A method for preparing a dot matrix biochip

Abstract

The application provides a preparation method of a dot array biochip. The application forms a nano-pore dot array on a chip substrate through nano-imprinting, uniformly forms a functional modification layer on the dot array surface through a hydrogel, and then performs etching treatment on the substrate by using a deflected ion beam; an angle between an ion beam incident direction and a surface of a slide stage is θ during etching, so as to perform selective etching on the functional modification layer; through the etching with the angle, the functional modification layer of the bottom and part of the sidewall of the nano-pore can be reserved, and the functional modification layer of other parts is etched clean by the ion beam; the ion source of the IBE is argon, which is stable in chemical property and has no obvious influence on the chemical composition of the material, so that the damage of chemical residues to the performance of the functional material is avoided; the IBE does not produce chemical reaction byproducts, so that the risk of surface residual particles and pollution is reduced; the ion beam energy of the IBE is adjustable, the relatively mild etching avoids damage to the substrate, and the influence on the lattice and surface state of the surface layer material is small.

Description

Technical Field

[0001] The present invention relates to the technical field of chip preparation, and in particular to a method for preparing a dot matrix biochip. Background Art

[0002] Dot-array biochips are microdevices that enable high-throughput analysis of large numbers of biological samples using a miniaturized array of dots. Each dot represents a "detection site" or "array point," which specifically binds to target molecules such as DNA, RNA, or proteins. Dot-array biochips are widely used in genetic analysis, disease detection, protein interaction studies, drug screening, and environmental monitoring.

[0003] The general structure of a dot matrix biochip consists of a substrate, a dot matrix structure, a surface chemical modification layer, and probe molecules. Selective modification is a key step in dot matrix biochip fabrication. Its purpose is to modify specific regions with functionalized materials. Selective modification technology immobilizes probe molecules to the modified regions, while unmodified regions remain unfixed. This helps reduce background noise, improve signal-to-noise ratio, and minimize nonspecific adsorption, thereby ensuring the independence and accuracy of the dot matrix chip. The surface functionalized material is evenly applied to the substrate through spin coating or spray coating. A selective surface is then created using a technique that retains the functionalized material on the bottom and sidewalls of the dot matrix while removing it from the top surface. This results in probe molecules being immobilized within the dot matrix, while areas outside the dot matrix are removed through other methods such as ultrasound. There are two main methods for creating a selectively modified surface. One is the dispensing process. After preparing a surface-treated substrate, a dispensing device sets the volume and spacing of the droplets. The reagents loaded in the dispensing device are then periodically applied to the substrate via a dispensing needle, forming a selectively modified microarray. The other method is to first form a hole lattice pattern with different heights on the substrate surface through micro-nano processing technology, perform uniform surface modification, and then use CMP (chemical mechanical polishing) to grind off the protruding parts of the pattern, retaining only the surface modification inside the holes. CMP combines chemical and physical methods to treat the surface. Specifically, under the action of abrasive particles and polishing liquid, the substrate and the polishing pad move relative to each other. With the help of the mechanical grinding action of nano-abrasive particles and the chemical corrosion action of the polishing liquid, the functionalized material is ground off from the protruding parts of the substrate surface, thereby achieving the purpose of removing the surface and retaining the material inside the lattice, and ultimately achieving selective modification.

[0004] The dispensing process has the following drawbacks: While it can rapidly form selectively modified dot structures, it also has significant drawbacks. Firstly, due to limitations in sample placement accuracy, it is difficult to achieve high-density dot arrays, making high-throughput testing impossible. Secondly, the deposited droplets are easily affected by factors such as substrate surface properties and ambient temperature and humidity, resulting in poor dot array uniformity, which in turn affects detection efficiency and results.

