels, so that each round can encode multiple lines.

Example 2: A microfluidic device

As shown in Figures 2 to 12, this embodiment provides a microfluidic device for implementing the oligonucleotide spatial coordinate encoding method described in Embodiment 1 (see Figure 1 for the process of synthesizing oligonucleotides using this device) ), 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 and a first coding chip from bottom to top 4. The first microfluidic chip 5, the first sample injection adapter plate 6 and the first upper fixing plate 7; the first microfluidic chip 5 is provided with a number of first injection ports 8, a number of first outlet The sample port 9 and several X flow channels 10; the first sample inlet ports 9 are arranged in pairs, and the pair of first sample inlet ports 9 are connected through the X flow channel 10; the first sample outlet ports 9 are in pairs A pair of first sample outlets 9 communicate with each other through the X flow channel 10; the first sample injection adapter plate 6 is provided with a number of first sample inlets 11 and a number of first sample outlets 12; The first sample inlet 11 corresponds to the first sample inlet 8 one-to-one; the first sample outlet 12 corresponds to the first sample outlet 9 one-to-one; an opening 13; the first opening 13 communicates with at least one first sample inlet 11 or a first sample outlet 12; the first opening 13 is provided with a first sealing cover plate 14; the second microfluidic The control module 2 is provided with a second base 15, a second coding chip 16, a second microfluidic chip 17, a second sample injection adapter plate 18 and a second upper fixing plate 19 in order from bottom to top; the second microfluidic The control chip 17 is provided with several second sample inlets 20, several second sample outlets 21 and several Y flow channels 22; the second sample inlets 20 are arranged in pairs, between the pair of second sample inlets 20 Connected through the Y flow channel 22; the second sample outlets 21 are arranged in pairs, and the pair of second sample outlets 21 are connected through the Y flow channel 22; the X flow channel 10 and the Y flow channel 22 are directly connected The intersection of the X flow channel 10 and the Y flow channel 22 forms a coding area 23; the second sample injection adapter plate 18 is provided with a number of second sample inlets 24 and a number of second sample outlets 25; The second sample inlet 24 corresponds to the second sample inlet 20 one-to-one; the second sample outlet 25 corresponds to the second sample outlet 21 one-to-one; the second upper fixing plate 19 is provided with a plurality of second openings 26; the second opening 26 communicates with at least one second sample inlet 24 or a second sample outlet 25; the second opening 26 is provided with a second sealing cover 27; the first coding chip 4 and the second The coding chip 16 is provided with a coding photographing area 28; the coding photographing area 28 is set at the center of the first coding chip 4 and the second coding chip 16; between the first sealing cover 14 and the first opening 13 There is a first sealing ring 29; a second sealing ring 30 is provided between the second sealing cover plate 27 and the second opening 26; bolts (eg M3 , M4, etc.) are fixed; the second base 15 and the second upper fixing plate 19 are fixed by bolts; the first microfluidic chip 5, the first sampling 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 (eg, fluororubber, neoprene, PDMS, etc.).

Preferably, the width of the X runner and the Y runner are 2 μm to 200 μm; the center distance between the X runner and the Y runner is 4 μm to 500 μm; the height of the X runner and the Y runner is 2 μm to 200 μm; The number of the first sample inlet, the first sample outlet, 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 hole diameter is 0.5mm ~ 5.0mm; the size of the bolt length is 3mm ~ 12mm.

