Chimeric carrier as well as preparation method and application thereof

Abstract

The invention relates to a chimeric vector as well as a preparation method and application thereof. The chimeric carrier comprises a supporting layer and at least one reaction site, the reaction site comprises holes penetrating through the supporting layer and a nucleic acid synthesis carrier embedded in the supporting layer through the holes, the nucleic acid synthesis carrier is a porous material, the average pore size is 30-3000 nm, the bending modulus of the supporting layer is smaller than or equal to 1000 MPa, and the molecular weight of the nucleic acid synthesis carrier is larger than or equal to 100. The contact angle of the supporting layer is larger than 90 degrees. The embedded carrier can apply a conventional ink-jet printer to perform sample loading operation like printing paper, a user does not need to use a high-precision automatic sample loading system, and the conventional ink-jet printer can be modified to serve as a liquid injection system, so that high-precision sample loading is realized.

Description

Technical Field

[0001] The present invention relates to the field of nucleic acid synthesis consumables, and particularly to a chimeric vector, a preparation method thereof and an application thereof. Background Art

[0002] With the development of genetic engineering and synthetic biology technologies, high-throughput and high-precision nucleic acid synthesis processes have attracted more and more attention. The traditional nucleic acid solid-phase synthesis technology mainly uses controlled pore glass (CPG) as a solid-phase carrier and conducts nucleic acid synthesis through chemical coupling. However, this process has certain defects. Generally, CPG is compounded with a thermoplastic material such as polyethylene to form a synthesis column, and then the synthesis column is installed in a multi-well plate. This method has a low throughput (up to a 384-well plate) on the one hand, a large amount of reagent consumption on the other hand, and requires the development of professional sample addition equipment, resulting in the inability to further reduce the cost of nucleic acid synthesis. Researchers have developed microfluidic chips and photolithography array technologies to improve nucleic acid synthesis methods. However, these advanced processes require expensive consumables and complex sample addition equipment, otherwise it is easy to cause cross-contamination problems.

[0003] An inkjet printer is a mature printing method, and the droplet size of its nozzle can reach 3.5 pL, which is much larger than that of a conventional automated liquid injection system (0.1 mL) in the medical device field. However, conventional multi-well plates or microfluidic and photolithography array technologies cannot be directly applied to inkjet printers.

[0004] Therefore, it is necessary to provide a chimeric vector, a preparation method thereof and an application thereof. Summary of the Invention

[0005] In order to improve, it is necessary to provide a chimeric vector, a preparation method thereof and an application thereof.

[0006] In the first aspect of the present application, a chimeric vector is provided, which comprises a support layer and at least one reaction site. The reaction site comprises a hole penetrating through the support layer and a nucleic acid synthesis carrier embedded in the support layer through the hole. The nucleic acid synthesis carrier is a porous material with an average pore diameter of 30 - 3000 nm, the flexural modulus of the support layer ≤ 1000 MPa, and the contact angle of the support layer > 90°.

[0007] The chimeric carrier of this solution is co-designed through the structures of the support layer and the porous nucleic acid synthesis carrier. The flexible hydrophobic (contact angle > 90°) support layer is used to limit the diffusion range of reagents. At the same time, with the help of the high surface area generated by the porous structure of the porous nucleic acid carrier in the through holes, on the one hand, a very small amount of reaction reagents can achieve efficient nucleic acid synthesis, and on the other hand, the support layer can serve as a physical barrier for reaction sites, thus enabling a high-density distribution of reaction sites (a chimeric carrier with the size of A4 paper can achieve more than 6000 reaction sites), so as to achieve the purpose of high-throughput synthesis; the flexible support layer allows the chimeric carrier to be loaded like printing paper using a conventional inkjet printer. Users do not need to use a high-precision automated loading system and can use a conventional inkjet printer modified as a liquid injection system to achieve high-precision sample addition.

