Solid phase carrier, its preparation method and application, and biochip

Abstract

This application relates to the field of biochip technology, and particularly to a solid-phase carrier, a preparation method and application thereof, and a biochip. It is used to solve the problem in the related art that when cutting the synthesized polymer from the carrier body, improper selection of conditions is likely to limit the reuse of the carrier body. This application provides a solid-phase carrier, including: a carrier body and a linking molecule, the linking molecule having a first linking end and a second linking end, and a first molecular segment connected between the first linking end and the second linking end; the first molecular segment is composed of N of the following formula (I) connected together, N is an integer greater than or equal to 2. In the first molecular segment, every two adjacent ones of the following formula (I) are connected through the X at the 3'-position in one formula (I) and the R at the 5'-position in another formula (I) to form the structure shown in the following formula (II);

Description

Technical Field

[0001] This application relates to the field of biochip technology, and particularly relates to a solid-phase carrier, a preparation method and application thereof, and a biochip. Background Art

[0002] The large-scale synthesis of polymers such as nucleic acids, polypeptides, and polysaccharides has important significance in the fields of life science, drug screening, etc.

[0003] A biochip is a microfluidic analysis unit and system constructed on the surface of a solid chip by using microelectronics, micro-machinery, chemistry, physics technology, and computer technology for the processes involved in life science research, such as sample preparation, chemical reaction, and analysis and detection, making them continuous, integrated, and miniaturized.

[0004] In practical applications, biochip technology can be widely used in many fields such as disease diagnosis and treatment, drug screening, excellent breeding of crops, forensic identification, food hygiene supervision, environmental detection, national defense, and aerospace. It will open up a brand-new way for humans to understand the origin, inheritance, development, and evolution of life, for the diagnosis, treatment, and prevention and control of human diseases, and provide a technical support platform for the new design of biological macromolecules and the rapid screening of lead compounds in drug development and pharmacogenomics research. Summary of the Invention

[0005] The main purpose of this application is to provide a solid-phase carrier, a preparation method and application thereof, and a biochip. It is used to solve the problem that when cutting the synthesized polymer from the carrier body in related technologies, improper selection of conditions is likely to limit the reuse of the carrier body.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, this application provides a solid-phase carrier, including: a carrier body and a linking molecule. The linking molecule has a first linking end and a second linking end, and a first molecular segment connected between the first linking end and the second linking end. The linking molecule is connected to the carrier body through the first linking end, and the second linking end is used to connect a polymer. The first molecular segment is formed by connecting N structures shown in the following formula (I), where N is an integer greater than or equal to 2. In the first molecular segment, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one structure shown in the following formula (I) and R at the 5'-position in another structure shown in the following formula (I) to form a structure shown in the following formula (II);

[0008]

[0009]

[0010] Among them, in formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S to form a corresponding P-S bond; N R1s are the same or different and are each selected from any one of H, a base, and nitropyrrole, N R2s are the same or different and are each selected from hydrogen or -OR4, and R3 and R4 are each selected from any one of hydrogen, an alkyl group, an acyl group, and a silyl group. In this way, when using this solid-phase carrier to prepare a polymer and separating the polymer from the solid-phase carrier, a specific oxide can be used to cleave the P-S bond. During the cleavage process, no bond cleavage occurs between the first connecting end and the carrier body, nor between the first molecular chain segment and the first connecting end, so that the activation of the closed short connecting molecule will not be caused, thereby enabling the repeated use of this solid-phase carrier and avoiding scrapping. Compared with the related art, when separating the polymer under alkaline conditions, if the concentration of the alkaline solution is too high, it is likely to damage the carrier body, and if the concentration of the alkaline solution is too low, the polymer cannot be completely separated. This can solve the problem in the related art that improper condition selection is likely to limit the repeated use of the carrier body. At the same time, by limiting the number of N, the distance between the polymer connection site and the surface of the carrier body and the density of the connection sites can also be adjusted, thereby being able to provide an appropriate reaction space for polymer synthesis and avoiding the problems of low reaction efficiency and low yield caused by steric effects during the reaction.

[0011] In one implementation manner of the first aspect, at least one R1 is a base, and the base is selected from any one of base A, base G, base C, base T, and base I; when the base is selected from any one of base A, base G, base C, and base I, the base is also connected to a protecting group. During the polymer synthesis process, it can also avoid the problem that the base participates in the reaction and is not conducive to the reaction site where the polymer is connected to the solid-phase carrier.

[0012] In one implementation manner of the first aspect, N R1s are each selected from any one of H and base T. H and base T do not react during the polymer synthesis process.

[0013] In one implementation manner of the first aspect, N is greater than or equal to 18 and less than or equal to 25. In this way, the first molecular chain segment can be an oligonucleotide sequence. In practical applications, the length of this connecting molecule can be adjusted by adjusting the size of N, thereby being able to adjust the distance of polymer synthesis and the density of reaction sites.

[0014] In an implementation of the first aspect, the linking molecule further includes a second molecular segment connected between the first linking end and the first molecular segment, and the first molecular segment is connected to the first linking end through the second molecular segment. The second molecular segment can be selected to provide a more suitable reaction site for the first molecular segment, avoiding directly connecting the first molecular segment to the carrier body, where the first molecular segment is prone to fall off due to steric effects and the like, resulting in low reaction efficiency and difficulty in providing a suitable reaction site density for polymer synthesis.

[0015] In an implementation of the first aspect, the first linking end and the carrier body are connected by a siloxane bond. The active group on the carrier body can be a hydroxyl group, and the second molecular segment can be a silylating reagent. During the preparation process, the length of the siloxane segment can be selected according to the required distance.

[0016] In an implementation of the first aspect, the linking molecule further includes a third molecular segment connected between the second linking end and the first molecular segment, and the first molecular segment is connected to the second linking end through the third molecular segment. The connection with the second linking end can be achieved by designing appropriate functional groups, avoiding directly forming the second linking end at the end of the first molecular segment, such as the 5'-position or 3'-position, which is not conducive to implementation.

[0017] In an implementation of the first aspect, when the linking molecule includes a second molecular segment and a third molecular segment; one end of the first molecular segment is connected to the second molecular segment through a 3'-position amino group, and the other end is connected to the third molecular segment through a 5'-position hydroxyl group.

[0018] In an implementation of the first aspect, it further includes: a first blocking group connected to the carrier body for blocking the first active group on the carrier body that is not connected to the first linking end. When the P-S bond is cleaved and the P-S bond is broken, the first blocking group and the first active group on the carrier body that is not connected to the first linking end may not break the bond. In this way, when using this solid-phase carrier, it is possible to prevent the first active group on the carrier body that is not connected to the first linking end from connecting with the polymer, which is not conducive to the polymer arranging in a certain order.

[0019] In an implementation of the first aspect, when the first active group is a hydroxyl group or an amino group, the first blocking group is a carboxyl group or an acid anhydride; when the first active group is a carboxyl group, the first blocking group is an amino group or an alkylamine.

[0020] In one implementation of the first aspect, the second connection end is connected to a second blocking group. By selecting a suitable second blocking group, when the bond between the second connection end and the second blocking group is broken, no reaction occurs at other positions of the linking molecule. In the case of synthesizing a polymer, the bond between the second blocking group and the second connection end is broken.

[0021] In one implementation of the first aspect, the shape of the carrier body is a sheet base or a microsphere.

[0022] In one implementation of the first aspect, the material of the carrier body is a composite material of one or more of glass, polymer, metal, and metal oxide.

[0023] In a second aspect, an embodiment of the present application provides a biochip, including a solid-phase carrier as described in the first aspect, and a polymer connected to the second connection end on the solid-phase carrier.

[0024] In one implementation of the second aspect, the biochip is a microfluidic chip.

[0025] In one implementation of the second aspect, the polymer includes at least one of nucleotides, polypeptides, and polysaccharides.

[0026] In a third aspect, an embodiment of the present application provides a method for preparing a solid-phase carrier, including:

[0027] Selecting a carrier body having a first active group, or forming a first active group on the carrier body by chemical or physical methods;

[0028] Bonding a linking molecule having a first connection end to the carrier body through the first connection end and the first active group;

[0029] The linking molecule further includes a second connection end, and a first molecular segment connected between the first connection end and the second connection end, and the second connection end is used to connect a polymer;

[0030] The first molecular segment is formed by connecting N structures shown in the following formula (I), N is an integer greater than or equal to 2. In the first molecular segment, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one structure shown in the formula (I) and R at the 5'-position in another structure shown in the formula (I) to form a structure shown in the following formula (II);

[0031]

[0032] Among them, in formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S to form a corresponding P-S bond; N R1s are the same or different and are each selected from any one of hydrogen, a base, and nitropyrrole, N R2s are the same or different and are each selected from hydrogen or -OR4, and R3 and R4 are each selected from any one of hydrogen, an alkyl group, an acyl group, and a silyl group.