[0005] The chemical mechanical polishing (CMP) process has the following defects: 1. Particle contamination on the array surface. CMP is a process that flattens or removes the surface of the material by organically combining the physical grinding action of micro-nano particles with the chemical corrosion action of the polishing liquid. However, residual particles in the abrasive or polishing liquid will adhere to the surface of the array, especially for deep holes and groove structures, and will fall inside and be difficult to completely remove, affecting subsequent processes. 2. Affecting the activity of functionalized materials. Since the components in the polishing liquid are complex and are composed of oxidants, chelating agents, surfactants, etc., it is not biocompatible and can easily lead to changes in the chemical properties of the functional groups (including but not limited to carboxyl groups, epoxy groups, etc.) in the functionalized materials, affecting specific reactions.

[0006] Based on the defects of the current dispensing process and chemical mechanical polishing (CMP) process for selectively modifying the chip surface, it is necessary to improve this. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing a dot matrix biochip to solve or at least partially solve the defects in the prior art.

[0008] The present invention adopts the following technical solutions:

[0009] The present invention provides a method for preparing a dot matrix biochip, comprising the following steps:

[0010] Nanopores arranged in an array are prepared on the surface of the substrate;

[0011] Coating a functional modification material on the surface of the substrate and in the nanopores to form a functional modification layer;

[0012] Placing the substrate with the functionalized modified layer on a slide stage of an ion etching machine and performing etching to obtain a dot matrix biochip;

[0013] During etching, the angle between the incident direction of the ion beam and the surface of the wafer stage is θ, so as to selectively etch the functionalized modification layer; 0°<θ<90°.

[0014] Preferably, the substrate formed with the functionalized modification layer is placed on the wafer stage of an ion etcher, and in the step of etching treatment, the etching process parameters are: the ion source is argon, the argon flow rate is 5-10 sccm, the screen voltage is 550-560 eV, the ion beam current is 110-120 mA, the wafer stage speed is 10-20 rpm, and the etching time is 10-20 s.

[0015] Preferably, the process of preparing arrayed nanopores on the substrate surface specifically comprises the following steps:

[0016] Providing a master, wherein the master surface has arrayed nanopores;

[0017] Coating a first embossing adhesive on the surface of the master, embossing the flexible substrate on the first embossing adhesive, UV curing, and demoulding to obtain a pattern on the flexible substrate opposite to that of the master, thereby forming a daughter plate;

[0018] Coating a tackifier on the surface of the substrate, heating and baking, then coating a second embossing adhesive, and heating and baking;

[0019] The daughter plate is printed on the second imprint adhesive, UV-cured, and demoulded to obtain the same array of nanopores as the mother plate on the second imprint adhesive, that is, the array of nanopores is prepared on the substrate surface.

[0020] Preferably, the material used for the functionalized modification layer includes but is not limited to acrylamide-based hydrogel containing functional groups; the functional groups include but are not limited to any one of carboxyl groups, epoxy groups, polysaccharide materials, biotin, hydroxylated PEG, and amino PEG.

[0021] Preferably, the material used for the functionalized modification layer is an acrylamide-based hydrogel containing a carboxyl group, and the method for preparing the functionalized modification layer comprises the following steps:

[0022] The acrylamide-based hydrogel containing carboxyl groups is mixed with fluorescein isothiocyanate to obtain a functionalized modified material;

[0023] The functional modification material is spin-coated on the substrate surface and in the nanopores, and baked at 80-100° C. for 10-15 minutes to form a functional modification layer.

[0024] Preferably, the volume mass ratio of the acrylamide hydrogel containing carboxyl groups to fluorescein isothiocyanate is (1-3) mL: (5-10) mg;

[0025] The method for preparing the acrylamide-based hydrogel containing carboxyl groups is as follows: adding partial sodium salt of poly(acrylamide-acrylic acid) to deionized water and stirring to obtain the acrylamide-based hydrogel containing carboxyl groups; the mass volume ratio of partial sodium salt of poly(acrylamide-acrylic acid) to water is (0.5-2) g: (100-150) mL.

[0026] Preferably, a tackifier is coated on the surface of the substrate, and the substrate is heated and baked at 115-120° C. for 3-4 minutes, and then a second embossing adhesive is coated on the substrate, and the substrate is heated and baked at 115-120° C. for 2-3 minutes.