[0008] Furthermore, it contains at least two of the said reaction sites, and the minimum intermediate distance between adjacent reaction sites is greater than or equal to 1.5 times the diameter of the reaction site. By defining the minimum distance between adjacent reaction sites ≥ 1.5 times the diameter, while ensuring a high-density distribution, a physical distance can be used to form an isolation buffer zone to further inhibit cross-contamination caused by liquid splashing or capillary diffusion.

[0009] Furthermore, the reaction sites are arranged in an array. The reaction sites arranged in an array have regular spatial coordinates, which is convenient for an automated liquid dispensing device (such as the inkjet system of a printer) to perform precise positioning and sample addition according to the coordinates.

[0010] Furthermore, the diameter of the reaction site is 0.5 - 3 mm. The above diameter range can ensure that the liquid from the nozzle of a conventional printer can completely cover the reaction site without overflowing to the surface of the support or other reaction sites.

[0011] Furthermore, the nucleic acid synthesis carrier contains CPG and an adhesive. Through the porous structure formed by CPG and the adhesive, while retaining the high specific surface area characteristics of CPG, the mechanical strength of the nucleic acid synthesis carrier is improved, and it is avoided that CPG is broken due to bending in the flexible support layer.

[0012] Furthermore, the pore diameter of the CPG is 20 - 350 nm. The above pore diameter range can allow phosphoramidite molecules to diffuse freely and provide enough anchor points for nucleotide immobilization, which can improve the reaction efficiency. Furthermore, the adhesive is a thermoplastic material. Thermoplastic adhesives such as polyethylene can be combined with the support layer by heat sealing after the nucleic acid synthesis carrier is prepared.

[0013] The second aspect of the present application provides a method for preparing the above-mentioned chimeric vector, comprising the following steps: preparing the nucleic acid synthesis vector, correspondingly providing the holes in the support layer, and embedding the nucleic acid synthesis vector into the holes.

[0014] In the above preparation method, the nucleic acid synthesis vector is prepared first, and then the process of punching holes in the support layer can ensure the matching of the holes and the nucleic acid synthesis vector.

[0015] The third aspect of the present application provides a nucleic acid synthesis method using the above-mentioned chimeric vector, comprising the following steps: applying a nucleic acid synthesis reagent to a reaction site through a liquid dispensing device.

[0016] Furthermore, the liquid dispensing device is an inkjet system of an inkjet printer. The inkjet system can generate liquids below 100 pL through principles such as piezoelectric / thermal bubble drive, which can ensure accurate sample addition, thus eliminating the need to use professional automatic sample addition equipment. Description of the Drawings

[0017] Figure 1 It is a schematic structural diagram of a chimeric vector in an embodiment according to the present invention.

[0018] Figure 2 It is a schematic structural diagram of a porous reaction layer in an embodiment according to the present invention.

[0019] Figure 3 It is a schematic enlarged partial structural diagram of a chimeric vector in an embodiment according to the present invention.

[0020] Figure 4 It is a schematic diagram of a DNA synthesis method in an embodiment according to the present invention.

[0021] Figure 5 It is a schematic structural diagram of a DNA synthesis system in an embodiment according to the present invention.

[0022] Figure 6 It is a schematic structural diagram of a DNA synthesis system in an embodiment according to the present invention.

[0023] Figure 7 It is a graph of the mass spectrometry detection and analysis results in an embodiment according to the present invention.

[0024] Figure 8 It is a graph of the mass spectrometry detection and analysis results in an embodiment according to the present invention.

[0025] 1 - chimeric vector; 11 - support layer; 12 - nucleic acid synthesis vector; 13 - reaction site; 2 - inkjet printer; 3 - rinsing system; 31 - holder; 32 - waste liquid collection box. Detailed Embodiments

[0026] To facilitate the understanding of this application, the following will provide a more comprehensive description of this application. This application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided to make the understanding of the disclosure of this application more thorough and comprehensive.