[0033] In this way, a solid-phase carrier can be prepared. When using this solid-phase carrier to prepare a polymer and separating the polymer from the solid-phase carrier, a specific oxide can be used to cleave the P-S bond. During the cleavage process, no bond cleavage occurs between the first linking end and the carrier body, nor between the first molecular chain segment and the first linking end, so that the activation of the closed short linking molecules will not be caused, thereby enabling the reuse of this solid-phase carrier and avoiding scrapping. Compared with the related art, when separating the polymer under alkaline conditions, if the concentration of the alkaline solution is too high, it is easy to damage the carrier body, and if the concentration of the alkaline solution is too low, the polymer cannot be completely separated. It can solve the problem that improper condition selection in the related art is likely to limit the reuse of the carrier body. At the same time, by limiting the number of N, the distance between the polymer linking site and the surface of the carrier body and the density of the linking sites can also be adjusted, thereby providing an appropriate reaction space for polymer synthesis and avoiding the problems of low reaction efficiency and low yield caused by steric effects during the reaction.

[0034] In one implementation manner of the third aspect, the bonding and connecting of the linking molecule having the first linking end to the carrier body through the first linking end and the first active group includes:

[0035] Select a first linking molecule having the first linking end and a third linking end; react the first linking molecule with the carrier body to connect the first linking molecule to the carrier body through the first linking end;

[0036] Select a monomer structure shown in formula (I), and connect N monomer structures through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form a structure shown in formula (II) to obtain the first molecular chain segment;

[0037] Modify the first end and the second end of the first molecular chain segment so that the modified first end of the first molecular chain segment is connected to the third linking end under the first reaction condition, and the modified second end of the first molecular chain segment has the second linking end; under the first reaction condition, the P-S bond does not break.

[0038] Alternatively, a monomer structure having a second reactive group at the 3'-position or 5'-position is selected, and the monomer structure having a second reactive group at the 3'-position or 5'-position is connected to the third linking end through the second reactive group under the second reaction conditions, and at the same time, the monomer structure having the structure shown in formula (I) is connected to the monomer structure having the second reactive group to form the structure shown in formula (II) through DNA synthesis to obtain the first molecular chain segment; under the third reaction conditions, the ends of the first molecular chain segment are modified so that the modified ends of the first molecular chain segment have the second linking end; in the second reaction conditions, the third reaction conditions, and DNA synthesis, the P-S bond does not break.

[0039] In one implementation manner of the third aspect, the modification of the first end and the second end of the first molecular chain segment so that the modified first end of the first molecular chain segment is connected to the third linking end under the first reaction conditions and the modified second end of the first molecular chain segment has the second linking end includes:

[0040] Modify the first end of the first molecular chain segment;

[0041] Make the modified first end of the first molecular chain segment connected to the third linking end under the first reaction conditions;

[0042] Under the fourth reaction conditions, modify the second end of the first molecular chain segment to generate the second linking end at the second end of the first molecular chain segment, and under the fourth reaction conditions, no reaction occurs at the remaining positions of the linking molecule.

[0043] In one implementation manner of the third aspect, the method of connecting N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the structure shown in formula (II) to obtain the first molecular chain segment includes:

[0044] Connect N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the first molecular chain segment by enzymatic synthesis or DNA synthesis.

[0045] In one implementation manner of the third aspect, in the case of connecting N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure by DNA synthesis, the oxidant used in the DNA synthesis is iodine, and the concentration of iodine is 0.005M to 0.01M. This can prevent the oxidation effect of the oxidant from being too strong and causing the already formed P-S bond to break.

[0046] In one implementation of the third aspect, modifying the second end of the first molecular segment under the fourth reaction conditions includes:

[0047] Selecting a second linking molecule having the second linking end and the fourth linking end, and connecting the second end of the first molecular segment to the fourth linking end of the second linking molecule under the fourth reaction conditions;

[0048] In the third reaction conditions, modifying the end of the first molecular segment includes:

[0049] Selecting a second linking molecule having the second linking end and the fourth linking end, and connecting the end of the first molecular segment to the fourth linking end of the second linking molecule under the third reaction conditions.

[0050] In a fourth aspect, an embodiment of the present application provides an application of the solid-phase carrier as described in the first aspect in polymer synthesis, including:

[0051] Contacting the solid-phase carrier with a monomer of the polymer, causing reactions between the second linking end on the solid-phase carrier and the monomer of the polymer, and between the monomers of the polymer, to generate the polymer and connect the polymer to the solid-phase carrier;

[0052] Cleaving the P-S bond on the solid-phase carrier using an oxidant to break the P-S bond, obtaining a solid-phase carrier with a fourth molecular segment connected to the carrier body and a fifth molecular segment containing the polymer, where the fourth molecular segment is a molecular segment with a 3'-position thiol group or a 5'-position phosphate group at the end;

[0053] Regenerating the solid-phase carrier with the fourth molecular segment connected using an enzyme synthesis method or a DNA synthesis method, and connecting a sixth molecular segment having the second linking end to the end of the fourth molecular segment to obtain the solid-phase carrier as described in the first aspect.

[0054] By introducing at least one P-S bond into the linking molecule, when preparing a polymer using this solid-phase support and separating the polymer from the solid-phase support, a specific oxide can be used to cleave the P-S bond. During the cleavage process, no bond cleavage occurs between the first linking end and the support body, nor between the first molecular chain segment and the first linking end, thus preventing the activation of the closed short linking molecules, enabling the reuse of this solid-phase support. During reuse, the solid-phase support linked with the fourth molecular chain segment is regenerated by using the enzyme synthesis method or the DNA synthesis method, that is, a P-S bond can be generated again in the linking molecule of the solid-phase support to achieve the regeneration of the solid-phase support. The method is simple and easy to implement. Compared with the related technology where when separating the polymer under alkaline conditions, if the concentration of the alkaline solution is too high, it is easy to damage the support body, and if the concentration of the alkaline solution is too low, the polymer cannot be completely separated, it can solve the problem that improper condition selection in the related technology easily limits the reuse of the support body.

[0055] In one implementation of the fourth aspect, the oxidant is selected from silver oxide or potassium permanganate with a concentration of 0.01M to 1M; the temperature during cleavage is 35°C to 45°C. Through experiments, it is found that under these conditions, quantitative cleavage of the P-S bond can be achieved within no more than 10 minutes, and no reaction occurs at other positions.

[0056] In one implementation of the fourth aspect, when regenerating the solid-phase support linked with the fourth molecular chain segment by using the DNA synthesis method, the oxidant used in the DNA synthesis method is iodine, and the concentration of iodine is 0.005M to 0.01M. This can prevent the oxidation effect of the oxidant from being too strong and causing the already formed P-S bond to break.

[0057] The present application provides a solid-phase carrier, a preparation method and application thereof, and a biochip. By introducing a P-S bond that is stable under polymer synthesis conditions but sensitive to specific oxides into the linking molecule, when using this solid-phase carrier to prepare a polymer and separating the polymer from the solid-phase carrier, a specific oxide can be used to cleave the P-S bond. During the cleavage process, no bond cleavage occurs between the first linking end and the carrier body, nor between the first molecular chain segment and the first linking end, so that activation of the closed short linking molecule will not occur, thereby enabling the reuse of this solid-phase carrier and avoiding scrapping. Compared with the related art, when separating the polymer under alkaline conditions, if the concentration of the alkaline solution is too high, it is easy to damage the carrier body, and if the concentration of the alkaline solution is too low, the polymer cannot be completely separated. The present application can solve the problem in the related art that improper condition selection easily limits the reuse of the carrier body. At the same time, by limiting the number of N, the distance between the polymer linking site and the surface of the carrier body and the density of the linking sites can also be adjusted, thereby providing an appropriate reaction space for polymer synthesis and avoiding the problem of low reaction efficiency and yield caused by steric effects during the reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 Structural diagram of a solid-phase carrier provided by the present application;

[0059] Figure 2 Structural diagram of another solid-phase carrier provided by the present application;

[0060] Figure 3 Structural diagram of another solid-phase carrier provided by the present application;

[0061] Figure 4 Structural diagram of another solid-phase carrier provided by the present application;

[0062] Figure 5 Structural diagram of another solid-phase carrier provided by the present application;

[0063] Figure 6 Structural diagram of another solid-phase carrier provided by the present application;

[0064] Figure 7 Structural diagram of the reaction equation for cleaving the P-S bond after polymer synthesis provided by the present application;

[0065] Figure 8 Structural diagram of another reaction equation for cleaving the P-S bond after polymer synthesis provided by the present application;

[0066] Figure 9 Flow chart of a preparation method of a solid-phase carrier provided by the present application;

[0067] Figure 10 Structural diagram of a reaction equation for the reaction of a carrier body with a first linking molecule provided by this application;

[0068] Figure 11 Structural diagram of a reaction equation for modifying the second end of a first molecular segment provided by this application;

[0069] Figure 12 Flow chart of another method for preparing a solid-phase carrier provided by this application;

[0070] Figure 13 Flow chart of yet another method for preparing a solid-phase carrier provided by this application;

[0071] Figure 14 Flow chart for cleaving P-S bonds after polymer synthesis and regenerating the solid-phase carrier after cleavage provided by this application. Detailed implementation manners

[0072] The embodiments of this application will be described in detail below in conjunction with the accompanying drawings.

