Biochip, preparation method therefor and use thereof

Abstract

A biochip, a preparation method therefor and the use thereof. The biochip is a double-sided porous nano-biochip, which has an anodized nanostructure on the surface thereof; and the surface of the chip can be chemically modified to provide, for RNA synthesis, linking molecules required for a synthesis reaction, thus enabling the attachment of monomers to the surface of the biochip. The biochip can be prepared by using an anodization method which is easily operated and is low cost, thereby lowering the difficulty of the preparation process, and providing synthetic products having a higher loading capacity at a lower price.

Description

Biochip and its preparation method and application

[0001] Priority information

[0002] This disclosure claims priority to and the benefits of patent application 2023112888291 filed on October 7, 2023 with the State Intellectual Property Office of China, and incorporates the entire text of which is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the technical field of biochip preparation, and in particular to a biochip and a preparation method and application thereof. Background Art

[0004] RNA is a long chain molecule formed by the condensation of ribonucleotides through phosphodiester bonds. It is an essential genetic information carrier for gene expression in living organisms and plays an important role in guiding protein synthesis. Chemically synthesized RNA is also an important research tool and is widely used in applications such as vaccine, nucleic acid drug development, gene function analysis, and gene therapy. The chemical synthesis method of RNA is similar to that of DNA. It generally adopts the solid-phase phosphoramidite triester method, which mainly includes 4 steps: deprotection, activation / coupling, capping, and oxidation, and the synthesis of nucleotide chains is achieved through multiple rounds of cyclic reactions (Sekine, M., Synthesis of Therapeutic Oligonucleotides, 2018, 41-65). However, due to the need to protect the 2'-OH of the RNA monomer with special groups during the synthesis process, the low coupling efficiency, and the easy decomposition of the product, the synthesis is relatively difficult compared to DNA.

[0005] Currently, mainstream commercial RNA synthesis relies mainly on first-generation column synthesis technology. Among them, the RNA synthesis column is mainly composed of a column tube, a sieve plate, and controlled porous glass (CPG). The reagents required for RNA synthesis flow through the CPG and link to the CPG surface, thereby achieving the fixation of RNA molecules on the solid phase carrier. However, the column synthesis flux is extremely low, with a maximum synthesis flux of only 1536, which is difficult to meet the needs of large-scale commercial RNA synthesis.

[0006] To commercialize and automate RNA chemical synthesis, reduce costs, and improve efficiency, chip-based microarray synthesis technology has become increasingly mainstream. Chip-based microarray synthesis is a second-generation synthesis technology, following column-based synthesis. It is a novel synthesis technique developed by integrating DNA hybridization probe technology with semiconductor industry technology. This technique typically uses micron-scale biochips as synthesis substrates, achieving high integration, high throughput, and high sensitivity, and holds significant promise for biosynthesis.

[0007] In contrast, the development time of chip-based RNA synthesis is shorter than that of column synthesis technology, but it shows great potential in improving throughput. Chip-based RNA synthesis is usually achieved through a microarray synthesizer (Lietard et al., JoVE, 2019, 150, e59936). This technology transfers the synthesis process to the micron-scale reaction array sites on the surface of the biochip to achieve the synthesis of RNA molecules. Therefore, biochips are also called microarray chips. Biochips usually use a silicon-based substrate with a pre-designed sequence array on the surface, and primer synthesis is completed by contacting the reaction reagent with the chip surface. At this stage, most RNA synthesis chips use photolithography processing technology (Lietard et al., CSBJ, 2019, 17: 862-868). The array processing of the chip surface is completed by high-cost photoresist and complex micro-nano processing technology. The higher the array processing precision, the more reaction sites available for synthesis, the greater the flux improvement, and the corresponding higher the processing difficulty requirement (Hughes et al., Nat. Biotechnol., 2001, 19(4), 342-347). At the same time, depending on the selected synthesis path, some biochips also involve optical and electrical control, and the degree of integration is extremely high, further increasing the difficulty of chip preparation technology (Maurer et al., PLoS One, 2006, 1(1), e34). In addition, due to the heterogeneity of chip surface modification, edge effects and other reasons, in actual applications, there will be phenomena such as lower than expected synthesis load, unstable synthesis products, and higher error rates. At the same time, most biochips currently use silicon-based materials. In order to meet the requirements of synthesis flux and load, silicon-based biochips require very precise structures, and the micro-nano processing technology used is also relatively difficult. Integrating a micro-reaction system from synthesis to signal detection on the surface of such a small-sized biochip is a challenge in terms of both process and cost.

[0008] On the other hand, the reactive area of ​​the biochip will directly affect the primer synthesis capacity. The larger the specific surface area of ​​the chip, the more reaction sites it can provide, and therefore the more synthetic RNA molecules it can carry. Based on this theoretical support, the specific surface area of ​​the chip can be effectively increased by constructing micro-nano structures on the surface of the biochip, thereby achieving high-capacity synthesis. At present, the micro-nano structures of mainstream commercial biochips are mostly etched on the surface using micro-nano processing technologies such as photolithography. However, the production process is very complicated and requires multiple thinning, cleaning, gluing, and etching, which damages the surface structure of the material to a certain extent, and the processing accuracy still needs to be improved. This technical path greatly increases the difficulty and cost of preparing biochips, severely limiting the development of commercial RNA synthesis chips. In addition, in practical applications, addressing the synthesis sites through different labeling methods to obtain the required RNA primer information is also a very important application link.