[0027] Preferably, the substrate includes any one of a sapphire substrate, a silicon substrate, a quartz glass substrate, a polyimide substrate, a polyethylene terephthalate substrate, and a polyethylene naphthalate substrate.

[0028] Preferably, the diameter of the nanopore is 200-300 nm and the height is 100-150 nm;

[0029] and / or, the thickness of the substrate is 500 to 1000 μm;

[0030] And / or, the thickness of the functional modification layer is 30-50 nm.

[0031] Preferably, 3°≤θ≤12°.

[0032] The method for preparing a dot matrix biochip of the present invention has the following beneficial effects compared to the prior art:

[0033] The preparation method of the dot matrix biochip of the present invention forms a nanopore dot matrix on the chip substrate by nanoimprinting, uniformly forms a functionalized modification layer on the dot matrix surface by hydrogel, and then etches the substrate with a deflected ion beam; during etching, the angle between the incident direction of the ion beam and the surface of the carrier stage is θ, so as to selectively etch the functionalized modification layer; 0°<θ<90°; the functionalized modification layer at the bottom and part of the side wall of the nanopore can be retained by etching with the angle, while the functionalized modification layer in other parts is etched cleanly by the ion beam; in IBE etching, the ion beam moves along the direction of the electric field, has good directionality, and is easy to achieve highly anisotropic etching, which is convenient for achieving selective modification Lay the foundation; compared with the dispensing process and chemical mechanical polishing (CMP) etching, IBE etching has no pollution residue and low damage etching. The ion source of IBE is argon, which has stable chemical properties. It is a purely physical etching process and has no obvious effect on the chemical composition of the material, avoiding the damage of chemical residues to the performance of the functionalized modification layer material; and IBE does not produce chemical reaction by-products, reducing the risk of residual particles and pollution on the surface; on the other hand, the IBE ion beam energy is adjustable, and the relatively mild etching avoids substrate damage, and has little effect on the lattice and surface state of the surface material; the preparation method of the dot matrix biochip of the present invention does not affect the properties of the functionalized modification layer material in the retained area and can maintain normal chemical activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive effort.

[0035] Figure 1 This is a schematic diagram of the present invention when a substrate having a functionalized modified layer is placed on a wafer stage of an ion etcher for etching;

[0036] Figure 2 This is an enlarged view of the present invention when etching a substrate having a functionalized modified layer;

[0037] Figure 3 Schematic diagram of coating a functional modification material on a substrate surface and in nanopores to form a functional modification layer according to the present invention;

[0038] Figure 4 A schematic diagram of the structure of the dot matrix biochip prepared by the present invention;

[0039] Figure 5 It is a structural schematic diagram of the sub-plate of the present invention;

[0040] Figure 6 This is a schematic diagram of the structure of nanopores with the same array arrangement as the master obtained on the second imprinting adhesive of the present invention;

[0041] Figure 7 The dot matrix biochip prepared according to the method in Example 1 presents a periodic fluorescence dot matrix signal graph under a fluorescence microscope;

[0042] Figure 8 The biochip prepared according to the method in Comparative Example 1 presents a fluorescence signal image of the entire surface under a fluorescence microscope. DETAILED DESCRIPTION

[0043] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0044] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, it does not need to be further defined or explained in subsequent drawings.

[0045] The following are detailed descriptions respectively. It should be noted that the order of description of the following embodiments is not intended to limit the preferred order of the embodiments. In addition, in the description of this application, the term "including" means "including but not limited to". Various embodiments of the present invention may be in the form of a range; it should be understood that the description in the form of a range is only for convenience and brevity and should not be understood as a rigid limitation on the scope of the invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single numerical values ​​within the range. For example, the range description from 1 to 6 should be considered to have specifically disclosed sub-ranges, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as single numbers within the numbered ranges, such as 1, 2, 3, 4, 5 and 6, which apply regardless of the range. In addition, whenever a numerical range is indicated in this article, it is meant to include any cited numbers (fractions or integers) within the indicated range.