[0073] This application provides a biochip, including a solid-phase carrier and a biopolymer connected to the surface of the solid-phase carrier, such as at least one of nucleotides, polypeptides, and polysaccharides. These biopolymers hybridize with target molecules in a labeled biological sample to be detected or directly synthesize a labeled molecule, and by using a specific instrument to quickly, parallelly, and efficiently detect and analyze the intensity of the hybridization (synthesis) label signal, the number of target molecules in the sample can be determined.

[0074] During the preparation process, the solid-phase carrier can be a silicon wafer subjected to surface functionalization treatment, and during the preparation process, the preparation technology of computer chips is simulated to arrange DNA fragments or protein molecules on the chip in sequence, so it is also called biochip technology.

[0075] In some embodiments, the biochip can be a microfluidic chip. A microfluidic chip integrates basic operation units such as sample preparation, reaction, separation, and detection in analytical processes such as biology, chemistry, and medicine onto a chip with a micron scale, and automatically completes the entire analytical process. For example, a microreactor is a commonly used structure for biochemical reactions in a lab-on-a-chip, such as microreactors for capillary electrophoresis, polymerase chain reaction, enzyme reaction, and DNA hybridization reaction.

[0076] According to the preparation method of the biochip, it can be known that the solid-phase carriers used for the synthesis of polymers such as nucleic acids, polypeptides, and polysaccharides generally need to meet three requirements: 1. It can provide reaction sites for polymer synthesis, that is, the surface of the solid-phase carrier can provide active groups that react with the polymer. 2. Provide cleavage sites for the synthesized polymer, that is, it can cut the synthesized polymer completely from the solid-phase carrier without any damage to the structure. 3. Provide a better reaction environment for the polymer synthesis reaction. For example, the density of the active groups used for the synthesis reaction and the distance from the solid-phase carrier should be appropriate to avoid problems such as low reaction efficiency and low yield due to steric effects when reacting with the polymer.

[0077] Therefore, the linking molecule after the surface functionalization of the solid-phase carrier generally consists of three parts: 1. The linker arm provides reaction sites with appropriate distance and density from the surface of the solid-phase carrier. 2. The tether provides a cleavage site for the separation of the synthesized polymer and the solid-phase carrier. 3. The protected group provides reactive groups for linking with the polymer during polymer synthesis.

[0078] For example, in the related art, in order to achieve the separation of the synthesized polymer and the surface of the solid-phase carrier, a linking molecule that is stable under polymer synthesis conditions (acidic conditions) but sensitive to alkali is usually used. After the polymer synthesis is completed, treatment under alkaline conditions can separate the polymer from the solid-phase carrier.

[0079] In one of the examples, it specifically includes the following steps:

[0080] Step 1): Add the hydroxylated carrier, the reagent HQDA (1,4-Bis(3,4-dicarboxyphenoxy)benzenedianhydride, 4,4'-diphenoxydiphthalic anhydride), DMAP (4-dimethylaminopyridine), and HBTU (O(7Azabenzotriazol1yl)N,N,N',N'-tetramethyluroniumhexafluophosphate) into a sealed bottle. Inject 35 μL of DIEA (Diisopropylethylamine) and 2.5 mL of acetonitrile (quickly) into the sealed bottle through a syringe. Shake at room temperature for 2 h. The reaction equation is shown in the following formula (1). After the reaction is completed, wash 3 times with 2.5 mL of acetonitrile each time and wash 3 times with 2.5 mL of dichloromethane each time. Dry at room temperature. In the following formula (1), the carrier is represented by ○.

[0081]

[0082] Step 2): Add the HQDA-modified carrier, DMT (4,4-Dimethoxytriphenylmethyl)-nucleotide monomer, DMAP, and HBTU into a sealed bottle. Add 35 μL of DIEA and 1.255 mL of acetonitrile (quickly) into the sealed bottle through a syringe. Shake at room temperature for 2 h. The reaction equation is shown in the following formula (2). After the reaction is completed, wash three times with 2.5 mL of acetonitrile each time and three times with 2.5 mL of dichloromethane each time. Dry at room temperature. In the following formula (2), B represents a base.

[0083]

[0084] Step 3): Synthesize the DNA sequence using the DNA synthesis method (steps such as de-DMT, coupling, capping, and oxidation). The equation is shown in the following formula (3). In the following formula (3), the DNA sequence is represented by to represent.

[0085]

[0086] Step 4): Cut the synthesized DNA sequence from the carrier using concentrated ammonia water. The reaction time is 5 - 15 minutes, the reaction temperature is 25 °C, and the reaction equation is shown in the following formula (4).

[0087]

[0088] According to the above example, in order to provide a suitable distance and reaction site for polymer synthesis, HQDA is used as a linking molecule. One carboxylic acid group in HQDA reacts with the hydroxyl group on the surface of the carrier body under acidic conditions to form an ester bond for connection, and the other carboxylic acid group of HQDA is used as an active reaction site to connect with the polymer. After the polymer synthesis is completed, while cutting the linkage between the polymer and HQDA under alkaline conditions, the ester bond between HQDA and the surface of the carrier body will also be broken.

[0089] It can be seen that in the related art, although a linker molecule that is stable under polymer synthesis conditions and sensitive to bases can provide a suitable distance and reaction sites for polymer synthesis and can achieve the separation of the polymer and the carrier body after the polymer synthesis is completed, there is a dilemma with this strategy. On the one hand, if harsh alkaline conditions (such as high concentration, high temperature, long time, etc.) are used to cleave the polymer after the polymer synthesis is completed, the polymer can be completely cleaved from the carrier body, but it will cause the activation of the closed short linker molecules, such as the activation of the hydroxyl groups with protecting groups on the carrier body, and even cause damage to the surface of the carrier body. On the other hand, if the polymer is cleaved under milder conditions, the synthesized polymer cannot be completely cleaved from the carrier body. This dilemma limits the reuse of the carrier body, resulting in a waste of resources.

[0090] Based on this, the present application provides a solid-phase carrier 10, such as Figure 1 and Figure 2 shown, comprising: a carrier body 1, and a linker molecule 2. The carrier body 1 can be a hard material, such as glass, silicon wafer, etc., or a soft material, such as a nylon film, etc., or a metal material, a polymer material, such as the most representative PolydiMethyl-Siloxane (PDMS), and other polymer materials, such as PolyMethylMethAcrylate (PMMA), PolyCarbonate (PC), etc. Of course, the carrier body 1 can also be a composite material of the above-mentioned various materials. In addition, according to the use of the carrier body 1 itself, the carrier body 1 can also be classified into a conductive material and an insulating material. According to whether the surface of the carrier body 1 has mesopores, the carrier body 1 can be further classified into a non-porous material and a porous material. In some embodiments, the shape of the carrier body can be a sheet substrate or a microsphere, etc.

[0091] The linker molecule 2 has a first linking end L1 and a second linking end L2, and a first molecular segment 21 connected between the first linking end L1 and the second linking end L2. The linker molecule 2 is connected to the carrier body 1 through the first linking end L1, and the second linking end L2 is used to connect the polymer 3. The first molecular segment 21 is formed by connecting N structures shown in the following formula (I), where N is an integer greater than or equal to 2. In the first molecular segment 21, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one of the structures shown in formula (I) and R at the 5'-position in the other structure shown in formula (I) to form a structure shown in formula (II).

[0092]

[0093] In formulas (I) and (II), X is selected from oxygen or sulfur, and in at least one structure represented by formula (II), X is selected from S. The N R1s are the same or different and are each independently selected from hydrogen, a base, and nitropyrrole. The N R2s are the same or different and are each independently selected from hydrogen or -OR4, and R3 and R4 are each independently selected from an alkyl group, an acyl group, and a silyl group.

[0094] Among them, in formula (1), R at the 5'-position can be any group capable of connecting with X to form a structure as shown in formula (II).

[0095] For example, when X is selected from oxygen, R can be a 5'-phosphoramidite group with or without a protecting group DMT attached. The oxygen at the 3'-position and the 5'-phosphoramidite can be connected in the form of a phosphite ester through DNA synthesis or enzymatic synthesis. When X is selected from S, R can also be a 5'-phosphoramidite group with or without a protecting group DMT attached. Similarly, the S at the 3'-position and the 5'-phosphoramidite can be connected in the form of a phosphorothioate ester through DNA synthesis or enzymatic synthesis.