[0009] In general, current biochips suffer from high integration density, complex processing, and low synthesis capacity per unit area. Therefore, the key to the development of RNA synthesis chips is how to overcome the high fabrication difficulty caused by micro-nanofabrication technology through technological innovation, and address the low synthesis yield caused by the difficulty in freely expanding chip reaction sites.

[0010] Summary of the Invention

[0011] The present disclosure aims to solve, at least to a certain extent, one of the technical problems in the related art. To this end, one purpose of the present disclosure is to provide a biochip and its preparation method and application. The biochip is a double-sided porous nanobiochip with an anodized nanostructure on the surface. The surface of the chip can be chemically modified to provide the connecting molecules required for the synthesis reaction of RNA synthesis, thereby connecting the monomers to the surface of the biochip. The biochip provided by the present disclosure can be prepared by an anodizing method that is simple to operate and low in cost, which simplifies the preparation process and provides a higher load of synthetic products at a lower price.

[0012] To this end, the present disclosure provides a first aspect of a biochip, comprising:

[0013] Metal aluminum layer;

[0014] Anodized aluminum layers, the anodized aluminum layers being disposed on both sides of the metal aluminum layer and connected to the metal aluminum layer via chemical bonds;

[0015] A chemically modified layer is located on a surface of the anodized aluminum layer away from the metal aluminum layer. The chemically modified layer includes a connecting molecule, and the connecting molecule can connect an RNA monomer to a terminal of the connecting molecule.

[0016] Currently, RNA sequences can be produced at a high throughput using microarray synthesis, but overall, the synthetic loading capacity of commercial biochips is still at a low level. The loading capacity of a biochip is closely related to the number of reaction sites loaded per unit area. In terms of processing technology, the preparation of biochips is mainly through micro-nano processing technology, so the density of its reaction sites is mainly determined by the processing precision. Therefore, under the existing technology, the technical difficulty and processing cost of increasing the product loading capacity by increasing the micro-nano processing precision are relatively high. In terms of reagent injection, the currently used biochip synthesis requires the introduction of excessive reaction reagents to ensure the full progress of the reaction, resulting in a huge waste of reagents and extremely low utilization rate, which greatly increases the cost of the synthesis reaction. Overall, the existing commercial biochip synthesis cost is extremely high, and the processing precision requirements are high, which is not conducive to the development of large-scale RNA synthesis.

[0017] The biochip provided by the present disclosure has an anodized aluminum layer, which is distributed on both sides of the metal aluminum layer. The anodized aluminum layer can be prepared by an anodizing method with simple operation and low cost. The prepared anodized aluminum layer is mainly formed by a combination of anodized aluminum nanochannels, thereby greatly improving the loading capacity of the biochip synthesis products, achieving nmol-level synthesis loading capacity at low cost, and the flux can be increased by 10-1000 times compared with traditional biochips, providing a new strategy for large-scale synthesis of biological macromolecules.

[0018] According to an embodiment of the present disclosure, the anodized aluminum oxide layer is a porous nanostructure, and the inner diameter of the pores in the porous nanostructure is 100-1000 nm, thereby providing more active sites for subsequent connection of linker molecules.

[0019] According to an embodiment of the present disclosure, the linker molecule has a functional group that can react with an amino group at its beginning and a hydroxyl group protected by an acid-labile protecting group at its end. After acid treatment, the linker molecule can remove the protecting group and expose the hydroxyl group for subsequent reactions and synthesis.

[0020] According to an embodiment of the present disclosure, the linker molecule is exo-nitrogen-phenyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide, exo-nitrogen-methyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide or exo-nitrogen-methyl-5-(diglycoloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide.

[0021] According to an embodiment of the present disclosure, the surface of the anodized aluminum layer away from the metal aluminum layer is treated with a silanization agent to introduce amino groups on the surface. The amino groups can react with the starting groups of the linker molecules, thereby connecting the linker molecules to the biochip.

[0022] According to an embodiment of the present disclosure, one end of the silanization molecule in the silanization agent is an alkoxy silicon group, which is used to connect to the anodized aluminum oxide layer; the other end of the silanization molecule is an amino group, which is used to connect to the connecting molecule.

[0023] According to an embodiment of the present disclosure, the silanization molecule in the silanization reagent is at least one of APTES (3-aminopropyltriethoxysilane), APTS (3-aminopropyltriethoxysilane), and MSTFA (N-methyl-N-trimethylsilane trifluoroacetamide).

[0024] According to an embodiment of the present disclosure, the biochip carries a code, and the code has a unique correspondence with the biochip, thereby achieving chip differentiation and identification, and the synthetic information carried on the chip corresponding to the code can be obtained by reading the code.

[0025] According to an embodiment of the present disclosure, the code is provided on a surface of each anodized aluminum layer away from the metal aluminum layer.

[0026] According to an embodiment of the present disclosure, the code includes at least one of numbers, symbols, graphics, and identification codes.

[0027] According to an embodiment of the present disclosure, the code is a QR code.

[0028] According to an embodiment of the present disclosure, the chip is in the shape of a sheet, a cuboid or a cylinder.

