Gene sequencing chip, preparation method and device, and chip storage method

Abstract

The present application provides a gene sequencing chip and a preparation method thereof, a gene sequencing device comprising the gene sequencing chip, and a storage method comprising the gene sequencing chip. The gene sequencing chip substrate has a first surface; a microwell array is located in the first surface; and a nanowire array is formed by nanowires surrounding each microwell in the microwell array. The gene sequencing chip of the present application expands the traditional planar sequencing reaction to three-dimensional space sequencing by constructing a high-density nanowire array, greatly improves the sequencing throughput, effectively improves the fluorescence intensity of the sequencing cluster, and improves the detection sensitivity.

Description

Technical Field

[0001] This disclosure relates to a gene sequencing chip and a method for preparing the same, including a gene sequencing device for the gene sequencing chip and a method for preserving the gene sequencing chip. Background Technology

[0002] For a long time, DNA sequencing technology has been one of the most commonly used techniques in molecular biology research, driving the rapid development of the field to a certain extent. The Human Genome Project, transcriptome analysis, microbial genome resequencing, and single nucleotide polymorphism (SNP) analysis have also promoted research and development in other biological fields. Each generation of sequencing technology signifies a new breakthrough in biological technologies such as gene chips, data analysis, surface chemistry, and bioengineering, which are then applied to sequencing, significantly reducing sequencing costs, improving sequencing efficiency, and propelling sequencing towards high throughput, low cost, high security, and commercialization.

[0003] Second-generation DNA sequencing technology, also known as next-generation sequencing technology, represents a revolutionary advancement over first-generation sequencing technology. Current technology platforms primarily include Roche / 454GS FLX, Illumina / Sol-exa Genome Analyzer, and HeliScope from Helicos BioSciences. TM Single Molecule Sequencer, the Polonator from DanaherMotion (USA), and ligation sequencing (which uses primers to locate nucleic acid information) are all available technologies, with platforms including Applied Biosystems / SOLiD. TM The sequencing principles used in the above technology platforms are all cyclic microarrays. Currently, the most mainstream sequencers on the market (Illumina, BGI) all require the use of sequencing chips. Each sequencing reaction is completed on the sequencing chip, so the throughput and other performance characteristics of the sequencing chip directly determine the sequencing effect.

[0004] All next-generation sequencing platforms require a gene library containing custom adapter sequences derived through extension or ligation. The constructed library is immobilized onto the sequencing chip surface. Synthesis sequencing uses a stepwise integration method with reversible fluorescence and terminating nucleotides for DNA sequencing. First, four nucleotides are simultaneously added to the sequencing chip. After nucleotide binding, the remaining DNA bases are washed away. The fluorescence signal in each gene cluster is read and recorded, and this process is repeated until the sequencing reaction is complete. To ensure that each sequencing unit's reaction proceeds independently and smoothly, the sequencing chip needs to be divided into hundreds of millions of independent reaction separators. Strict requirements are placed on parameters such as the slope angle of the microwell array to support DNA molecule immobilization and prevent crosstalk noise. Furthermore, cost is one of the most important factors in high-throughput sequencing; therefore, high-density sequencing reaction clusters can effectively reduce sequencing costs. Thus, the process of constructing high-density reaction units becomes crucial, and surface modification of the chip to reduce noise and improve sequencing accuracy is another key technology. Summary of the Invention

[0005] This invention provides a gene sequencing chip that solves or alleviates one or more technical problems in the prior art by constructing micropore arrays and nanowire arrays.

[0006] As one aspect of the present invention, a gene sequencing chip is provided, comprising:

[0007] The substrate has a first surface;

[0008] A micropore array is located within the first surface;

[0009] The nanowire array is formed by nanowires that surround each micropore in the micropore array.

[0010] In some possible implementations, the nanowire array is spaced apart.

[0011] In some possible implementations, the interior of the micropores in the micropore array is hydrophilic, while the spacers between the micropores are hydrophobic.

[0012] In some possible implementations, at least 1 G reads of throughput are achieved within an area of ​​25 mm × 65 mm.

[0013] In some possible implementations, the nanowires are connected to adapters required for sequencing.

[0014] In some possible implementations, the diameter of the nanowire is less than 100 nanometers.

[0015] As another aspect of the present invention, a method for preparing a gene sequencing chip is provided, comprising:

[0016] A microporous material layer is formed on the surface of the first substrate, and a microporous array is patterned thereon.