[0096] In some embodiments, at least one R1 is a base, and the base is selected from any one of base A, base G, base C, base T, and base I. That is, the structure represented by formula (I) can be a nucleotide monomer structure. Among them, when the base is selected from base A, base G, base C, base T, and base T, the structural formula of formula (I) is as shown below. When the base is selected from any one of base A, base G, base C, and base I, the base is also connected with a protecting group. That is, during the synthesis of polymer 3, the problem that the base participates in the reaction and is not conducive to the reaction site where polymer 3 is connected to the solid-phase carrier 10 can also be avoided.

[0097]

[0098] Based on this, in some embodiments, the N R1s are each independently selected from H and base T that are stable under the polymer 3 synthesis conditions and do not react.

[0099] In some embodiments, N is greater than or equal to 15 and less than or equal to 50. That is, the first molecular chain segment 21 can be an oligonucleotide sequence. In practical applications, by adjusting the size of N, the length of the linking molecule 2 can be adjusted, so that the distance and the density of the reaction sites for the synthesis of polymer 3 can be adjusted.

[0100] In other embodiments, N is greater than or equal to 18 and less than or equal to 25.

[0101] It can be known from the fact that before connecting the above-mentioned linking molecule 2 to the carrier body 1, the active groups on the carrier body 1 can be hydroxyl groups, amino groups, etc., that the first linking end L1 can be any group that can form a stable bond with a hydroxyl group or an amino group under the synthesis conditions of the polymer 3 and does not break the bond when the P-S bond is cleaved to break the P-S bond.

[0102] In one case, as Figure 1 and Figure 2 shown, the linking molecule 2 may only include a first linking end L1, a second linking end L2, and a first molecular chain segment 21 connected between the first linking end L1 and the second linking end L2. In this case, the first linking end L1 and the second linking end L2 may respectively form the 3'-position and 5'-position at both ends of the first molecular chain segment 21. In some examples, when the active group on the carrier body 1 is a hydroxyl group, the first linking end L1 may be an amino group formed at the 3'-position at one end of the first molecular chain segment 21, and the second linking end L2 may be a hydroxyl group or a carboxyl group formed at the 5'-position at the other end of the first molecular chain segment 21.

[0103] In another case, as Figure 3 and Figure 4 shown, in addition to the first linking end L1, the second linking end L2, and the first molecular chain segment 21 described above, the linking molecule 2 may further include: a second molecular chain segment 22 connected between the first linking end L1 and the first molecular chain segment 21, and the first molecular chain segment 21 is connected to the first linking end L1 through the second molecular chain segment 22. In this case, since the bond between the first molecular chain segment 21 and the first linking end L1 does not break when the P-S bond is cleaved to break the P-S bond, it can be known that when the P-S bond is cleaved to break the P-S bond, the bond between the second molecular chain segment 22 and the first linking end L1, and the bond between the first molecular chain segment 21 and the second molecular chain segment 22 also do not break.

[0104] Both ends of the second molecular chain segment 22 may be groups that can form a stable bond with the first linking end L1 and one end of the first molecular chain segment 21 (such as the first end Y1) under the synthesis conditions of the polymer 3 and do not break the bond when the P-S bond is cleaved to break the P-S bond.

[0105] In this case, the reuse of the solid-phase carrier can be achieved. Meanwhile, when connecting the linking molecule 2 to the carrier body 1, the second molecular segment 22 can be selected to provide a more suitable reaction site for the first molecular segment 21, avoiding directly connecting the first molecular segment 21 to the carrier body 1, which may cause the first molecular segment 21 to easily fall off due to steric effects, etc., resulting in low reaction efficiency and difficulty in providing a suitable reaction site density for the synthesis of the polymer 3.

[0106] Based on this, in some embodiments, the second molecular segment 22 can be a molecular segment capable of providing a connection distance for the first molecular segment 21 on the surface of the carrier body 1.

[0107] In some embodiments, the first connection end L1 and the carrier body 1 are connected by a siloxane bond. At this time, the active group on the carrier body 1 can be a hydroxyl group, and the second molecular segment 22 can be a silanization reagent. During the preparation process, the length of the siloxane segment can be selected according to the required distance.

[0108] In some embodiments, the solid-phase carrier 10 further includes: a first blocking group connected to the carrier body 1 for blocking the first active group on the carrier body 1 that is not connected to the first connection end L1. Here, in the case of cleaving the P-S bond to break the P-S bond, no bond breakage may occur between the first blocking group and the first active group on the carrier body 1 that is not connected to the first connection end L1. In this way, the activation of the blocked short linking molecule will not be caused, thereby avoiding damage to the carrier body 1.

[0109] In this embodiment, by blocking the first active group on the carrier body 1 that is not connected to the first connection end L1, when using the solid-phase carrier 10, it is possible to prevent the first active group on the carrier body 1 that is not connected to the first connection end L1 from connecting to the polymer 3, which is not conducive to the polymer 3 arranging in a certain order. Meanwhile, according to the above preparation process of the solid-phase carrier 10, in the case of using a silanization reagent to react with the hydroxyl groups on the surface of the carrier body 1 to generate a siloxane segment, if the first active group on the carrier body 1 that is not connected to the first connection end L1 is not blocked, the first active group on the carrier body 1 that is not connected to the first connection end L1 will further react subsequently, which is not conducive to the linking molecule 2 connecting to the carrier body 1 in a certain order, and thus not conducive to the synthesis of the polymer 3.

[0110] Among them, the first active groups on the carrier body 1 that are not connected to the first connection end L1 can be hydroxyl groups, amino groups, carboxyl groups, etc. Appropriate first blocking groups can be selected. For example, when the active groups on the carrier body 1 that are not connected to the first connection end L1 are hydroxyl groups or amino groups, the first blocking group can be a carboxyl group or an acid anhydride. When the first active group on the carrier body 1 that is not connected to the first connection end is a carboxyl group, the first blocking group can be an amino group or an alkylamine, etc.

[0111] During the preparation process, when the first molecular chain segment 21 is connected to the carrier body 1, the second connection end L2 can be directly formed at the end of the first molecular chain segment 21, or can be connected through the first molecular chain segment 21 to other molecules to form the second connection end L2 at the end of the other molecules. Or, the first molecular chain segment 21 is connected to other molecules that implicitly contain the second connection end L2 (such as the second connection end L2 can be generated through a certain chemical reaction) to form the second connection end L2 for connecting the polymer 3 in the connecting molecule 2.

[0112] Regardless of which of the above situations, in some embodiments, when the above connecting molecule 2 includes the second connection end L2, the second connection end L2 is connected or not connected to the second blocking group. When the second connection end L2 is connected to the second blocking group, by selecting an appropriate second blocking group, when the second connection end L2 and the second blocking group are broken, no reaction occurs at the remaining positions of the connecting molecule 2. And in the case of synthesizing a polymer, the second blocking group and the second connection end are broken.

[0113] In some embodiments, such as Figure 5 and Figure 6 shown, the connecting molecule 2 further includes: a third molecular chain segment 23 connected between the second connection end L2 and the first molecular chain segment 21, and the first molecular chain segment 21 is connected to the second connection end L2 through the third molecular chain segment 23. When the P-S bond is cleaved and the P-S bond is broken, the connection bonds between the third molecular chain segment 23 and the second connection end L2, and between the first molecular chain segment 21 and the third molecular chain segment 23 may not be broken.

[0114] In this embodiment, by introducing a third molecular chain segment 23 between the second connection end L2 and the first molecular chain segment 21, on the one hand, it is possible to achieve connection with the second connection end L2 by designing appropriate functional groups, avoiding the problem that the second connection end L2 is directly formed at the end of the first molecular chain segment 21, such as the 5'-position or 3'-position, which is not conducive to implementation. On the other hand, after the polymer 3 is synthesized, by breaking the P-S bond, the third molecular chain segment 23 and the polymer 3 can be cut off together, and then the polymer 3 is separated from the third molecular chain segment 23 under alkaline conditions. Compared with directly using alkaline conditions to separate the polymer 3 from the solid-phase carrier 10 in the related art, it is possible to avoid the problem that the alkaline conditions damage the surface-functionalized solid-phase carrier 10, which is not conducive to the reuse of the solid-phase carrier 10. Compared with directly connecting the polymer 3 to the first molecular chain segment 21 through the second connection end L2, it can also provide a more suitable connection group for the connection of the polymer 3, and when the polymer 3 is DNA and polymerase amplification of DNA needs to be achieved, it can avoid the influence of the direct connection of the first molecular chain segment 21 to the polymer 3 on the sequencing of the DNA sequence.