[0027] In addition, the terms "first" and "second" are only used for descriptive purposes and cannot be construed as indicating or implying relative importance or implicitly specifying the quantity of the indicated technical features. Thus, the features defined with "first" and "second" may explicitly or implicitly include at least one of such features. In the description of the application, the meaning of "a plurality" is at least two, such as two, three, etc., unless otherwise specifically defined. In the description of this application, the meaning of "several" is at least one, such as one, two, etc., unless otherwise specifically defined.

[0028] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which this application belongs. The terms used in the specification of this application herein are only for the purpose of describing specific embodiments and are not intended to limit this application. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0029] In this application, among the technical features described in an open-ended manner, it includes a closed technical solution composed of the listed features, and also includes an open technical solution containing the listed features.

[0030] In this application, regarding numerical ranges, unless otherwise specified, the above numerical ranges are considered continuous and include the minimum and maximum values of the range, as well as each value between such minimum and maximum values. Further, when the range refers to integers, it includes each integer between the minimum and maximum values of the range. In addition, when multiple ranges are provided to describe features or characteristics, these ranges can be combined. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all sub-ranges subsumed therein.

[0031] In this application, regarding the percentage content, unless otherwise specified, for solid-liquid mixing and solid-solid mixing, it refers to the mass percentage, and for liquid-liquid mixing, it refers to the volume percentage.

[0032] In this application, regarding the percentage concentration, unless otherwise specified, it all refers to the final concentration. The final concentration refers to the proportion of the added component in the system after adding this component.

[0033] The temperature parameters in this application, unless otherwise specified, allow for both constant temperature treatment and treatment within a certain temperature range. The constant temperature treatment allows the temperature to fluctuate within the accuracy range controlled by the instrument.

[0034] The "particles" mentioned in this application, or substances with a defined particle size distribution, do not necessarily have a spherical shape and may be irregular. They can be primary particles or secondary particles. The size of irregular particles is calculated as the average of their maximum and minimum diameters.

[0035] Please refer to Figure 1-3 , this embodiment provides a chimeric vector 1, which includes a support layer 11 and reaction sites 13. The reaction sites 13 include holes (not shown in the drawing) that penetrate the support layer 11 and in which a nucleic acid synthesis vector 12 is embedded. The nucleic acid synthesis vector 12 is a porous material with an average pore diameter of 300 nm. The nucleic acid synthesis vector 12 is obtained by compounding CPG and polyethylene. The pore diameter of CPG is 100 nm, and the flexural modulus is 535 MPa. The contact angle of the support layer 11 is 111°. Figure 1 It contains hundreds of reaction sites and is distributed in a rectangular array. The diameter of the reaction sites is 1 mm, the minimum intermediate distance between the reaction sites is 1.5 mm, and the thickness of the chimeric vector 1 is 0.2 mm.

[0036] In other embodiments, the average pore diameter of the nucleic acid synthesis vector 12 can be selected from 30, 80, 150, 250, 500, 1000, 1500, 2000, and 3000 nm; the material of the support layer 11 can be any one or a combination of polypropylene, polystyrene, polycarbonate, polyamide, or other thermoplastic materials. The flexural modulus can be selected between 50, 100, 200, 400, 600, 800, and 1000 MPa according to the selected material and preparation process. The contact angle of the support layer 11 is related to the selected material, preparation process, and modification process and can be selected between 91°, 100°, 110°, 120°, 130°, 140°, and 150°; the pore diameter of CPG can be selected between 20, 50, 100, 150, 200, 250, 300, and 350 nm; the diameter of the reaction sites 13 can be selected between 0.5 mm, 0.8 mm, 1.2 mm, 1.8 mm, 2.5 m, and 3 mm; the ratio of the minimum intermediate distance between adjacent reaction sites 13 to the diameter of the reaction sites 13 can be selected between 1.6 times, 2 times, 2.5 times, and 4 times; the thickness of the chimeric vector 1 can be selected between 0.1 mm, 0.15 mm, 0.25 mm, and 0.3 mm.