[0046] The present invention provides a method for preparing a dot matrix biochip, comprising the following steps:

[0047] S1. preparing an array of nanopores on the surface of a substrate;

[0048] S2, coating a functional modification material on the substrate surface and in the nanopores to form a functional modification layer;

[0049] S3, placing the substrate with the functionalized modified layer on a slide stage of an ion etcher and performing etching to obtain a dot matrix biochip;

[0050] During etching, the angle between the incident direction of the ion beam and the surface of the wafer stage is θ, so as to selectively etch the functionalized modification layer; 0°<θ<90°.

[0051] The preparation method of the dot matrix biochip of the present invention comprises the following steps: preparing arrayed nanopores 11 on a substrate 10; coating a functionalized modification material on the surface of the substrate 10 and within the nanopores 11 to form a functionalized modification layer 12; placing the substrate with the functionalized modification layer 12 on a stage 1 of an ion etcher for etching; the present invention adopts ion beam etching (IBE) to selectively etch the functionalized modification layer 12. Ion beam etching (IBE) is an etching process that uses an ion beam to directly bombard the surface of a material to achieve removal; it has significant advantages in some specific applications and is particularly suitable for directional etching and low-damage etching of materials. Ion beam etching has the following advantages: highly anisotropic etching. In IBE etching, the ion beam moves along the direction of the electric field and has good directionality, making it easy to achieve highly anisotropic etching, laying the foundation for selective modification. In IBE etching, there is no pollution residue and low damage etching. The ion source of IBE is argon gas, which has stable chemical properties. It is a purely physical etching process and has no significant effect on the chemical composition of the material, avoiding the damage of chemical residues to the performance of the functionalized modified layer material. In addition, IBE does not produce chemical reaction byproducts, reducing the risk of residual particles and contamination on the surface. On the other hand, the energy of the IBE ion beam is adjustable, and the relatively mild etching avoids damage to the substrate and has little effect on the lattice and surface state of the surface material. The preparation method of the dot matrix biochip of the present invention uses a deflected ion beam to etch the substrate, without affecting the properties of the functionalized modified layer material in the retained area, and can maintain normal activity.

[0052] Specifically, refer to Figure 1 As shown, a substrate 10 formed with a functionalized modified layer 12 is placed on the stage 1 of an ion etcher for etching. Since the angle of the stage 1 of the IBE can be adjusted from 0 to 180 degrees, the angle between the incident direction of the ion beam and the surface of the stage 1 is 0 to 90 degrees. During etching, the angle between the incident direction of the ion beam and the surface of the stage 1 is controlled to be θ, that is, the angle between the substrate surface and the ion beam is θ, 0°<θ<90°. By controlling the size of the angle θ, the functionalized modified layer 12 on the surface of the substrate 10 and in the nanopore 11 is selectively etched. Further, referring to Figure 2 As shown, for a single nanopore 11, the diameter of the nanopore 11 is l, and according to the triangle relationship, d=l×tanθ, where d is the etching depth of the sidewall of the nanopore 11. The bottom and part of the sidewall of the nanopore 11 can be retained by etching with an angle, while the functionalized modified layer 12 of the other parts is cleaned by the ion beam etching. Figure 4 As shown, it is a schematic diagram of the structure of the dot matrix biochip prepared after etching.

[0053] In some embodiments, a substrate having a functionalized modification layer is placed on a wafer stage of an ion etcher, and in the etching step, the etching process parameters are: the ion source is argon (Ar), the argon flow rate is 5 to 10 sccm, the screen voltage is 550 to 560 eV, the ion beam current is 110 to 120 mA, the wafer stage speed is 10 to 20 rpm, and the etching time is 10 to 20 s.

[0054] In the above embodiment, the wafer stage can rotate along the central axis, the central axis is perpendicular to the surface of the wafer stage, and the rotation speed of the wafer stage is 10 to 20 rpm. The rotation of the wafer stage greatly improves the uniformity of etching and ultimately achieves the etching purpose.