[0017] A metal mask material layer is deposited on the surface of the micropore array, and a metal mask is formed by patterning.

[0018] The micropore array with the metal mask is etched to form a nanowire array surrounding each micropore in the micropore array.

[0019] In some possible implementations, the fabrication method further includes surface modification of the micropore array between forming the micropore array and forming the metal mask, such that the micropore spacing portion of the micropore array is hydrophobic.

[0020] In some possible implementations, the surface modification includes partially depositing silica within the micropores for hydrophilic modification and coating the spacer portion of the micropores with a silane coupling agent layer for hydrophobic modification.

[0021] As another aspect of the present invention, a gene sequencing device is provided, which includes the above-described gene sequencing chip.

[0022] In another aspect of the present invention, a method for preserving a gene sequencing chip is provided, the gene sequencing chip comprising an array of microwells spaced apart, the preservation method comprising:

[0023] Preparation of water-soluble prepolymers;

[0024] The water-soluble prepolymer is coated onto the surface of the gene sequencing chip;

[0025] The prepolymer is polymerized into a water-soluble polymer.

[0026] In some possible implementations, the preservation method further includes removing the water-soluble polymer from the micropore spacers of the gene sequencing chip.

[0027] In some possible implementations, the water-soluble polymer is an acrylamide prepolymer.

[0028] In some possible implementations, the preparation of water-soluble prepolymers includes prepolymerizing N-(5-azidoacetamidopentyl)acrylamide and acrylamide separately at 50°C.

[0029] The gene sequencing chip of the present invention, by constructing a high-density nanowire array and the difference in hydrophilicity and hydrophobicity inside and outside the micropore array, extends the traditional planar sequencing reaction to three-dimensional spatial sequencing, greatly improving the sequencing throughput, while effectively increasing the fluorescence intensity of the sequencing cluster and improving the detection sensitivity.

[0030] The above overview is for illustrative purposes only and is not intended to be limiting in any way. Further aspects, embodiments, and features of this disclosure will become readily apparent from the accompanying drawings and the following detailed description, in addition to the illustrative aspects, embodiments, and features described above. Attached Figure Description

[0031] In the accompanying drawings, unless otherwise specified, the same reference numerals throughout the various drawings denote the same or similar parts or elements. These drawings are not necessarily drawn to scale. It should be understood that these drawings depict only some embodiments disclosed in this disclosure and should not be construed as limiting the scope of this disclosure.

[0032] Figure 1 This is a schematic diagram of a micropore array for a gene sequencing chip according to an embodiment of the present invention;

[0033] Figure 2 This is a schematic diagram of the arrangement shape of the micropore array of a gene sequencing chip according to an embodiment of the present invention.

[0034] Figure 3 This is a schematic diagram of a nanowire array for a gene sequencing chip according to an embodiment of the present invention.

[0035] Figure 4 A cross-sectional scanning electron microscope image of the micropores of a gene sequencing chip according to an embodiment of the present invention;

[0036] Figure 5 A cross-sectional scanning electron microscope image of nanowires in a gene sequencing chip according to an embodiment of the present invention;

[0037] Figure 6 A schematic diagram of a mask pattern for preparing a gene sequencing chip according to an embodiment of the present invention;

[0038] Figure 7 A flowchart of a method for preparing a gene sequencing chip according to an embodiment of the present invention;

[0039] Figure 8 This is a comparison diagram of the preservation effect of a gene sequencing chip according to an embodiment of the present invention. Detailed Implementation

[0040] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of this disclosure. Therefore, the drawings and description are to be considered exemplary in nature and not restrictive.

[0041] This invention provides a gene sequencing chip, comprising a high-density three-dimensional nanowire array. Specifically, the gene sequencing chip of this invention includes a substrate, a micropore array formed on the surface of the substrate, and a nanowire array formed around each micropore in the micropore array.

[0042] The substrate used in this invention can be a substrate commonly used in the field of gene sequencing chips, such as a glass substrate.

[0043] like Figure 1 As shown, the micropore array of the gene sequencing chip of the present invention is formed on the substrate surface, typically in the form of a circular micrometer-scale micropore array, with the micropores spaced apart. Furthermore, the micropores are hydrophilic, while the spacers between them are hydrophobic, arranged in an alternating dense packing. Each micropore forms a reaction unit, and the reaction unit contains a sequencing reagent reaction chamber.