[0029] According to an embodiment of the present disclosure, the chip is in the shape of a square sheet with a side length of 1-3 mm.

[0030] According to an embodiment of the present disclosure, the thickness of the metal aluminum layer is 0.3-0.5 mm.

[0031] According to an embodiment of the present disclosure, the thickness of the anodized aluminum oxide layer is 5-1000 μm.

[0032] A second aspect of the present disclosure provides a method for producing a biochip according to the first aspect, the method comprising the following steps:

[0033] (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip;

[0034] (2) The anodized aluminum chip is sequentially subjected to pretreatment, silanization treatment, and chemical modification to obtain the biochip.

[0035] The biochip preparation method disclosed herein uses aluminum as the substrate, avoiding the complex manufacturing processes associated with silicon-based chips. Instead, it utilizes a simple, low-cost anodization method to create an ordered microarray of pores with a precision of 10-1000 nm. Surface modification then provides the linker molecules required for RNA synthesis, thereby attaching monomers to the surface of the anodized aluminum chip. This method is simple to operate, simplifies the production process, and provides a higher loading capacity of synthetic products at a lower cost.

[0036] According to an embodiment of the present disclosure, step (1) further includes encoding the anodized aluminum chip. The encoding process enables the biochip to have an identification function, and the synthetic information carried on the chip corresponding to the code can be obtained by reading the code.

[0037] According to an embodiment of the present disclosure, the encoding process includes printing the code on the anodized aluminum chip by laser etching.

[0038] According to an embodiment of the present disclosure, the encoding process further includes printing the QR code on the anodized aluminum chip in a dotted QR code mode.

[0039] According to an embodiment of the present disclosure, the encoding process includes performing double-sided mirror coding on the anodized aluminum chip.

[0040] According to an embodiment of the present disclosure, the pretreatment method in step (2) includes a physical method or a chemical method.

[0041] According to an embodiment of the present disclosure, the physical method includes at least one of ultrasound and heat treatment.

[0042] According to an embodiment of the present disclosure, the chemical method includes at least one of acid reagent treatment, alkaline reagent treatment, and organic reagent treatment.

[0043] According to an embodiment of the present disclosure, the acid reagent includes at least one selected from sulfuric acid, hydrochloric acid, and nitric acid.

[0044] According to an embodiment of the present disclosure, the alkaline reagent includes at least one selected from sodium hydroxide and potassium hydroxide.

[0045] According to an embodiment of the present disclosure, the organic reagent includes at least one selected from acetone, isopropanol, and acetonitrile.

[0046] According to an embodiment of the present disclosure, the silanization treatment in step (2) includes immersing the pretreated anodized aluminum chip in an organic solvent containing a silanization agent.

[0047] According to an embodiment of the present disclosure, the silanization agent includes at least one selected from APTES, APTS, and MSTFA.

[0048] According to an embodiment of the present disclosure, the organic solvent is acetone, isopropyl alcohol, toluene or anhydrous ethanol.

[0049] According to an embodiment of the present disclosure, the organic solvent is anhydrous ethanol.

[0050] According to an embodiment of the present disclosure, the chemical modification in step (2) includes connecting the linker molecule to the surface of the anodized aluminum chip.

[0051] A third aspect of the present disclosure provides use of the biochip described in the first aspect or the biochip prepared according to the method described in the second aspect in RNA synthesis.

[0052] The biochip described in the first aspect or the biochip prepared according to the method described in the second aspect has a simple production process, low price, high synthetic load and throughput, is reusable, has recognition function, and can be used in RNA synthesis.

[0053] A fourth aspect of the present disclosure provides a method for RNA synthesis, comprising the following steps:

[0054] S1: providing the biochip described in the first aspect or the biochip prepared according to the method described in the second aspect;

[0055] S2: Provides 4 types of synthesis columns, used for adding ribonucleotides A, U, C, and G respectively;

[0056] S3: Determine the target ribonucleotide to be added according to the RNA sequence to be synthesized, and sort the biochip into the synthesis column corresponding to the target ribonucleotide;

[0057] S4: performing deprotection, activation, capping, and oxidation treatments on the biochip in the synthesis column to link the target ribonucleotide to the biochip;

[0058] S5: Based on the RNA sequence, determine the next ribonucleotide connected to the target ribonucleotide, and repeat steps S3-S4 until a ribonucleotide that is completely consistent with the RNA sequence is connected to the biochip.

[0059] The RNA synthesis method provided by the present disclosure is based on the biochip provided in the first aspect of the present disclosure or the biochip prepared according to the method described in the second aspect of the present disclosure, and can achieve an RNA synthesis load at the nmol level. At the same time, the flux can be increased by 10-1000 times compared with traditional biochips, greatly reducing the cost of commercial RNA synthesis and providing a new strategy for the synthesis of biological macromolecules.

[0060] According to an embodiment of the present disclosure, the biochip carries a code corresponding to the RNA sequence to be synthesized.

[0061] According to an embodiment of the present disclosure, step S3 further includes: identifying the code carried by the biochip, and determining the target ribonucleotide to be added based on the RNA sequence corresponding to the code, and sorting the biochip into a synthesis column corresponding to the target ribonucleotide.

[0062] According to an embodiment of the present disclosure, the method further includes the following steps:

[0063] S6: Cutting the RNA from the biochip to obtain the RNA.