[0055] In some embodiments, the value of θ can be adjusted according to actual conditions. For example, the value of θ can be 1°, 5°, 10°, 15°, 20°, 25°, 30°, 35°, 40°, 45°, 50°, 55°, 60°, 65°, 70°, 75°, 80°, 85°, 89°. Furthermore, 3°≤θ≤12°.

[0056] In some embodiments, the nanopores arranged in an array are prepared on the surface of a substrate, specifically comprising the following steps:

[0057] S11, providing a master plate, wherein the master plate has arrayed nanopores on its surface;

[0058] S12, coating a first embossing adhesive on the surface of the master, embossing the flexible substrate on the first embossing adhesive, UV curing, and demolding to obtain a pattern on the flexible substrate opposite to that of the master, thereby forming a daughter plate;

[0059] S13, coating a tackifier on the surface of the substrate, heating and baking, and then coating a second embossing adhesive, heating and baking;

[0060] S14, imprinting the daughter plate on the second imprinting adhesive, UV curing, and demoulding, to obtain the same array of nanopores as the mother plate on the second imprinting adhesive, that is, preparing the array of nanopores on the substrate surface.

[0061] Specifically, the present invention uses the high-precision mass-production ultraviolet nanoimprint lithography equipment of Qingdao Tianren Micro-Nano Technology Co., Ltd. to obtain nanopores with the same array arrangement as the master on the second imprint resin; wherein, the preparation method of the daughter plate is specifically as follows:

[0062] S111. Providing a master having an array of nanopores on its surface; performing a cleaning process on the master: using an SPM solution, i.e., a mixture of 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3 to obtain an SPM solution; immersing the master in the SPM solution for 15 minutes, then rinsing with deionized water, drying, and setting aside;

[0063] S112, coating the first embossing adhesive on the master after cleaning in S111, specifically using a spin coating method to spin coat the first embossing adhesive; the first embossing adhesive spin coating process: dripping the first embossing adhesive on the master, and spin coating at a speed of 4000 r / min for 60 seconds;

[0064] Using nanoimprint equipment, a flexible substrate such as a PET film is imprinted on the first imprinting glue, UV-cured, and demolded. The pattern opposite to the master is replicated on the PET film to obtain a daughter plate. Specifically, ultraviolet light with a wavelength of 365 nanometers is used for curing, and the ultraviolet light intensity is 300-350 mj / cm 2 .

[0065] like Figure 5 As shown, it is a structural schematic diagram of the daughter plate 2, including a flexible substrate 21, and an array of bosses 22 formed after the first embossing glue is cured. It can be understood that the bosses 22 are adapted to the nanopores on the mother plate and also to the nanopores 11 on the second embossing glue 13.

[0066] Specifically, the surface of the master has nanopores arranged in an array, and the master material is a silicon wafer, which can be formed on the master by using a photolithography process to form the nanopores arranged in an array.

[0067] After the daughter plate is made, you can start imprinting the target substrate, which includes the following steps:

[0068] S13, first perform a tackifying treatment on the substrate, add a tackifier to the surface of the substrate, and spin-coat it at a speed of 3000 r / min for 30 seconds, then bake it on a hot plate at a temperature of 115-120° C. for 3-4 minutes; after baking, wait for the substrate to cool down, and then spin-coat a second embossing adhesive, add the second embossing adhesive on the tackifier, spin-coat it at a speed of 2000 r / min for 60 seconds, and bake it on a hot plate at 115-120° C. for 2-3 minutes;

[0069] S14. Use nanoimprint equipment to imprint the daughter plate on the second imprint adhesive, UV-curing, and demolding to obtain arrayed nanopores with the same pattern as the mother plate on the second imprint adhesive, that is, nanopores arranged in an array are prepared on the substrate surface; specifically, ultraviolet light with a wavelength of 365 nanometers is used for curing, and the ultraviolet light intensity is 300-350 mj / cm 2 .

[0070] After creating a nanopore array on a substrate using nanoimprinting, the aforementioned method can also be used to fabricate an array of nanopores on the substrate using methods such as UV lithography and inkjet printing. Nanoimprinting is a more cost-effective method for creating nanopore arrays on a substrate, facilitating industrial mass production.