[0044] The arrangement pattern of the aforementioned micropore array can be set as needed, for example, as shown in the figure. Figure 1 and Figure 2 As shown, the arrays are arranged in a hexagonal shape, with intervals between each hexagonal array. According to one specific embodiment, to facilitate counting and positioning during sequencing result readout, each hexagonal array pattern contains 96 microwells, with each microwell and its interval forming a cycle.

[0045] According to a specific implementation method, such as Figure 4 As shown, the diameter of the micropores is typically 0.3-5 μm, preferably 1-3 μm, and the spacing between the micropores is 1-3 μm. Preferably, a certain slope angle can be formed within the micropores, for example, >87°.

[0046] According to one embodiment, the micropores have a silica layer, which gives them hydrophilicity; while the spacers of the micropores have a silane coupling agent layer, which gives them hydrophobicity.

[0047] like Figure 3 As shown, in the gene testing chip of the present invention, nanowires are disposed around each micropore in the above-described array structure, thereby forming a nanowire array spaced apart corresponding to the micropore array. Specifically, as... Figure 5 As shown, the lateral dimension of the nanowires can be below 100 nm, preferably 50-80 nm. This invention does not limit the longitudinal dimension of the nanowires, but in actual processes, the height of the nanowires can be 1-2 μm. Using this nanowire array, the adapters required for sequencing can be connected to each nanowire, thereby extending the sequencing reaction, which traditionally occurs in a planar plane, to three-dimensional conditions. Sequencing reagent reaction chambers are formed inside each micropore-formed reaction unit, and these uniformly distributed nanowires surround the reaction chambers, ensuring both the reaction environment within the reaction unit and preventing crosstalk between reaction units.

[0048] According to one specific embodiment, the nanowires are connected with adapters required for sequencing, providing conditions for the sequencing reaction. In this case, each nanowire forms a sequencing cluster, thereby reducing the traditional 300nm-level sequencing cluster to below 100nm, significantly increasing sequencing throughput. Furthermore, vertical stacking, compared to planar distribution, can more effectively enhance the fluorescence intensity of the sequencing clusters, improving detection sensitivity.

[0049] Based on the above high-density nanowire array, the gene sequencing chip of the present invention can achieve a throughput of at least 1G reads within an area of ​​25mm×65mm.

[0050] like Figure 7 As shown, according to another aspect of the present invention, a method for preparing the above-mentioned gene sequencing chip is provided, comprising the steps of forming a microporous array, modifying the microporous array, forming a metal mask, and forming a nanowire array.

[0051] The above-described micropore array formation steps include sequentially coating a protective adhesive layer on the substrate surface, etching to form the desired micropore array, depositing a silicon dioxide layer on top of this, and then etching to remove the silicon dioxide at the micropore spacing portions, while retaining the silicon dioxide inside the micropores and on the sidewalls. At this point, the formed micropore size is within the range described above.

[0052] According to one specific embodiment, the fabrication of a high-density microporous array includes the following steps.

[0053] Pre-cleaning

[0054] First, the substrate (e.g., a glass substrate) is pre-cleaned, and this pre-cleaning step can be performed using standard cleaning methods conventional in the art.

[0055] Micropore array patterning

[0056] An adhesive layer is applied to the surface of the pre-cleaned glass substrate. Specifically, this adhesive layer can typically be achieved by spin-coating a single layer of conventional adhesive, such as first applying OC adhesive, then applying two layers of PS TG-S509 adhesive, followed by exposure through a mask (mask pattern as shown). Figure 6 As shown, the target micropore pattern is formed by development. This micropore pattern is formed by patterning an adhesion layer on the substrate surface, without involving etching of the substrate.

[0057] Preferably, PS TG-S509 adhesive is spin-coated onto the substrate after OC coating at 300 rpm, pre-baked at 90°C for 2 minutes, and the spin-coating of PS TG-S509 adhesive is repeated once. The target micropore pattern is formed by exposure through a mask. Specifically, the exposure intensity is 200 mJ, the exposure gap is 100 μm, the exposure time is 15 seconds, development with developer is 45 seconds, and curing is performed at 230°C for 30 minutes to obtain a micron-scale micropore array.

[0058] Micropore array modification

[0059] SiO2 is deposited on the surface of the micropore array and removed from the micropore gaps by mask etching, while the SiO2 on the inner sidewalls of the pores is retained.