[0064] According to an embodiment of the present disclosure, step S6 further includes cutting the RNA from the biochip by an aminolysis reagent.

[0065] According to an embodiment of the present disclosure, the ammonolysis reagent includes at least one selected from ammonia water, ammonia gas, and methylamine.

[0066] According to an embodiment of the present disclosure, the method further includes the following steps:

[0067] S7: isolating and purifying the RNA.

[0068] According to an embodiment of the present disclosure, the separation and purification method includes at least one selected from PAGE, PAGE Plus, and HPLC.

[0069] The present disclosure has the following beneficial effects compared to the prior art:

[0070] The biochip provided by the present disclosure is an aluminum-based chip, which solves the problems of high brittleness and easy wear of traditional silicon-based chips, ensures the reuse of chips, and reduces production costs. In addition, it is distributed with anodized aluminum layers on both sides, avoiding the complex manufacturing process of conventional silicon-based chips. The anodization method can achieve the preparation of ordered microarray holes with a precision of 10-1000nm. At the same time, the anodized aluminum layer increases the reactive area of ​​the biochip, achieving the synthesis of nmol-level RNA at low cost, and the throughput can be increased by 10-1000 times compared with traditional biochips, significantly reducing the cost of commercial RNA synthesis, and providing a new strategy for large-scale and large-scale synthesis of biological macromolecules.

[0071] Additional aspects and advantages of the present disclosure will be given in part in the description that follows and, in part, will be obvious from the description that follows, or will be learned through practice of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0072] The above and / or additional aspects and advantages of the present disclosure will become apparent and readily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0073] FIG1 shows the appearance of a biochip provided by the present disclosure;

[0074] FIG2 shows a structural diagram of a biochip provided by the present disclosure;

[0075] Figure 3 shows a schematic diagram of the structure of the anodized aluminum layer in the biochip.

[0076] Detailed Description of the Invention

[0077] The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be understood as limiting the present disclosure.

[0078] Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features being referred to. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. Throughout the present disclosure, "plurality" means at least two, such as two, three, etc., unless otherwise specifically defined.

[0079] The endpoints of the ranges and any values ​​disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoints of each range, the endpoints of each range and individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered to be specifically disclosed herein.

[0080] In order to make the present disclosure more easily understood, certain technical and scientific terms are specifically defined below. Unless otherwise clearly defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by those skilled in the art to which the present disclosure belongs.

[0081] In this document, the terms “include” or “comprising” are open expressions, that is, including the contents specified in the present disclosure, but not excluding other contents.

[0082] As used herein, the terms "optionally," "optional," or "optionally" generally mean that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

[0083] According to an embodiment of the present disclosure, a biochip is provided. The chip appearance is shown in FIG1 and the structure is shown in FIG2 , including:

[0084] A metal aluminum layer 102;

[0085] Anodized aluminum layers 101 and 103, the anodized aluminum layers are provided on both sides of the metal aluminum layer 102 and are connected to the metal aluminum layer 102 via chemical bonds;

[0086] A chemically modified layer is located on a surface of the anodized aluminum layer away from the metal aluminum layer 102 . The chemically modified layer includes a linker molecule capable of linking an RNA monomer to a terminal end of the linker molecule.

[0087] According to the specific embodiments of the present disclosure, the biochip can be processed into any shape and size, such as sheet, cuboid, cylinder, etc., according to the actual production application and in conjunction with the needs of the synthesizer. Preferably, the chip is a square sheet with a side length of 1-3 mm, more preferably, a side length of 2 mm. Preferably, the thickness of the metal aluminum layer is 0.3-0.5 mm, and the thickness of the anodized aluminum layer is 5-1000 μm. The anodized aluminum layer is a porous nanostructure, which is formed by a combination of anodized aluminum nanochannels 104, see Figure 3, and the parameters such as the pore depth, pore diameter, channel angle, and pore density of the nanochannel can be adjusted according to the actual production application. Preferably, the inner diameter of the pores in the porous nanostructure is 100-1000 nm, the pore array is highly ordered and evenly distributed, the pore surface and inner wall are relatively smooth and have hydroxyl active sites for subsequent connection of linking molecules, thereby solving the problem of low loading of synthetic products in current biochips.

[0088] According to a specific embodiment of the present disclosure, the biochip carries a code, and the code has a unique corresponding relationship with the biochip. The code provides a specific signal for the chip, and achieves one code per chip, so that the chip has an identifiable characteristic. By reading the code, the synthetic information carried on the chip corresponding to the code can be obtained, and it can be transported to the corresponding synthetic column for synthetic reaction. The specific signal can be an optical signal, a magnetic signal, an identification code, an RFID tag, etc., or a combination of such characteristic codes can be used for marking and identification. The code includes but is not limited to numbers, symbols, graphics, and identification codes. Preferably, the code is a QR code. Preferably, the front and back surfaces of the biochip carry the same QR code mark, and the QR code can be recognized by a detector to distinguish different synthetic information of multiple chips, or to quickly find the target chip from multiple chips for sorting.