[0071] In some embodiments, the material used for the functionalized modification layer includes but is not limited to acrylamide-based hydrogels containing functional groups; the functional groups include but are not limited to any one of carboxyl groups, epoxy groups, polysaccharide materials, biotin, hydroxylated PEG (polyethylene glycol), and amino PEG.

[0072] In some embodiments, the polysaccharide material is, for example, agarose, dextran, or the like.

[0073] In some embodiments, the hydrogel may also be other types of hydrogels, which may be selected based on actual conditions.

[0074] In some embodiments, the first embossing adhesive and the second embossing adhesive are conventional commercially available UV embossing adhesives, such as IPNR-PC1000 and IPNR-PC2000 from Imprint, or German Micro Resist nano-embossing adhesives such as mr-NIL6000 series, mr-UVCur06, and mr-UVCur21.

[0075] In some embodiments, the tackifier is a conventional commercially available tackifier, for example, the micro-nano lithography tackifier SurPass, model SurPass 3000 and SurPass 4000, produced by DisChem-CHEMISTRYFOR ADVANCED LITHOGRAPHY can be used.

[0076] In some embodiments, the material used for the functionalized modification layer is an acrylamide-based hydrogel containing carboxyl groups, and the method for preparing the functionalized modification layer comprises the following steps:

[0077] S21, acrylamide hydrogel containing carboxyl groups and fluorescein isothiocyanate (molecular formula: C 21 H 11 NO5S) to obtain a functional modified material;

[0078] S22, spin-coating the functional modification material on the substrate surface and inside the nanopores, and baking at 80-100° C. for 10-15 minutes to form a functional modification layer.

[0079] In some embodiments, the volume-to-mass ratio of the acrylamide-based hydrogel containing carboxyl groups to fluorescein isothiocyanate is (1-3) mL: (5-10) mg.

[0080] In some embodiments, the preparation method of the acrylamide-based hydrogel containing carboxyl groups is as follows: adding partial sodium salt of poly(acrylamide-acrylic acid) to deionized water and stirring to obtain the acrylamide-based hydrogel containing carboxyl groups; the mass volume ratio of partial sodium salt of poly(acrylamide-acrylic acid) to water is (0.5-2) g: (100-150) mL.

[0081] In some embodiments, a tackifier is coated on the surface of the substrate, and the substrate is heated and baked at 115-120° C. for 3-4 minutes, and then a second embossing adhesive is coated and heated and baked at 115-120° C. for 2-3 minutes.

[0082] In some embodiments, the substrate 10 includes a hard substrate such as a sapphire substrate, a silicon substrate, a quartz glass substrate, etc.; and also includes a flexible substrate such as a polyimide (PI) substrate, a polyethylene terephthalate (PET) substrate, a polyethylene naphthalate substrate, etc.

[0083] In some embodiments, the diameter of the nanopore 11 is 200-300 nm, the height (ie, depth) is 100-150 nm, and the distance between any two adjacent nanopores is 500-600 nm.

[0084] In some embodiments, the thickness of the substrate 10 is 500-1000 μm.

[0085] In some embodiments, the thickness of the functionalized modification layer 12 is 30-50 nm.

[0086] For further reference, Figures 3 to 6 As shown, a tackifier is coated on the surface of the substrate 10, and then a second embossing adhesive is coated and heated and baked; the daughter plate is embossed on the second embossing adhesive, UV-cured, and demolded to obtain nanopores 11 with the same array arrangement as the mother plate on the second embossing adhesive 13, as shown in FIG. Figure 6 Then the functionalized modification material is coated on the surface of the substrate 10, the surface of the second imprinted adhesive 13, and the nanopores 11 to form a functionalized modification layer 12, as shown. Figure 3 As shown, the substrate 10 with the functionalized modified layer 12 is placed on the wafer stage of the ion etching machine, and the functionalized modified layer is selectively etched to obtain the final product. Figure 4 The biological dot array chip shown.