[0115] According to the above, in the first molecular chain segment 21, the first molecular chain segment 21 can be connected to the second molecular chain segment 22 and the third molecular chain segment 23 through the 3'-position of the first end Y1 and the 5'-position of the second end Y2 respectively, as Figure 7 shown. When the first molecular chain segment 21 is connected to the second molecular chain segment 22 through the 3'-position of the first end Y1 and connected to the third molecular chain segment 23 through the 5'-position of the second end Y2, after the P-S bond is broken, the first molecular chain segment 21 is broken into a first sub-chain segment 211 connected to the carrier body 1 and a second sub-chain segment 212 connected to the third molecular chain segment 23. The end of the first sub-chain segment 211 is a 5'-phosphate group, and the end of the second sub-chain segment 212 is a 3'-mercapto group. In this case, the first molecular chain segment 21 can be connected to the second molecular chain segment 22 through the amino group at the 3'-position and connected to the third molecular chain segment 23 through the hydroxyl group at the 5'-position. During the preparation process, it can be synthesized by the DNA synthesis method in the direction from the 3'-position to the 5'-position, the first molecular chain segment 21 is synthesized on the carrier body 1, and at the same time, the 3'-position in the first molecular chain segment 21 is connected to the second molecular chain segment 22. After the P-S bond is broken, the P-S bond can also be reintroduced into the linker molecule 2 according to the DNA synthesis method in the direction from the 3'-position to the 5'-position to realize the regeneration of the solid-phase carrier, or the P-S bond can be reintroduced into the linker molecule 2 according to the base pairing principle and by the enzyme synthesis method to regenerate the solid-phase carrier.

[0116] As Figure 8As shown in the figure, when the first molecular chain segment 21 is connected to the second molecular chain segment 22 through the 5'-position of the first end Y1 and to the third molecular chain segment 23 through the 3'-position of the second end Y2, after the P-S bond is broken, the first molecular chain segment 21 is broken into a third sub-chain segment 211 connected to the carrier body 1 and a fourth sub-chain segment 212 connected to the third molecular chain segment 213. The end of the third sub-chain segment 211 is a 3'-mercapto group, and the end of the fourth sub-chain segment 212 is a 5'-phosphate group. In this case, the first molecular chain segment 21 can be connected to the second molecular chain segment 22 through the amino group at the 5'-position and to the third molecular chain segment 23 through the hydroxyl group at the 3'-position. During the preparation process, although it is also possible to perform synthesis by the DNA synthesis method in the direction from the 3'-position to the 5'-position, compared with the above-mentioned case where the first molecular chain segment 21 is connected to the second molecular chain segment 22 through the amino group at the 3'-position and to the third molecular chain segment 23 through the hydroxyl group at the 5'-position, the preparation raw materials are not easily obtained and the synthesis is difficult. Therefore, in this case, the base pairing principle can be adopted, and the solid-phase carrier can be regenerated by reintroducing the P-S bond into the linker molecule 2 through the enzyme synthesis method.

[0117] In the solid-phase carrier 10 provided in the embodiment of the present application, by introducing a P-S bond that is stable under the synthesis conditions of the polymer 3 but sensitive to a specific oxide into the linker molecule 2, when using this solid-phase carrier 10 to prepare the polymer 3 and separating the polymer 3 and the solid-phase carrier 10, a specific oxide can be used to cleave the P-S bond. During the cleavage process, by ensuring that the linkage bonds between the first linking end L1 and the carrier body 1 and between the first molecular chain segment 2 and the first linking end L1 do not break, the activation of the closed short linker molecule will not occur, thereby enabling the reuse of this solid-phase carrier 10 and avoiding scrapping. Compared with the related art, when separating the polymer 3 under alkaline conditions, if the concentration of the alkaline solution is too high, it is easy to damage the carrier body 1, and if the concentration of the alkaline solution is too low, the polymer 3 cannot be completely separated. The present application can solve the problem in the related art that improper condition selection is likely to limit the reuse of the carrier body 1. At the same time, by limiting the number of N, the distance between the polymer 3 connection site and the surface of the carrier body 1 and the density of the connection sites can also be adjusted, thereby providing an appropriate reaction space for the synthesis of the polymer 3 and avoiding the problems of low reaction efficiency and low yield caused by steric effects during the reaction.

[0118] Some embodiments of the present application provide a method for preparing a solid-phase carrier, as Figure 9 shown, including:

[0119] S101. Select a carrier body 1 with a first active group on its surface, or form a first active group on the surface of the carrier body 1 by chemical or physical methods.

[0120] For example, a first active group can be formed on the surface of the carrier body 1 by methods such as chemical reagent activation, plasma treatment, or deposition. The first active group can be a hydroxyl group, an amino group, etc.

[0121] S102. Connect the linking molecule 2 having a first linking end L1 to the carrier body 1 by bonding the first linking end L1 and the first active group on the carrier body 1. The linking molecule 2 further includes a second linking end L2 and a first molecular segment 21 connected between the first linking end L1 and the second linking end L2. The second linking end L2 is used to connect to a polymer. The first molecular segment 21 is formed by connecting N structures shown in the following formula (I), where N is an integer greater than or equal to 2. In the first molecular segment 21, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one structure shown in formula (I) and R at the 5'-position in another structure shown in formula (I) to form a structure shown in the following formula (II).

[0122]

[0123]

[0124] In formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S. The N R1s are the same or different and are each selected from any one of H, a base, and nitropyrrole. The N R2s are the same or different and are each selected from hydrogen or -OR4, and R3 and R4 are each selected from any one of an alkyl group, an acyl group, and a silyl group.

[0125] Wherein, R can be any group capable of connecting with X to form a structure shown in formula (II).

[0126] In some embodiments, connecting the linking molecule 2 having a first linking end L1 to the carrier body 1 by bonding the first linking end L1 and the first active group on the carrier body 1 includes:

[0127] As Figure 10 shown, select a first linking molecule 210 having a first linking end L1 and a third linking end L3, react the first linking molecule 210 with the carrier body 1, and connect the first linking molecule 210 to the carrier body 1 through the first linking end L1.

[0128] Here, taking the first active group on the carrier body 1 as a hydroxyl group as an example, according to the fact that the first linking end L1 can be connected to the carrier body 1 through a siloxane bond, it can be known that the first linking molecule 210 can be a silanizing reagent. By bonding the first linking molecule 210 and the hydroxyl group through a siloxane bond, the first linking molecule 210 can be connected to the carrier body 1. At this time, the third linking end L3 can be any group capable of connecting to the first molecular segment 21, or a group implicitly containing a group capable of connecting to the first molecular segment 21. For example, the third linking end L3 can be an epoxy group. After connecting the first linking molecule 210 to the carrier body 1, the third linking end L3 can be converted into a hydroxyl group by hydrochloric acid activation.

[0129] Here, it should be noted that when connecting the first linking molecule 210 to the carrier body 1, the density of the first linking molecule 210 connected to the carrier body 1 can be controlled by controlling the concentration, contact area, etc. of the first linking molecule 210. After the connection is completed, the first active groups on the carrier body 1 that are not connected to the first linking molecule 210 can be blocked by connecting a first blocking group to avoid subsequent reactions, which is not conducive to the subsequent connection of the polymer 3 to the linking molecule 2.

[0130] According to the different first active groups on the carrier body 1, the first blocking group can be different. When the first active group on the carrier body 1 is a hydroxyl group or an amino group, the first blocking group can be a carboxyl group or an acid anhydride. When the first active group on the carrier body 1 is a carboxyl group, the first blocking group can be an amino group or an alkylamine.

[0131] Select a monomer structure shown in formula (1), and connect N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form a structure shown in formula (II) to obtain the first molecular segment 21.

[0132] It can include: connecting N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the first molecular segment 21 by an enzyme synthesis method or a DNA synthesis method.

[0133] At this time, the first molecular segment 21 can be prepared by a synthesis method on other carriers.

[0134] Modify the first end Y1 and the second end Y2 of the first molecular segment 21 so that the modified first end Y1 of the first molecular segment 21 is connected to the third linking end L3 under the first reaction conditions, and the modified second end Y2 of the first molecular segment 21 has a second linking end L2; under the first reaction conditions, the P-S bond does not break.

[0135] Among them, the modification of the first end Y1 and the second end Y2 of the first molecular chain segment 21 can occur before connecting the modified first end Y1 of the first molecular chain segment 21 to the third connecting end L3 under the first reaction conditions. At this time, by connecting the modified first end Y1 of the first molecular chain segment 21 to the third connecting end L3 under the first reaction conditions, the carrier body 1 can be connected to the linking molecule 2.

[0136] In some embodiments, the modification of the first end Y1 and the second end Y2 of the first molecular chain segment 21 includes:

[0137] Modifying the first end Y1 of the first molecular chain segment 21.