[0037] The above chimeric vector can be prepared by the following method: 1. Mix a certain proportion of CPG powder with polymer materials (thermoplastic materials such as PE / PP / HDPE / PVC / PS or cellulose). The mixing mass ratio is: 1:10 (in other embodiments, it can be selected between 1:1, 1:5, 1:100, 1:500, and 1:1000). Then load the evenly mixed powder into a mold and form the required nucleic acid synthesis carrier by hot pressing, such as those with diameters of 1 mm, 1.5 mm, 2.0 mm, etc.

[0038] 2. Cut out holes of corresponding sizes (such as diameters of 1 mm, 1.5 mm, 2.0 mm, etc.) on the support layer (made of materials such as PE / PP / HDPE / PVC / PS) by punching or laser melting.

[0039] 3. Load the nucleic acid synthesis carrier prepared in step 1 onto the support layer with the cut-out holes.

[0040] 4. By means of thermal lamination, embed the nucleic acid synthesis carrier into the support layer to finally form a chimeric carrier with good flexibility.

[0041] Please refer to Figure 4-5 , this embodiment provides a nucleic acid synthesis method using the above chimeric carrier 1. Load the chimeric carrier 1 into the paper inlet of an inkjet printer 2, replace the ink of the inkjet printer 2 with a reagent for nucleic acid synthesis, and different reagents can be sprayed onto the corresponding reaction sites 13 as needed. Then, wash the printed chimeric carrier 1 through a washing system 3, and the washing system 3 includes a holder 31 for fixing the chimeric carrier 1 and a waste liquid collection box 32.

[0042] The following will detect the effect of the above chimeric carrier for nucleic acid synthesis through examples, where Figure 6 is a schematic diagram of the principle of the nucleic acid synthesis method.

[0043] Example 1: This example provides a nucleic acid synthesis method.

[0044] DNA sequence: 5‘-GCTAGATCGA-3‘; Molecular weight: 2980.000 ± 0.1% (or ± 10 Da).

[0045] Implementation process: (1) Deprotection of the chimeric carrier Wash the chimeric carrier with a deprotection reagent until no obvious orange color is visible to the naked eye. Then wash it twice with acetonitrile, and then bake it in a vacuum drying oven at 85 °C for 2 minutes to remove the volatile reagents.

[0046] (2) Addition of monomer reagent Load the dried chimeric carrier onto an inkjet printer and add 0.1 μL of monomer reagent to the corresponding reaction sites for coupling reaction according to the set sequence.

[0047] (3) Capping After the coupling reaction is completed, wash the chimeric carrier with a capping reagent and then let it stand for 2 minutes. This step is to use the capping reagent to block the unreacted sites.

[0048] (4)Oxidation Wash the chimeric carrier with an iodine-containing oxidation reagent and then let it stand for 2 minutes. Oxidize the phosphoramidite to a structurally stable phosphoamide. Finally, wash it twice with acetonitrile.

[0049] Repeat steps 1 - 4 until the product of the desired length is obtained, and then perform ammonolysis with ammonia water. After the ammonolysis is completed, dissolve the cleaved end product with deionized water, and then perform downstream mass spectrometry detection and analysis. The mass spectrometry results are shown in Figure 7 , and after calculation, the average length deviation is -0.109%.

[0050] Example 2: This example provides a nucleic acid synthesis method.

[0051] DNA sequence: 5‘-GCTAGATCGA-3‘; Molecular weight: 2980.000 ± 0.1% (or ± 10 Da).

[0052] Implementation process: (1)Deprotection of chimeric carrier Wash the chimeric carrier with a deprotection reagent until no obvious orange color is visible to the naked eye. Then wash it twice with acetonitrile, and then bake it in a vacuum drying oven at 85 °C for 2 minutes to remove the volatile reagents.

[0053] (2)Addition of monomer reagent Load the dried chimeric carrier onto an inkjet printer, and add 0.3 μL of the monomer reagent to the corresponding reaction sites according to the set sequence for the coupling reaction.