[0060] According to one specific implementation, the SiO2 layer thickness can be controlled at 600-700 nm, followed by inductively coupled plasma (ICP) etching or reactive ion etching (RIE) dry etching to reduce the micropore inner diameter to 1-3 μm, increase the hole spacing to 1-3 μm, and ensure the slope angle is >87°. Figure 4 As shown.

[0061] A silane coupling agent, preferably a diamino-functionalized silane coupling agent such as KH-900, is deposited on the surface of a microporous array with a SiO2 layer, with a thickness of 100-300 nm. Further, the silane coupling agent within the micropores is removed, while the silane coupling agent in the micropore spacers is retained. The deposition and patterning of the silane coupling agent layer can be performed using conventional methods in the art. Thus, the micropore interior with the SiO2 layer is hydrophilic, while the micropore spacers with the silane coupling agent layer are hydrophobic.

[0062] Metal mask formation

[0063] A metal mask material layer is deposited on the surface of the surface-modified substrate with a microporous array. The metal mask material layer of this invention may include sequentially depositing a Mo film, an Al film, and another Mo film, or a Ti film, an Al film, and another Ti film to form a 300-500 nm Mo-Al-Mo or Ti-Al-Ti coating, forming a metal protective layer. Photoresist is spin-coated onto the surface of the metal mask material layer at 300 rpm, pre-baked at 90°C for 2 minutes, and the spin-coating is repeated once. The target pattern is formed through masking and exposure. The exposure intensity is 200 mJ, the exposure gap is 100 μm, the exposure time is 15 seconds, development is performed with developer for 45 seconds, and curing is carried out at 230°C for 30 minutes.

[0064] See Figure 7 After the above steps, the metal mask material layer forms a patterned metal protective layer. For example, through ICP etching, the portion of the metal mask material layer corresponding to the micro-hole spacing is patterned to form a metal hard mask with a pattern corresponding to the nanowire pattern. There is no residue in the pattern. The photoresist protective layer is then peeled off to form a complete metal mask.

[0065] Nanowire array formation

[0066] Dry etching, such as ICP etching, is performed on the substrate with the aforementioned metal mask to complete the nanowire patterning, such as... Figure 5 , Figure 7 As shown. Specifically, the metal mask layer and silicon dioxide layer are etched, for example, the metal mask layer is etched by ICP for 100s and the SiO2 layer is etched by ICP for 300s to form a nanowire array of the required size.

[0067] The method for preparing the gene sequencing chip of the present invention may further include a adapter coupling step. The adapter coupling step of the present invention may employ a 1:1 EDC:NHS treatment, reacting the adapter primers with -COOH on the chip surface, thereby covalently linking the adapters within the chip micropores.

[0068] Furthermore, the preparation method of the gene sequencing chip of the present invention may also include a chip packaging step, for example, GE 6585 adhesive can be uniformly coated around the chip with a width of 1.2 mm, covered with a cover plate, baked at 100°C for 5 min, then the temperature is programmed to rise to 150°C and baked for 10 min to form a 100 μm thick box.

[0069] According to another aspect of the present invention, a gene sequencing device is provided, comprising the above-described gene sequencing chip.

[0070] According to another aspect of the present invention, a method for preserving a gene sequencing chip is provided, comprising: preparing a water-soluble prepolymer; coating the surface of the gene sequencing chip with the water-soluble prepolymer; and polymerizing the prepolymer into a water-soluble polymer.

[0071] According to one specific implementation, the above-mentioned storage method may include:

[0072] N-(5-azidoacetamidopentyl)acrylamide and acrylamide were prepolymerized at 50°C for 5 min.

[0073] The prepolymerized product is coated onto the surface of a chip substrate and fully polymerized at 35°C for 2 hours. The copolymer outside the micropores is then removed by ashing or polishing, leaving only the portion inside the micropores to completely cover the primers that have been coupled on the chip.

[0074] Before use, chips preserved using the above preservation method can have the water-soluble copolymer removed (e.g., washed away with deionized water).

[0075] According to one specific embodiment, chips coated with a water-soluble polymer of N-(5-azidoacetamidopentyl)acrylamide and acrylamide and stored were tested every month to verify the connector efficiency. Simultaneously, chips without the aforementioned water-soluble polymer coating were used as a control. The measured efficiencies are shown below. Figure 8 .like Figure 8As shown, the chip protected by the aforementioned water-soluble copolymer according to the present invention maintains a relatively consistent connector efficiency after 1-12 months of storage, indicating good storage life. In contrast, the chip without the aforementioned water-soluble copolymer protection shows a connector efficiency reduction of over 50% after 1-12 months of storage. Therefore, the chip storage method according to the present invention can effectively extend the storage life of the chip.