[0138] Taking the 3'-hydroxyl group at the first end Y1 of the first molecular chain segment 21 as an example, according to the group type of the first connecting end L1, the 3'-hydroxyl group at the first end Y1 of the first molecular chain segment 21 can be converted into a group that can be connected to the first connecting end L1 under the first reaction conditions. At this time, taking the first connecting end L1 as a hydroxyl group as an example, the 3'-hydroxyl group at the first end Y1 of the first molecular chain segment 21 can be converted into a 3'-amino group through a silylation reaction.

[0139] Connecting the modified first end Y1 of the first molecular chain segment 21 to the third connecting end L3 under the first reaction conditions.

[0140] At this time, still taking the first connecting end L1 as a hydroxyl group as an example, the 3'-amino group at the first end Y1 of the first molecular chain segment 21 is connected to the hydroxyl group under acidic conditions.

[0141] Under the fourth reaction conditions, the second end Y2 of the first molecular chain segment 21 is modified to generate a second connecting end L2 at the second end Y2 of the first molecular chain segment 21, and no reaction occurs at other positions of the linking molecule under the fourth reaction conditions.

[0142] In the case where the 3'-amino group at the first end Y1 of the first molecular chain segment 21 is connected to the hydroxyl group as described above, the 5'-phosphate group at the second end Y2 of the first molecular chain segment 21 can be modified to generate a second connecting end L2 at the second end Y2 of the first molecular chain segment 21. For example, in the case where the second connecting end L2 is a hydroxyl group, the 5'-phosphate group can be directly converted into a hydroxyl group, or another linking molecule having a second connecting end L2 or implicitly containing a second connecting end L2 can be connected to the second end Y2 of the first molecular chain segment 21.

[0143] In some embodiments, under the fourth reaction conditions, the modification of the second end Y2 of the first molecular chain segment 21 includes:

[0144] Such as Figure 11As shown, a second linking molecule 220 having a second linking end L2 and a fourth linking end L4 is selected, and the second end Y1 of the first molecular segment 21 is linked to the fourth linking end L4 of the second linking molecule 220 under a fourth reaction condition.

[0145] Compared with directly converting the second end Y2 of the first molecular segment 21 into the second linking end L2, a second linking molecule 220 with a suitable functional group can be selected according to the type of group of the desired second linking end L2, avoiding the problem that it is not conducive to implementation when directly converting the second end Y2 of the first molecular segment 21 into the second linking end L2.

[0146] In some other embodiments, as Figure 12 and Figure 13 shown, a monomer structure having a second active group at the 3'-position or 5'-position is selected, and the monomer structure having a second active group at the 3'-position or 5'-position is linked to the third linking end L3 through the second active group under a second reaction condition, and at the same time, a monomer structure having a structure shown in formula (I) is linked to the monomer structure having a second active group to form a structure shown in formula (II) by DNA synthesis method to obtain the first molecular segment 21; under a third reaction condition, the end of the first molecular segment 21 is modified so that the end of the modified first molecular segment 21 has a second linking end L2; in the second reaction condition, the third reaction condition, and the DNA synthesis method, the P-S bond does not break.

[0147] In this embodiment, different from the above preparation of the first molecular segment 21 by the method of synthesis on other carriers, the above first molecular segment 21 is directly synthesized on the carrier body 1 by DNA synthesis method. In this process, except for the first monomer structure being linked to the first linking end L1 and the end of the first molecular segment 21 being modified, the remaining reactions all go through reaction steps such as activation, coupling, capping, and oxidation in the DNA synthesis method.

[0148] In this case, by selecting appropriate reaction conditions, such as in the second reaction condition, the third reaction condition, and the DNA synthesis method, making the P-S bond not break, the connection between the carrier body 1 and the linking molecule 2 can be achieved.

[0149] Here, still taking the example that the first molecular segment 21 is connected to the first linker L1 through the amino group at the 3'-position, the above second active group can be the amino group at the 3'-position. At this time, modifying the end of the first molecular segment 21 can be modifying the 5'-position of the end of the first molecular segment 21. Similar to the above modification of the 5'-position of the second end of the first molecular segment 21, the 5'-position can also be modified by directly converting the phosphate group at the 5'-position into the second linker L2, or by connecting other linker molecules to modify the 5'-position.

[0150] In some embodiments, under the third reaction conditions, modifying the end of the first molecular segment 21 includes:

[0151] As Figure 12 and Figure 13 shown, select a second linker molecule 220 having a second linker L2 and a fourth linker L4, and connect the end of the first molecular segment 21 to the fourth linker L4 of the second linker molecule 220 under the third reaction conditions.

[0152] In this embodiment, compared with directly converting the end of the first molecular segment 21 into the second linker L2, a second linker molecule 220 with appropriate functional groups can be selected according to the type of group of the second linker L2 to be obtained, avoiding the problem that it is not conducive to implementation when directly converting the end of the first molecular segment 21 into the second linker L2.

[0153] Some embodiments of the present application provide an application of the solid-phase carrier as described above in polymer synthesis, including:

[0154] As Figure 14 shown, contact the solid-phase carrier 10 with the monomer of the polymer 3, so that the second linker L2 on the solid-phase carrier 10 reacts with the monomer of the polymer 3 and between the monomers of the polymer 3 to generate the polymer 3, and connect the generated polymer 3 to the solid-phase carrier 10.

[0155] Among them, the polymer 3 can be at least one of nucleic acid, polypeptide and polysaccharide, or any other high molecular polymer that can be polymerized under acidic conditions.

[0156] Use an oxidant to cleave the P-S bond on the solid-phase carrier 10, break the P-S bond, and obtain a solid-phase carrier 10 with a fourth molecular segment 24 connected to the carrier body 1 and a fifth molecular segment 25 containing the polymer 3. The fourth molecular segment 24 is a molecular segment with a thiol group at the 3'-end or a phosphate group at the 5'-end. The reaction equation can be seen in Figure 7 shown.

[0157] Taking the above-mentioned first molecular chain segment 21 as an example, which is connected to the second molecular chain segment 22 through the 3'-position amino group and connected to the third molecular chain segment 23 through the 5'-position hydroxyl group, the S in the broken P-S bond is located on the fifth molecular chain segment 25 containing the polymer 3 with a 3'-position mercapto group at the end.

[0158] On the contrary, in the case where the first molecular chain segment 21 is connected to the second molecular chain segment 22 through the 5'-position amino group and connected to the third molecular chain segment 23 through the 3'-position hydroxyl group, the S in the broken P-S bond is located on the fourth molecular chain segment 24 with a 3'-position mercapto group at the end.

[0159] Among them, in order to avoid damage to the solid-phase carrier 10 during the cleavage process, optionally, the oxidant can be selected from silver oxide or potassium permanganate with a concentration of 0.01 M to 1 M. The temperature during cleavage can be 35 °C to 45 °C. It is found through experiments that under these conditions, quantitative cleavage of the P-S bond can be achieved within no more than 10 minutes, and no reaction occurs at other positions.

[0160] The solid-phase carrier 10 connected with the fourth molecular chain segment 24 is regenerated by an enzyme synthesis method or a DNA synthesis method, and the sixth molecular chain segment 26 with a second connection end L2 is connected to the end of the fourth molecular chain segment 24 to obtain the solid-phase carrier 10 including the carrier body 1 and the connecting molecule 2. The connecting molecule 2 includes a first connection end L1 and a second connection end L2, and the first molecular chain segment 21 connected between the first connection end L1 and the second connection end L2. The connecting molecule 2 is connected to the carrier body 1 through the first connection end L1, and the second connection end L2 is used to connect the polymer 3. The first molecular chain segment 21 is formed by connecting N structures shown in the following formula (I), and N is an integer greater than or equal to 2. In the first molecular chain segment 21, every two adjacent structures shown in the following formula (I) are connected through the X at the 3'-position in one of the structures shown in formula (I) and the R at the 5'-position in the other structure shown in formula (I) to form a structure shown in formula (II).

[0161]

[0162] In formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S. The N R1s are the same or different and are selected from any one of H, a base, and nitropyrrole. The N R2s are the same or different and are selected from hydrogen or -OR4, and R3 and R4 are selected from any one of an alkyl group, an acyl group, and a silyl group.

[0163] Thus, a cycle is formed, and the solid-phase carrier 10 can be reused to avoid scrapping.