[0054] (3)Capping After the coupling reaction is completed, wash the chimeric carrier with a capping reagent and then let it stand for 2 minutes. This step is to use the capping reagent to block the unreacted sites.

[0055] (4)Oxidation Wash the chimeric carrier with an iodine-containing oxidation reagent and then let it stand for 2 minutes. Oxidize the phosphoramidite to a structurally stable phosphoamide. Finally, wash it twice with acetonitrile.

[0056] Repeat steps 1 - 4 until the product of the desired length is obtained, and then perform ammonolysis with ammonia water. After the ammonolysis is completed, dissolve the cleaved end product with deionized water, and then perform downstream mass spectrometry detection and analysis. The mass spectrometry results are shown in Figure 8 , and after calculation, the average length deviation is 0.059%.

[0057] According to the results of Examples 1-2, it can be seen that the chimeric carrier of the present solution can achieve high-throughput and high-precision nucleic acid synthesis through a conventional inkjet printer. This is because the chimeric carrier of the present solution is designed through the structural cooperation of the support layer and the porous nucleic acid synthesis carrier. The flexible hydrophobic (contact angle > 90°) support layer restricts the diffusion range of reagents. At the same time, with the help of the high surface area generated by the porous structure of the porous nucleic acid carrier in the through holes, on the one hand, a very small amount of reaction reagents can achieve efficient nucleic acid synthesis, and on the other hand, the support layer can serve as a physical barrier for reaction sites, thus enabling a high-density distribution of reaction sites (a chimeric carrier with the size of A4 paper can achieve more than 6000 reaction sites), so as to achieve the purpose of high-throughput synthesis; the flexible support layer allows the chimeric carrier to be loaded using a conventional inkjet printer similar to printing paper. Users need to use a high-precision automated loading system, which can be modified from a conventional inkjet printer as a liquid injection system to achieve high-precision sample addition.

[0058] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered as the scope described in this specification.

[0059] The above-described embodiments only represent several implementation manners of the present invention, and the description is relatively specific and detailed, but it cannot be understood as a limitation on the scope of the invention patent. It should be noted that for those of ordinary skill in the art, without departing from the concept of the present invention, several modifications and improvements can be made, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the invention patent should be subject to the appended claims.

Claims

1. A chimeric vector, characterized in that It comprises a support layer and at least one reaction site, wherein the reaction site comprises a hole penetrating the support layer and a nucleic acid synthesis carrier embedded in the support layer through the hole, the nucleic acid synthesis carrier is a porous material with an average pore size of 30-3000nm, the bending modulus of the support layer is ≤1000MPa, and the contact angle of the support layer is greater than 90°.

2. The chimeric vector according to claim 1, characterized in that At least two reaction sites are included, and the minimum intermediate distance between adjacent reaction sites is greater than or equal to 1.5 times the diameter of the reaction site.

3. The chimeric vector according to claim 2, characterized in that The reaction sites are distributed in an array.

4. The chimeric vector according to claim 1, characterized in that The diameter of the reaction site is 0.5-3 mm.

5. The chimeric vector according to claim 1, characterized in that The nucleic acid synthesis carrier comprises CPG and a binder.

6. The chimeric vector according to claim 5, characterized in that The pore size of the CPG is 20-350 nm.

7. The chimeric vector according to claim 5, characterized in that The adhesive is a thermoplastic material.

8. The method for preparing a chimeric vector according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: preparing the nucleic acid synthesis carrier, arranging the holes corresponding to the support layer, and embedding the nucleic acid synthesis carrier into the holes.

9. The method for synthesizing a nucleic acid of a chimeric vector according to any one of claims 1 to 7, characterized in that: The method comprises the following steps: applying nucleic acid synthesis reagents to the reaction site through a liquid dispensing device.

10. The method for synthesizing nucleic acid of a chimeric vector according to claim 9, characterized in that: The liquid dispensing device is an inkjet system of an inkjet printer.

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