Example 3: A method of synthesizing oligonucleotides

This example provides a method for synthesizing oligonucleotides, which is performed on the microfluidic device for oligonucleotide synthesis of Example 2 using the oligonucleotide spatial coordinate encoding method of Example 1 Synthesis of oligonucleotide, the synthetic target is an oligonucleotide whose nucleotide sequence is shown in SEQ ID NO: 1 (AAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACACACGACGCTCTTCCGATCTNNNNNNNNNNGAGTGATTGCTTGTGACGCCTTNNNNNNNNNNNNNNNNNNNNT30VN), first according to the oligonucleotide spatial coordinate encoding method of Embodiment 1, preset nucleosides The oligonucleotide whose acid sequence is shown in SEQ ID NO: 1 is formed by connecting oligo1, oligo2 and oligo3, wherein, oligo1 is the general head sequence shown in nucleotide sequence as shown in SEQ ID NO: 2 (TTAAGCAGTGGTATCAACGCAGAGTACGTCTCTTTCCCTACAC), oligo2 and oligo3 are coding sequences, respectively including 4 groups (ie n=4) different oligonucleotide sequence fragments X and 4 groups (ie i=4) different oligonucleotide sequence fragments Y, and according to this preset, synthetic 4 groups of different oligonucleotide sequence fragments X1 to X4 (nucleotide sequences are shown in SEQ ID NO: 3 to SEQ ID NO: 6, ACGACGCTCTTCCGATCTGCTTACGCAGGAGTGATTGC, ACGACGCTCTTCCGATCTCTACAGAGCGGAGTGATTGC, ACGACGCTCTTCCGATCTGATCTGGTCCGAGTGATTGC, ACGACGCTCTTCCGATCTTACAGCGTTAGAGTGATTGC) and 4 groups of different oligonucleotide sequence fragments Y1 to Y4 (the nucleotide sequences are shown in SEQ ID NO: 7 to SEQ ID NO: 10, TGTGACGCCTTCCGATTGGATNNNNNNNNNNT30VN, TGTGACGCCTTAGGCCTCATANNNNNNNNNNNNT30VN, TGTGACGCCTTTTGTACGGCCNNNNNNNNNNNT30VN, TGTGACGCCTTTACTATCGGANNNNNNNNNNNNT30VN), and then the microfluidic device for oligonucleotide synthesis of Example 2 was used Carry out the connection of oligonucleotides (see Figure 13 for the connection process), and the specific steps are as follows:

1. Prepare the substrate, perform surface treatment on the substrate, and then connect the universal head sequence Oligo1;

2. Build X-direction microchannels on the substrate;

3. Apply the Oligo2 coding sequence by pumping the X-direction microchannel by a positive pressure pump, and connect the oligonucleotide sequence fragment X1 to the head sequence through a ligation reaction;

4. Disassemble the X-direction microfluidic channel and rebuild a new Y-direction microfluidic channel. The fluid direction of the new microfluidic channel and the previous microfluidic channel are arranged in an orthogonal direction (the first microfluidic module and the second microfluidic The fluid control module shares a base, and the disassembly and reconstruction of the X-direction micro-channel and the Y-direction micro-channel on the same base are realized by bolts);

5. Apply the Oligo3 coding sequence through the microfluidic channel, and connect the oligonucleotide sequence fragment Y1 to the oligonucleotide sequence fragment X1 through a ligation reaction;

6. Repeat steps 4 and 5 until the number of coding rounds is reached, disassemble the micro-channel, and obtain the Oligo coding array on the substrate;

Among them, Oligo1, Oligo2 and Oligo3 were synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The reagent used for oligonucleotide ligation was T4 DNALigase (Novizan, Cat. No. N103-01), and the oligonucleotide ligation was room temperature (25 °C);

The specific parameters of the microfluidic device for oligonucleotide synthesis of Example 2 are: the width of the X flow channel and the Y flow channel is 50 μm; the size of the center distance between the X flow channel and the Y flow channel is 100 μm; The dimensions of the channel and the Y channel height are 50 μm; the first inlet, the first outlet, the first sample inlet, the first sample outlet, the second inlet, the second outlet, the second sample The number of inlets and the second sample outlet is 48, and the aperture is 1.0mm; the first microfluidic chip, the first sampling adapter plate, the first upper fixing plate, the second microfluidic chip, the second sampling adapter The plate and the second upper fixing plate are made of PDMS material; the bolts are made of M4, and the length is 8mm.

In the process of oligonucleotide synthesis, the X channel and the Y channel are respectively connected with 4 oligonucleotide sequence fragments X and 4 oligonucleotide sequence fragments Y, and theoretically there are 16 hybridization points with clear boundaries , therefore, after passing the fluorescent hybridization probe into the Y channel, the local coded photographing area (ie, one-half coded photographing area) after oligonucleotide synthesis is photographed by confocal microscope, and the observation results are shown in Figure 14. It can be seen from Fig. 14 that the confocal microscope captured 8 hybridization spots with clear boundaries on the local code photographed area after oligonucleotide synthesis, and the results were in line with expectations.

Obviously, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation manner. For those of ordinary skill in the art, changes or modifications in other different forms can also be made on the basis of the above description. There is no need and cannot be exhaustive of all implementations here. And the obvious changes or changes derived from this are still within the protection scope of the present invention.