[0089] According to a specific embodiment of the present disclosure, after the unique code is added to the biochip, the surface can be silanized and chemically modified to meet the RNA synthesis requirements, so that the chip surface can be connected to the RNA monomer reagent through a specific molecular structure, ensuring that there are enough reaction sites on the chip surface and that the sites are evenly distributed. Before silanization and chemical modification, the chip with the code added can be placed in an organic reagent. The type of organic reagent can be a reagent with low water content such as acetone, isopropanol, toluene, etc. Preferably, anhydrous ethanol is selected as the reaction reagent. Subsequently, a silanization reagent such as APTES, APTS, or MSTFA is added to react with the hydroxyl groups exposed on the chip surface and connect to the chip surface. This process can adjust the spacing of the reaction sites and optimize the density of the reaction sites on the chip surface by adjusting the concentration of the silanization reagent, the time and temperature of the silanization treatment, and the type of reagent. After the silanization process is completed, the biochip is cleaned with acetone and then chemically modified to connect the connecting molecules on the chip surface. The linker molecule is used to initiate RNA synthesis reaction. The starting end of the linker molecule is a functional group that can react with an amino group; the middle end can be a group having any one or more functional groups selected from ester group, thioester group, o-nitrobenzyl group, coumarin group, hydroxyl group, sulfhydryl group, sulfhydryl group, carboxyl group, aldehyde group, amino group, amine group, amide group, alkenyl group, and alkynyl group; the terminal end is a hydroxyl group protected by an acid-labile protecting group, preferably 4,4'-dimethoxytrityl (DMT). After treatment with an acid (such as a mixed acid reagent of dichloroacetic acid, trichloroacetic acid, or trifluoroacetic acid and an organic solvent), the protecting group can be removed to expose the hydroxyl group for subsequent reaction synthesis. Preferred linker molecules are exo-nitrogen-phenyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide, exo-nitrogen-methyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide or exo-nitrogen-methyl-5-(diglycoloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide.

[0090] According to an embodiment of the present disclosure, the present disclosure further provides a method for preparing the aforementioned biochip, the method comprising the following steps:

[0091] (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip;

[0092] (2) The anodized aluminum chip is sequentially subjected to pretreatment, silanization treatment, and chemical modification to obtain the biochip.

[0093] According to a specific embodiment of the present disclosure, step (1) further includes encoding the anodized aluminum chip. The encoding process can be performed by laser etching or a dotted QR code mode to print the QR code on the anodized aluminum chip. As a unique identification signal, the QR code corresponds one-to-one to the chip to which it belongs, that is, each chip carries a different QR code. In order to ensure the recognition rate of the chip during use, the chip can be double-sided mirrored, that is, the surface of the anodized aluminum layer has the same and mirror-image QR code mark. The code point marking range can be adjusted according to the chip size, and the marking range can be changed by adjusting the code point diameter and spacing. Preferably, the marking range is 1mm×1mm.

[0094] According to a specific embodiment of the present disclosure, the anodized aluminum chip can be cut after step (1), that is, the size and geometric shape of the chip prepared by the anodizing method can be adjusted by processing. For example, a square porous nano biochip with a side length of 1-3 mm is obtained by laser cutting. Preferably, the chip size is determined to be 2mm×2mm. In order to ensure the accuracy of cutting and improve the yield, while avoiding destroying the nanostructure of the anodized aluminum layer and destroying the burrs on the cutting edge of the coding site, the cutting cross section should clearly show the nanopores of the upper and lower surface anodized aluminum layers and the intermediate metal aluminum layer.

[0095] According to a specific embodiment of the present disclosure, the pretreatment method in step (2) includes a physical method or a chemical method, or a method combining physical and chemical methods. The physical method includes but is not limited to ultrasonic and heat treatment. The chemical method includes but is not limited to the use of acid solutions such as sulfuric acid, hydrochloric acid, nitric acid, mixed acid, alkaline solutions such as sodium hydroxide and potassium hydroxide, or organic reagents such as acetone, isopropanol, and acetonitrile for cleaning. The pretreatment is, on the one hand, to clean the chip surface and remove impurities loaded on the chip surface during coding and cutting; on the other hand, to increase the surface activity of the chip, expose more hydroxyl groups, and increase silanization and subsequent reaction sites.

[0096] According to a specific embodiment of the present disclosure, the silanization treatment in step (2) includes immersing the pretreated anodized aluminum chip in an organic solvent containing a silanization agent, wherein the silanization agent may be APTES, APTS, or MSTFA, and the organic solvent may be acetone, isopropanol, toluene, or anhydrous ethanol with a low water content. The silanization treatment can uniformly introduce amino groups on the chip surface, and the spacing between synthesis sites can be controlled by APTES to adjust subsequent steric hindrance. Chemical modification includes connecting the connecting molecule to the surface of the anodized aluminum chip. The structure of the connecting molecule is as described above. When RNA synthesis is performed, when the connecting molecule carried on the chip contacts the reaction reagent, a monomer is obtained from the reaction reagent and connected to the end of the connecting molecule. After each round of injection synthesis, the chain will be extended to form an RNA molecule connected to the chip surface through the connecting molecule. It should be noted that the preparation order of the chip is preferably encoding first and then modifying, so as to protect the modified two-dimensional code chip surface and synthesis site, which is beneficial to the subsequent synthesis effect.