[0164] In this process, when the solid-phase carrier 10 linked with the fourth molecular segment 24 is regenerated by the enzymatic synthesis method, a nucleotide segment 100 that forms base pairing with the first molecular segment 21 (represented by TTTTTTTTTTTTTTTTTTTTN*TTTTT), and a molecular segment 101 containing at least one P-S bond (represented by *TTTTT) can be used. Under the action of T4 ligase, the molecular segment 101 containing at least one P-S bond is linked to the end of the fourth molecular segment 24. The reaction equation is as Figure 14 shown. In this process, the molecular segment containing at least one P-S bond can be directly linked to the terminal P of the fourth molecular segment 24 through the sulfur at the 3'-position, or the molecular segment 101 containing at least one P-S bond can be linked to the terminal P of the fourth molecular segment 24 through the hydroxyl group at the 3'-position, and the 3'-position S and the 5'-position P are linked between any two adjacent monomer structures in the molecular segment 101 containing at least one P-S bond.

[0165] When the solid-phase carrier 10 linked with the fourth molecular segment 24 is regenerated by the DNA synthesis method, similar to the enzymatic synthesis method, the formation position of the P-S bond can be the same as the position of the P-S bond before breakage, or different from the position of the P-S bond before breakage. For example, compared with before breakage, the P-S bond moves backward by two monomer structures.

[0166] In some embodiments, when the solid-phase carrier 10 linked with the fourth molecular segment 24 is regenerated by the DNA synthesis method, the oxidant used in the DNA synthesis method is iodine, and the concentration of iodine is 0.005M - 0.01M. It can also prevent the oxidation effect of the oxidant from being too strong and causing the formed P-S bond to break.

[0167] To more clearly illustrate the embodiments of the present application, the solid-phase carrier provided by the present application will be described exemplarily below through specific preparation methods and regeneration methods.

[0168] Step A. Preparation of the solid-phase carrier

[0169] Step 1) Treatment of the glass carrier: Immerse the glass carrier in a concentrated sulfuric acid and potassium dichromate cleaning solution for 24h, wash it with tap water and deionized water respectively, and thoroughly rinse the residual acid on the surface with a 95% ethanol solution, and then dry it.

[0170] Step 2) Silanization treatment: Place the cleaned carrier in an aqueous solution of 5% 3-(2,3-epoxypropoxy)propyltrimethoxysilane (pH = 5.5 - 5.8), maintain the reaction at 90°C for 1h, then wash it with ethanol and dry it.

[0171] Step 3) Enclosed reaction: Place the silylated carrier into the reaction solution which is a 1:1 mixture of reagent A (10% acetic anhydride anhydrous, 10% 2,6-dimethylpyridine, 80% tetrahydrofuran) and reagent B (17% N-methylimidazole, 83% tetrahydrofuran), react at room temperature for 30 minutes, then wash with acetonitrile and dry.

[0172] Step 4) Hydroxyl activation: Treat the carrier obtained from the reaction in step 3) above with 1 mol / L hydrochloric acid at room temperature for 1 h, wash with deionized water and dry.

[0173] Step 5) Ligation reaction of the first molecular segment 21: Place the carrier obtained from the reaction in step 4) above into a reaction solution of 2 μM DNA (such as 3’-NH2-TTTTTTTTTTTTTT TTTTTTT*TTTTT-5’, where T* is a 3’-thionucleotide (i.e., 3’-position thionucleotide), and the rest are ordinary nucleotides), 0.1 M 1-(3-aminopropyl)-3-ethylcarboxylic acid hydrochloride, and 0.1 M 2-morpholinoethanesulfonic acid, place at 37 °C for 4 hours, then wash with 2×SSC (Saline Sodium Citrate) + 1% SDS (Sodium Dodecyl Sulfate) and 0.2×SSC + 0.1% SDS washing solutions for 5 minutes respectively, rinse with distilled water and dry.

[0174] Step 6) Ligation reaction of succinic acid: Place the carrier obtained from the reaction in step 5) above into a mixed solvent of 0.1 M succinic acid, 0.01 M 4-dimethylaminopyridine, 0.1 M 1-(3-aminopropyl)-3-ethylcarboxylic acid hydrochloride in 22 ml of a mixed solvent of triethylamine and anhydrous pyridine with a volume ratio of 10:1, shake on a shaker at room temperature for 16 h. Wash with deionized water and dry; proceed to step B.

[0175] Step B: Pack the carrier treated in step A into a synthesis column, install it on a DNA synthesizer to synthesize an oligonucleotide with a specific sequence, obtain polymer 3, and proceed to step C.

[0176] Step C: React the polymer 3 synthesized in step B with 0.25 M silver nitrate aqueous solution at 40 °C for 5 minutes; then separate the carrier from the liquid, and proceed to steps D and E respectively.

[0177] Step D: Collect the liquid separated in step C, add 0.25 M hydrazine aqueous solution and mix well for 3 minutes; then, react the reaction solution in 0.1 M NaCO3-CH3OH solution at 60 °C for 3 h, and finally the reaction solution is dried and purified (such as polyacrylamide gel electrophoresis, or high performance liquid chromatography, etc.) to obtain a polymer, which is an oligonucleotide with a specific sequence here.

[0178] Step E: Regeneration of the sulfur-containing nucleoside sequence linker molecule: Wash the vector obtained in Step C with deionized water and dry it; regenerate the vector linker molecule according to the following (1) or (2).

[0179] (1) Add 0.2 μM of 5’-AAAAAAAAAAAAAAAAAAAAAAA, 0.5 μM of 5’-TTTTT*, 20 U of T4 ligase and its buffer solution to the vector obtained in Step C, and react at 37 °C for 5 minutes and at 15 °C for 10 minutes (repeat three times): 5’-AAAAAAAAAAAAAAAAAAAAAAA hybridizes with the oligonucleotide sequence containing a phosphate group at the end linked to the vector, and under the action of T4 ligase, completes the ligation reaction with 5’-TTTTT*, extending the oligonucleotide sequence on the vector by 5 nucleotides; treat the above vector according to Step 6) in Step A to achieve the regeneration and reuse of the linker molecule;

[0180] (2) Add 20 U of calf intestinal alkaline phosphatase and its buffer solution to the vector obtained in Step C, and react at 37 °C for 30 minutes to convert the phosphate group of the oligonucleotide sequence containing a 5’-terminal phosphate group in the vector obtained in Step C into a hydroxyl group, and synthesize the required sequence by phosphoramidite DNA synthesis method (mix 0.1 M phosphoramidite monomer acetonitrile solution and 0.5 M tetrazole acetonitrile solution and add them to the above vector, react at room temperature for 10 minutes, wash with acetonitrile, then add an oxidant (0.25% iodine, 2% water, 22.75% pyridine, 75% tetrahydrofuran, react at room temperature for 10 minutes, wash with acetonitrile, synthesize one nucleotide; (replace or not replace the nucleotide monomer) repeat the above reaction to complete the synthesis reaction of 5 nucleotides, including at least one sulfur-containing nucleoside, extending the oligonucleotide sequence on the vector by 5 nucleotides (the synthesis of the above 5 nucleotides can also be completed by a commercial DNA synthesizer); treat the above vector according to Step 6) in Step A to achieve the regeneration and reuse of the linker molecule.

[0181] In summary, the embodiments of the present application provide a solid-phase carrier, a preparation method and an application thereof. By connecting a linking molecule 2 having a polymer-linkable group to the carrier body 1 and introducing a P-S bond that is stable under the polymer 3 synthesis conditions but sensitive to a specific oxide into the linking molecule 2, after the synthesis of the polymer 3 is completed, the P-S bond is cleaved using the specific oxidant, and thus the separation of the polymer 3 and the carrier body 1 can be achieved. During this process, no bond cleavage occurs between the first linking end L1 and the carrier body 1, nor between the first molecular segment 2 and the first linking end L1. Therefore, after the cleavage is completed, the regeneration of the solid-phase carrier 10 can be achieved by continuing to introduce the P-S bond into the linking molecule 2, thereby realizing the reuse of the solid-phase carrier 10 and avoiding scrapping. On the other hand, compared with the separation of the polymer 3 under alkaline conditions in the related art, using an oxidant with milder conditions will not cause the activation of the blocked short linking molecules, nor will it damage the surface of the carrier body 1. At the same time, when R3 and R4 in the structures shown in the above formulas (I) and (II) are both blocking groups for blocking hydroxyl groups, the hydroxyl groups blocked by these blocking groups will not be activated either, further realizing the reuse of the solid-phase carrier. In addition, according to the above, N is an integer greater than or equal to 2, and it can be known that by controlling the number of N, the density of the reaction sites for the synthesis of the polymer 3 and the distance from the surface of the carrier body 1 can also be adjusted, so as to provide an appropriate reaction space for the synthesis of the polymer 3 and avoid the problems of low reaction efficiency and low yield caused by steric effects during the reaction.

[0182] As described above, the above are only specific embodiments of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application can easily think of changes or substitutions, which should all be covered within the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claimed rights.