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Claims (10)

1. A method for encoding oligonucleotides in spatial coordinates, said method comprising the steps of:

the method comprises the following steps: presetting the whole sequence of the oligonucleotide to be coded as n + i segments of oligonucleotide sequence segments, and synthesizing n groups of different oligonucleotide sequence segments X and i groups of different oligonucleotide sequence segments Y according to the presetting;

step two: setting an oligonucleotide synthesis array formed by orthogonally setting an X flow channel and a Y flow channel, so that a coding region for connecting oligonucleotide sequence fragments is formed at the intersection of the X flow channel and the Y flow channel;

step three: respectively applying oligonucleotide sequence segments X and oligonucleotide sequence segments Y to each X flow channel and each Y flow channel of the oligonucleotide synthesis array by wheel pairs, so that the oligonucleotide sequence segments X and the oligonucleotide sequence segments Y are gradually connected in a coding region to obtain an oligonucleotide complete sequence to be coded;

wherein the number of the X runners is n; the number of the Y flow channels is i; when oligonucleotide sequence fragment X and oligonucleotide sequence fragment Y are applied in rounds, the same oligonucleotide sequence fragment is applied to the coding region in the same flow channel, and different oligonucleotide sequence fragments are applied to the coding region in different flow channels, so that multiple rows can be coded in each round.

2. A microfluidic device for oligonucleotide synthesis, comprising a first microfluidic chip and a second microfluidic chip; a plurality of X flow channels are arranged on the first micro-fluidic chip; a plurality of Y flow channels are arranged on the second micro-fluidic chip; the X runner and the Y runner are arranged orthogonally; and the intersection of the X runner and the Y runner forms a coding region.

3. The microfluidic device for oligonucleotide synthesis according to claim 2, wherein the microfluidic device comprises a first microfluidic module and a second microfluidic module; the first microfluidic module is sequentially provided with a first substrate, a first coding chip, a first microfluidic chip, a first sample introduction adapter 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, and the pair of first sample inlets are communicated through an X flow channel; the first sample outlets are arranged in pairs, and the pair of first sample outlets are communicated through an X runner; the first sample introduction adapter plate is provided with a plurality of first sample inlets and a plurality of first sample outlets; the first sample inlets correspond to the first sample inlets one to one; the first sample outlets correspond to the first sample outlets one to one; a plurality of first openings are formed in the first upper fixing plate; the first opening is communicated with at least one first sample inlet or first sample outlet; a first sealing cover plate is arranged on the first opening; the second microfluidic module is sequentially provided with a second substrate, a second coding chip, a second microfluidic chip, a second sample introduction adapter plate and a second upper fixing plate from bottom to top; the second microfluidic chip is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets are arranged in pairs, and the pair of second sample inlets are communicated through a Y flow channel; the second sample outlets are arranged in pairs, and the pair of second sample outlets are communicated through a Y flow channel; the second sample introduction adapter plate is provided with a plurality of second sample inlets and a plurality of second sample outlets; the second sample inlets correspond to the second sample inlets one by one; the second sample outlets correspond to the second sample outlets one to one; a plurality of second openings are formed in the second upper fixing plate; the second opening is communicated with at least one second sample inlet or second sample outlet; and a second sealing cover plate is arranged on the second opening.

4. The microfluidic device according to claim 3, wherein the first coding chip and/or the second coding chip has a coded camera area.

5. The microfluidic device according to claim 4, wherein the encoded imaging region is located at the center of the first encoding chip and/or the second encoding chip.

6. The microfluidic device according to any of claims 3 to 5, wherein a first sealing ring is disposed between the first sealing cover plate and the first opening; and a second sealing ring is arranged between the second sealing cover plate and the second opening.

7. The microfluidic device according to any of claims 3 to 6, wherein the first substrate and the first upper fixing plate are fixed by bolts; the second base and the second upper fixing plate are fixed through bolts.

8. The microfluidic device according to any of claims 3 to 6, wherein the first microfluidic chip, the first sample adapter plate, the first upper fixing plate, the second microfluidic chip, the second sample adapter plate and the second upper fixing plate are made of a light-transmitting material.

9. A method for synthesizing oligonucleotides, wherein the method comprises performing oligonucleotide synthesis on the microfluidic device for oligonucleotide synthesis according to any one of claims 2 to 8 using the oligonucleotide spatial coordinate encoding method according to claim 1.

10. Use of the method for spatial coordinate encoding of oligonucleotides according to claim 1 or the microfluidic device for oligonucleotide synthesis according to any one of claims 1 to 8 or the method for oligonucleotide synthesis according to claim 9 for oligonucleotide synthesis.

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