[0097] According to an embodiment of the present disclosure, the present disclosure also provides a method for synthesizing RNA molecules, which is based on the aforementioned biochip for RNA synthesis. Generally speaking, the synthesis methods of RNA and DNA are similar, both using the solid-phase phosphoramidite triester method. Through multiple rounds of liquid injection cycle reactions, RNA monomers are connected one by one in sequence to the connecting molecules on the surface of the chip to form an RNA chain. Each cycle mainly includes four steps: deprotection, activation, capping and oxidation. In a specific embodiment, according to the flux requirements, the chip encoding method, encoding range and sorting scheme are formulated, and the corresponding identifier, sorter, and the drive device for controlling the movement of the chip and the injection of liquid are adjusted. The monomers synthesized in this round are identified by the identifier and screened by the sorter, and the chip is distributed to the corresponding synthesis column via a transport device for chemical reaction synthesis. The reaction order follows the four steps of each cycle, and the reactions are carried out in sequence until one cycle is completed. Through program setting, according to the required sequence, the cycle is repeated until the synthesis is completed, and finally a biochip containing the required RNA primer product can be collected.

[0098] According to the specific embodiments of the present disclosure, the synthetic monomers of RNA need to protect the 2'-OH group, and the selected protective group must be removed after the synthesis is completed. The selected protective group can be an ether protective group or an acetal protective group, such as: tert-butyldimethylsilyl (TBDMS), triisopropylsiloxymethyl (TOM), 1-(4-chlorophenyl)-4-ethoxypyridin-4-yl (Cpep) and pivaloyloxymethyl (PivOM), etc. The selection of its protective group follows the principles of high stability and easy removal. Preferably, pivaloyloxymethyl (PivOM) is selected as the protective group for 2'-OH. The selected RNA monomer can be:

[0099] (1) RNA modification monomer A, 2'-O-PivOM-5'-O-DMT-Adenosine (N-PAC) 3'-CEP

[0100] (2) RNA modification monomer U, 2'-O-PivOM-5'-O-DMT-Uridine 3'-CEP

[0101] (3) RNA modification monomer C, 2'-O-PivOM-5'-O-DMT-Cytidine(N-Ac)3'-CEP

[0102] (4) RNA modification monomer G, 2'-O-PivOM-5'-O-DMT-Guanosine (N-iprPAC) 3'-CEP.

[0103] According to specific embodiments of the present disclosure, the method further includes separating the chip from the synthetic product it carries. Specifically, the RNA molecules can be cleaved from the chip through an aminolysis reaction. The aminolysis reagent can be ammonia water, ammonia gas, methylamine, or any combination thereof. After the aminolysis reaction, the cleaved RNA can be separated and purified using methods such as PAGE, PAGE Plus, and HPLC.

[0104] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. All reagents or instruments used without specifying the manufacturer are commercially available conventional products.

[0105] Example 1 Preparation of biochip

[0106] (1) Anodizing both sides of the metal aluminum layer to obtain an anodized aluminum chip;

[0107] (2) Coding and cutting of anodized aluminum chips. Using laser printing technology, a 1mm×1mm QR code is printed in an orderly manner on the base material of the anodized aluminum chip. Using positioning cutting technology, the material is processed into a 2mm×2mm×0.45mm microchip with the QR code point position as the center. Each chip center carries a specific QR code. The coded and cut chips are pretreated with acetone and then plasma cleaned;

[0108] (3) Silanization treatment and chemical modification.

[0109] Silanization treatment: Prepare 100 mL of 50% silanization reagent and divide it into two 100 mL beakers. Place the cleaned QR code chip in each beaker, sonicate for 30 minutes, clean it with acetone five times, and place it in an 80°C drying oven for 10 minutes before taking it out.

[0110] The silanization reagent is a 1 vol.% (APTES:PTES = 1:1) solution in anhydrous ethanol. For a total volume of 100 mL, the components are 500 μL of APTES, 500 μL of PTES, and 99 mL of anhydrous ethanol.

[0111] Chemical modification: 1000 mg of the linker molecule, 800 mg of HATU, and 2000 μL of DIPEA were added to 100 mL of acetonitrile. The mixture was shaken and divided into two 50 mL centrifuge tubes. 2000 silanized QR code chips were placed in each centrifuge tube. The mixture was stirred overnight in a vertical stirrer. After the reaction was complete, the chips were collected and washed three times with acetonitrile and acetone, respectively. After drying, the chips were collected in centrifuge tubes.

[0112] Example 2 Synthesis of RNA

[0113] (1) Prepare synthetic monomers, reagents and corresponding reaction pools

[0114] Prepare four single-base reaction pools, which are used to add ribonucleotide A, U, C and G monomers, as well as the reaction reagents required for each round of reaction (deprotection, capping, oxidation and washing).