[0076] In summary, compared with the prior art, the present invention has the following beneficial effects:

[0077] (1) Compared with traditional chips, the gene testing chip of the present invention consists of thousands of nanowires in each reaction unit, and the sequencing clusters are formed on several adjacent nanowires in a three-dimensional distribution, which greatly improves the throughput.

[0078] (2) The gene testing chip preparation method of the present invention does not require nanoimprinting and glass etching, but can be achieved by traditional semiconductor processing methods. The entire process is fast and can effectively reduce costs.

[0079] (3) Compared with similar products in the prior art, the sequencing reaction clusters of the gene testing chip of the present invention are changed from the bottom of the micropore to nanowires, and from two-dimensional to three-dimensional, which effectively increases the sequencing cluster density per unit area and the signal strength per unit area, thereby improving the signal-to-noise ratio and resolution.

[0080] (4) The chip preservation method of the present invention can effectively protect the connectors connected to the chip and greatly extend the chip's shelf life.

[0081] The foregoing disclosure provides many different implementations or examples for carrying out different structures of this disclosure. To simplify the disclosure, specific examples of components and arrangements have been described above. Of course, these are merely examples and are not intended to limit the scope of this disclosure. Furthermore, reference numerals and / or letters may be repeated in different examples; such repetition is for simplification and clarity and does not in itself indicate a relationship between the various implementations and / or arrangements discussed.

[0082] The above are merely specific embodiments of this disclosure, but the scope of protection of this disclosure is not limited thereto. Any person skilled in the art can easily conceive of various variations or substitutions within the technical scope disclosed in this disclosure, and these should all be included within the scope of protection of this disclosure. Therefore, the scope of protection of this disclosure should be determined by the scope of the claims.

Claims

1. A gene sequencing chip, characterized in that, include: The substrate has a first surface; A micropore array is located within the first surface; The nanowire array is formed by nanowires that surround each micropore in the micropore array. The micropores in the micropore array are hydrophilic inside, the micropore spacing is hydrophobic, and the nanowires are connected to adapters required for sequencing.

2. The gene sequencing chip according to claim 1, characterized in that, The nanowire array is spaced apart.

3. The gene sequencing chip according to claim 1, characterized in that, It has a throughput of at least 1 G reads within an area of ​​25 mm × 65 mm.

4. The gene sequencing chip according to claim 1, characterized in that, The diameter of the nanowire is less than 100 nm.

5. A method for preparing a gene sequencing chip, characterized in that, include: A microporous material layer is formed on the surface of the first substrate, and a microporous array is patterned thereon. A metal mask material layer is deposited on the surface of the micropore array, and a metal mask is formed by patterning. The micropore array with the metal mask is etched to form a nanowire array surrounding each micropore in the micropore array; Between forming the micropore array and forming the metal mask, the surface of the micropore array is modified so that the interior of the micropores is hydrophilic, and the spacer portions are hydrophobic. The nanowires are connected to adapters required for sequencing.

6. The preparation method according to claim 5, characterized in that, The surface modification includes depositing silica in the micropores to perform hydrophilic modification and coating the spacer portion of the micropores with a silane coupling agent layer to perform hydrophobic modification.

7. A gene sequencing device, characterized in that, Includes gene sequencing chips according to any one of claims 1 to 4.

8. A method for preserving a gene sequencing chip according to any one of claims 1 to 4, wherein the gene sequencing chip comprises an array of microwells spaced apart, characterized in that, The storage method includes: Preparation of water-soluble prepolymers; The water-soluble prepolymer is coated onto the surface of the gene sequencing chip; The prepolymer is polymerized into a water-soluble polymer.

9. The preservation method according to claim 8, characterized in that, It also includes a water-soluble polymer for removing the micropore spacers in the gene sequencing chip.

10. The preservation method according to claim 8 or 9, characterized in that, The water-soluble polymer is an acrylamide prepolymer.

11. The preservation method according to claim 8 or 9, characterized in that, The preparation of water-soluble prepolymers involves prepolymerizing N-(5-azidoacetamidopentyl)acrylamide and acrylamide separately at 50°C.

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