Claims

1. A solid-phase carrier, characterized in that, Comprising: A carrier body; A linking molecule having a first linking end and a second linking end, and a first molecular segment connected between the first linking end and the second linking end, wherein the linking molecule is connected to the carrier body through the first linking end, and the second linking end is for connecting a polymer; The first molecular segment is formed by connecting N structures shown in the following formula (I), N is an integer greater than or equal to 2. In the first molecular segment, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one structure shown in the formula (I) and R at the 5'-position in another structure shown in the formula (I) to form a structure shown in the following formula (II); Formula (I) Formula (II) Wherein, in formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S to form a corresponding P-S bond; N R1s are the same or different and are each selected from any one of hydrogen, a base, and nitropyrrole, N R2s are the same or different and are each selected from hydrogen or -OR4, and R3 and R4 are each selected from any one of an alkyl group, an acyl group, and a silyl group.

2. The solid-phase carrier according to claim 1, wherein At least one R1 is a base, and the base is selected from any one of base A, base G, base C, base T, and base I; When the base is selected from any one of base A, base G, base C, and base I, the base is further connected with a protecting group.

3. The solid-phase carrier according to claim 2, characterized in that, N R1s are each selected from any one of H and base T.

4. The solid-phase carrier according to claim 1, wherein N is greater than or equal to 18 and less than or equal to 25.

5. The solid-phase carrier according to claim 1, wherein The linking molecule further includes a second molecular segment connected between the first linking end and the first molecular segment, and the first molecular segment is connected to the first linking end through the second molecular segment.

6. The solid-phase carrier according to claim 5, wherein The first linking end and the carrier body are connected through a siloxane bond.

7. The solid-phase carrier according to any one of claims 1 to 4, wherein The linking molecule further includes a third molecular segment connected between the second linking end and the first molecular segment, and the first molecular segment is connected to the second linking end through the third molecular segment.

8. The solid-phase carrier according to claim 5 or 6, wherein The linking molecule further includes a third molecular segment connected between the second linking end and the first molecular segment, and the first molecular segment is connected to the second linking end through the third molecular segment.

9. The solid-phase carrier according to claim 8, wherein When the linking molecule includes a second molecular segment and a third molecular segment; One end of the first molecular segment is connected to the second molecular segment through an amino group at the 3'-position, and the other end is connected to the third molecular segment through a hydroxyl group at the 5'-position.

10. The solid-phase carrier according to any one of claims 1 to 6, characterized in that, Further comprising: A first blocking group connected to the carrier body for blocking a first active group on the carrier body that is not connected to the first linking end.

11. The solid-phase carrier according to claim 10, wherein when the first active group is a hydroxyl group or an amino group, the first blocking group is a carboxyl group or an acid anhydride; when the first active group is a carboxyl group, the first blocking group is an amino group or an alkylamine.

12. The solid-phase carrier according to any one of claims 1 to 6, characterized in that, The second linking end is connected with a second blocking group.

13. The solid-phase carrier according to any one of claims 1 to 6, wherein the shape of the carrier body is a sheet base or a microsphere.

14. The solid-phase carrier according to claim 13, wherein the material of the carrier body is a composite material of one or more materials selected from glass, polymer, metal and metal oxide.

15. A biochip, characterized in that, Comprising the solid-phase carrier according to any one of claims 1 to 14, and a polymer connected to the second linking end on the solid-phase carrier.

16. The biochip according to claim 15, wherein, The biochip is a microfluidic chip.

17. The biochip according to claim 15, wherein The polymer comprises at least one of nucleotides, polypeptides and polysaccharides.

18. A method for preparing a solid-phase carrier, characterized in that, Comprising: selecting a carrier body having a first active group, or forming a first active group on the carrier body by chemical or physical methods; bonding and connecting a linking molecule having a first linking end to the carrier body through the first linking end and the first active group; the linking molecule further comprises a second linking end, and a first molecular segment connected between the first linking end and the second linking end, and the second linking end is used for connecting a polymer; the first molecular segment is formed by connecting N structures shown in the following formula (I), N is an integer greater than or equal to 2, in the first molecular segment, every two adjacent structures shown in the following formula (I) are connected through X at the 3'-position in one structure shown in the formula (I) and R at the 5'-position in another structure shown in the formula (I) to form a structure shown in the following formula (II); Formula (I) Formula (II) wherein, in formula (I) and formula (II), X is selected from oxygen or sulfur, and in at least one structure shown in formula (II), X is selected from S to form a corresponding P-S bond; N R1s are the same or different, and are each selected from any one of H, a base and nitropyrrole, N R2s are the same or different, and are each selected from hydrogen or -OR4, and R3 and R4 are each selected from hydrogen, an alkyl group, an acyl group and a silyl group.

19. The preparation method according to claim 18, wherein, The bonding and connecting the linking molecule having a first linking end to the carrier body through the first linking end and the first active group comprises: selecting a first linking molecule having the first linking end and a third linking end; reacting the first linking molecule with the carrier body, and connecting the first linking molecule to the carrier body through the first linking end; selecting a monomer structure shown in formula (I), and connecting N monomer structures through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form a structure shown in formula (II) to obtain the first molecular segment; Modify the first end and the second end of the first molecular segment so that the modified first end of the first molecular segment is connected to the third linking end under the first reaction conditions, and the modified second end of the first molecular segment has the second linking end; under the first reaction conditions, the P-S bond does not break. Or, Select a monomer structure having a second reactive group at the 3'-position or 5'-position, so that the monomer structure having a second reactive group at the 3'-position or 5'-position is connected to the third linking end through the second reactive group under the second reaction conditions, and at the same time, a monomer structure having the structure shown in formula (I) is connected to the monomer structure having the second reactive group to form the structure shown in formula (II) by DNA synthesis method to obtain the first molecular segment; under the third reaction conditions, modify the end of the first molecular segment so that the modified end of the first molecular segment has the second linking end; under the second reaction conditions and the third reaction conditions, and in the DNA synthesis method, the P-S bond does not break.

20. The preparation method according to claim 19, wherein The modification of the first end and the second end of the first molecular segment so that the modified first end of the first molecular segment is connected to the third linking end under the first reaction conditions and the modified second end of the first molecular segment has the second linking end includes: Modify the first end of the first molecular segment; Connect the modified first end of the first molecular segment to the third linking end under the first reaction conditions; Under the fourth reaction conditions, modify the second end of the first molecular segment to generate the second linking end at the second end of the first molecular segment, and under the fourth reaction conditions, no reaction occurs at the remaining positions of the linking molecule.

21. The preparation method according to claim 19 or 20, characterized in that, The step of connecting N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the structure shown in formula (II) to obtain the first molecular segment includes: Connect N monomer structures in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the first molecular segment by enzymatic synthesis method or DNA synthesis method.

22. According to the preparation method described in claim 19, wherein, When N monomer structures are connected in sequence through the 3'-position of one monomer structure and the 5'-position of another monomer structure to form the first molecular segment by DNA synthesis method, the oxidant used in the DNA synthesis method is iodine, and the concentration of iodine is 0.005M to 0.01M.

23. According to the preparation method described in claim 20, wherein, The modification of the second end of the first molecular segment under the fourth reaction conditions includes: Select a second linking molecule having the second linking end and the fourth linking end, and connect the second end of the first molecular segment to the fourth linking end of the second linking molecule under the fourth reaction conditions; The modification of the end of the first molecular segment under the third reaction conditions includes: Select a second linking molecule having the second linking end and the fourth linking end, and link the end of the first molecular segment to the fourth linking end of the second linking molecule under the third reaction condition.

24. Use of a solid phase carrier as described in any one of claims 1 to 14 in polymer synthesis, characterized in that, Comprising: Contact the solid support with the monomer of the polymer, such that the second linking end reacts with the monomer of the polymer and between the monomers of the polymer to form the polymer, and link the polymer to the solid support; Use an oxidant to cleave the P-S bond on the solid support, causing the P-S bond to break, to obtain a solid support having a fourth molecular segment linked thereto and a fifth molecular segment containing the polymer, wherein the fourth molecular segment is a molecular segment having a 3'-thiol group at the end or a 5'-phosphate group at the end; Regenerate the solid support having the fourth molecular segment linked thereto by enzymatic synthesis or DNA synthesis, and link a sixth molecular segment having the second linking end to the end of the fourth molecular segment to obtain the solid support according to any one of claims 1 to 14.

25. The use of the solid support according to claim 24 in polymer synthesis, characterized in that The oxidant is selected from silver oxide or potassium permanganate having a concentration of 0.01 M to 1 M; The temperature of the cleavage is 35°C to 45°C.

26. The use of the solid support according to claim 24 in polymer synthesis, characterized in that In the case of regenerating the solid support having the fourth molecular segment linked thereto by DNA synthesis, the oxidant used in the DNA synthesis is iodine, and the concentration of iodine is 0.005 M to 0.01 M.

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