[0115] During coupling,

[0116] Adenylate and tetrazole with 5'-OH protected by DMT, 2'-OH protected by PivOM, and 3'-OH protected by phosphoramidite were added to the first reaction pool;

[0117] The second reaction pool was added with uridine monophosphate and tetrazole whose 5'-OH was protected by DMT, 2'-OH was protected by PivOM, and 3'-OH was protected by phosphoramidite;

[0118] The third reaction pool is added with cytidine monophosphate and tetrazole whose 5'-OH is protected by DMT, 2'-OH is protected by PivOM, and 3'-OH is protected by phosphoramidite;

[0119] The fourth reaction pool is added with guanosine monophosphate and tetrazole whose 5'-OH is protected by DMT, 2'-OH is protected by PivOM, and 3'-OH is protected by phosphoramidite;

[0120] - During deprotection, trichloroacetic acid in dichloromethane was added to all four reaction cells as a deprotection reagent;

[0121] - During capping, acetic anhydride / pyridine / tetrahydrofuran and N-methylimidazole acetonitrile solution was added to all four reaction cells as capping reagents;

[0122] - During oxidation, 0.01 M iodine solution was added to all four reaction cells as an oxidant;

[0123] - During cleaning, acetonitrile was added to all four reaction cells as cleaning solution;

[0124] (2) Prepare equipment and software programs

[0125] Using a high-throughput RNA synthesis apparatus, wherein the apparatus comprises,

[0126] - A QR code reader that can identify the QR code information carried by the chip and generate a signal;

[0127] -Chip sorter, which can sort chips;

[0128] - A driving device that drives the chip to move;

[0129] - a reagent liquid driving device capable of introducing and withdrawing the reagent liquid from the synthesis reaction cell;

[0130] - A central processing unit, which is capable of receiving the signal generated by the QR code recognizer when recognizing the QR code, and issuing instructions to control the chip sorter to sort the chips, or controlling the driving device to move the chips;

[0131] - A memory for storing the sequence of the RNA molecules to be synthesized, the correspondence between the chip and the QR code, the correspondence between the RNA molecules to be synthesized and the chip, and the reactions that each solid phase carrier has undergone.

[0132] In addition, a control program is set according to the target RNA sequence to be synthesized, and the control program can determine the sorting and movement scheme of the chip according to the predetermined RNA sequence;

[0133] (3) RNA synthesis

[0134] First, all the chips are mixed, and the QR code pre-printed on the chip is identified by the identifier. The central processing unit controls the chip sorter to sort the chips according to the identification signal of the identifier, and moves each chip to the corresponding target reaction pool according to the preset control program. Then, the deprotection reagent is introduced into the reaction pool for deprotection through the control driver, and the deprotection reagent is drawn out after the reaction is completed; the cleaning liquid is introduced to clean the chip, and then the coupling reagent is introduced according to the monomer to be coupled corresponding to the reaction pool; the coupling reagent is drawn out after the reaction is completed; the cleaning liquid is introduced to clean the chip, and finally the capping reagent, oxidizing reagent and cleaning liquid are introduced in sequence for capping, oxidation and cleaning respectively. At this point, a cycle is completed, and the coupling of one base is completed. According to the predetermined RNA synthesis sequence information, the above steps are repeated cyclically until the RNA molecule is synthesized;

[0135] (4) RNA isolation and purification

[0136] After RNA synthesis is complete, all synthesized chips can be collected together and aminolyzed to isolate and purify all synthesized RNA molecules. Alternatively, all chips can be identified using a QR code, sorted and collected, and then aminolyzed to isolate and purify the target RNA molecules.

[0137] Example 3 Comparative Example

[0138] A 100 nm silicon oxide wafer (silicon wafer) was selected as the control group. The specific steps for synthesizing RNA molecules are as follows:

[0139] (1) Using laser printing technology, a 1mm×1mm QR code is printed in an orderly manner on the substrate material of the silicon oxide chip; using positioning cutting technology, the material is processed into a 2mm×2mm×0.45mm microchip with the QR code point position as the center. After the silicon wafer is coded and cut, the chip is pre-treated with acetone and then plasma cleaned;

[0140] (2) Preparation of biochip:

[0141] The processed and cleaned silicon wafer was subjected to silanization treatment and chemical modification, and the specific operation was the same as experimental step (3) in Example 1;

[0142] (3) RNA synthesis:

[0143] The silicon wafer chemically modified in step (2) was used to perform RNA synthesis experiments. The specific experimental steps and operation methods were the same as those in Example 2.

[0144] After summarizing the experimental results and comparing them, it was found that the chemically modified porous nanobiochip can be used for RNA synthesis, and the synthesis quality is better than that of silicon wafers, and the synthesis load is orders of magnitude higher than that of silicon wafers. The above examples and experimental results all show that the biochip proposed in this disclosure can be used for RNA synthesis, and the synthesis effect is more advantageous than the current commercial mainstream RNA synthesis effect. This further shows that the chemically modified identifiable porous nanobiochip proposed in this disclosure is highly feasible and has broad application prospects.

[0145] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and features of different embodiments or examples, unless they are mutually inconsistent.

[0146] Although the embodiments of the present disclosure have been shown and described above, it is understood that the above embodiments are illustrative and are not to be construed as limitations on the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

1. A biochip, characterized in that: include: Metal aluminum layer; Anodized aluminum layers, the anodized aluminum layers are disposed on both sides of the metal aluminum layer and are connected to the metal aluminum layer through chemical bonds; A chemically modified layer is located on a surface of the anodized aluminum layer away from the metal aluminum layer, and the chemically modified layer includes a connecting molecule, and the connecting molecule can connect an RNA monomer to a terminal of the connecting molecule.

2. The biochip according to claim 1, characterized in that: The anodized aluminum layer is a porous nanostructure, and the inner diameter of the pores in the porous nanostructure is 100-1000 nm.

3. The biochip according to claim 1, characterized in that: The linking molecule has a starting end that is a functional group that can react with an amino group and a terminal end that is a hydroxyl group protected by an acid-labile protecting group; Optionally, the linker molecule is exo-nitrogen-phenyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide, exo-nitrogen-methyl-5-(succinyloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide or exo-nitrogen-methyl-5-(diglycoloxy)-6-dimethyl-7-oxabicyclo[2.2.1]heptane-2-3-dicarboximide.

4. The biochip according to claim 3, characterized in that: The surface of the anodized aluminum layer away from the metal aluminum layer is treated with a silanization agent to introduce amino groups on the surface; Optionally, one end of the silanized molecule in the silanization agent is an alkoxysilyl group, which is used to connect to the anodized aluminum oxide layer; the other end of the silanized molecule is an amino group, which is used to connect to the connecting molecule; Optionally, the silanization molecule in the silanization agent is at least one of APTES, APTS, and MSTFA.

5. The biochip according to claim 1, characterized in that: The biochip carries a code, and the code has a unique corresponding relationship with the biochip; Optionally, the code is disposed on a surface of each anodized aluminum layer away from the metal aluminum layer; Optionally, the code includes at least one of a number, a symbol, a graphic, and an identification code; Optionally, the code is a QR code.

6. The biochip according to claim 1, characterized in that: The chip is in the shape of a sheet, a cuboid or a cylinder; Optionally, the chip is in the shape of a square sheet with a side length of 1-3 mm; Optionally, the thickness of the metal aluminum layer is 0.3-0.5 mm; Optionally, the thickness of the anodized aluminum layer is 5-1000 μm.

7. A method for preparing the biochip according to any one of claims 1 to 6, characterized in that: The method comprises the following steps: (1) performing anodizing treatment on both sides of the metal aluminum layer to obtain an anodized aluminum chip; (2) The anodized aluminum chip is sequentially subjected to pretreatment, silanization treatment and chemical modification to obtain the biochip.

8. The method according to claim 7, characterized in that Step (1) further includes encoding the anodized aluminum chip; Optionally, the coding process includes printing the code on the anodized aluminum chip by laser etching; Optionally, the encoding process further comprises printing a two-dimensional code on the anodized aluminum chip in a dotted two-dimensional code mode; Optionally, the encoding process includes double-sided mirror coding on the anodized aluminum chip.

9. The method according to claim 7, characterized in that: The pretreatment method in step (2) includes a physical method or a chemical method; Optionally, the physical method comprises at least one of ultrasound and heat treatment; Optionally, the chemical method comprises at least one of an acid reagent treatment, an alkali reagent treatment, and an organic reagent treatment; Optionally, the acid reagent includes at least one selected from sulfuric acid, hydrochloric acid, and nitric acid; Optionally, the alkaline reagent includes at least one selected from sodium hydroxide and potassium hydroxide; Optionally, the organic reagent includes at least one selected from acetone, isopropanol, and acetonitrile.

10. The method according to claim 7, characterized in that The silanization treatment in step (2) comprises immersing the pretreated anodized aluminum chip in an organic solvent containing a silanization agent; Optionally, the silanization agent comprises at least one selected from APTES, APTS, and MSTFA; Optionally, the organic solvent is acetone, isopropanol, toluene or anhydrous ethanol; Optionally, the organic solvent is anhydrous ethanol.

11. The method according to claim 7, characterized in that The chemical modification in step (2) includes connecting the linking molecule to the surface of the anodized aluminum chip.

12. Use of the biochip according to any one of claims 1 to 6 or the biochip prepared according to the method according to any one of claims 7 to 11 in RNA synthesis.

13. A method for RNA synthesis, characterized in that: The method comprises the following steps: S1: providing a biochip according to any one of claims 1 to 6 or a biochip prepared according to the method according to any one of claims 7 to 11; S2: Provides 4 types of synthetic columns, used to add ribonucleotides A, U, C, and G respectively; S3: determining the target ribonucleotide to be added according to the RNA sequence to be synthesized, and sorting the biochip into a synthesis column corresponding to the target ribonucleotide; S4: performing deprotection, activation, capping and oxidation treatment on the biochip in the synthesis column so as to connect the target ribonucleotide to the biochip; S5: Based on the RNA sequence, determine the next ribonucleotide connected to the target ribonucleotide, and repeat steps S3-S4 until the ribonucleotide that is completely consistent with the RNA sequence is connected to the biochip.

14. The RNA synthesis method according to claim 13, characterized in that: The biochip carries a code corresponding to the RNA sequence to be synthesized; Optionally, step S3 further comprises: identifying the code carried by the biochip, and according to the RNA sequence corresponding to the code, The target ribonucleotide to be added is determined, and the biochip is sorted into a synthesis column corresponding to the target ribonucleotide.

15. The RNA synthesis method according to claim 13, characterized in that: The method further comprises the following steps: S6: cutting the RNA from the biochip to obtain the RNA; Optionally, step S6 further comprises cleaving the RNA from the biochip by an aminolysis reagent; Optionally, the ammonolysis reagent includes at least one selected from aqueous ammonia, ammonia gas, and methylamine.

16. The RNA synthesis method according to claim 15, characterized in that: The method further comprises the following steps: S7: separating and purifying the RNA; Optionally, the separation and purification method comprises at least one selected from PAGE, PAGE Plus, and HPLC.

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