[0087] The following further illustrates the method for preparing the dot matrix biochip of the present application using specific examples. This section further illustrates the present invention with reference to specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the techniques employed in the examples are conventional techniques well known to those skilled in the art. Unless otherwise specified, the reagents, methods, and equipment employed in the present invention are conventional in the art.

[0088] In the following examples, the first embossing adhesive is mr-UVCur06, the second embossing adhesive is mr-UVCur21, and the tackifier is SurPass 4000; poly(acrylamide-acrylic acid) partial sodium salt, i.e., poly(acrylamide-co-acrylic acid) partial sodium salt, was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., CAS No.: 62649-23-4, molecular formula: C9H 12 NNaO5.

[0089] Example 1

[0090] The present invention provides a method for preparing a dot matrix biochip, comprising the following steps:

[0091] S1. Nanopores arranged in an array are prepared on the surface of a silicon wafer substrate with a thickness of 675 μm. The specific preparation method is as follows:

[0092] S11. Providing a master having an array of nanopores on its surface; performing a cleaning process on the master: using an SPM solution, namely, mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3 to obtain an SPM solution; soaking the master in the SPM solution for 15 minutes, then rinsing with deionized water, drying, and setting aside;

[0093] S12. Coating a first imprinting adhesive on the master after cleaning in S11, specifically using a spin coating method to spin-coat the first imprinting adhesive; the first imprinting adhesive spin coating process: dripping the first imprinting adhesive on the master, and spin coating at a speed of 4000 r / min for 60 seconds; using a nanoimprinting device, imprinting a flexible substrate PET film on the first imprinting adhesive, UV curing, demolding, and replicating a pattern opposite to that of the master on the PET film, thus obtaining a daughter plate; using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity of 300 mj / cm 2 ;

[0094] S13, adding a tackifier to the surface of the substrate, and spin-coating at a speed of 3000 r / min for 30 seconds, followed by baking at 115°C for 3 minutes; after baking, wait for the substrate to cool down, and then spin-coat a second imprinting adhesive, adding the second imprinting adhesive to the tackifier, spin-coating at a speed of 2000 r / min for 60 seconds, and baking at 115°C for 2 minutes;

[0095] S14. Use nanoimprint equipment to imprint the daughter plate on the second imprint adhesive, UV-curing, and demolding to obtain an array of nanopores with the same pattern as the mother plate on the second imprint adhesive, that is, an array of nanopores is prepared on the substrate surface; use ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300mj / cm 2The nanopore has a diameter of 200 nm and a height (i.e., depth) of 100 nm. The distance between any two adjacent nanopores is 500 nm.

[0096] S2, coating a functional modification material on the substrate surface, the second imprinting adhesive surface, and the nanopores to form a functional modification layer;

[0097] The material used for the functionalized modification layer is an acrylamide-based hydrogel containing a carboxyl group. The preparation method of the functionalized modification layer includes the following steps:

[0098] S21. Mixing 1 mL of an acrylamide-based hydrogel containing carboxyl groups with 5 mg of fluorescein isothiocyanate to obtain a functionalized modified material. The acrylamide-based hydrogel containing carboxyl groups is prepared by adding 1 g of a partial sodium salt of poly(acrylamide-acrylic acid) to 100 mL of deionized water and stirring to obtain the acrylamide-based hydrogel containing carboxyl groups.

[0099] S22, dropwise adding a functional modification material onto the substrate surface, the second imprinting adhesive surface, and the nanopores, and spin coating at a speed of 3000 rpm for 60 seconds, and baking at 80° C. for 10 minutes to form a functional modification layer; the thickness of the functional modification layer is 30 nm;

[0100] S3, placing the substrate with the functionalized modified layer on a slide stage of an ion etcher and performing etching to obtain a dot matrix biochip;

[0101] Among them, the angle between the incident direction of the ion beam and the surface of the wafer stage during etching is θ, so as to selectively etch the functionalized modification layer; θ is 8°; the etching process parameters are: the ion source is argon (Ar), the argon flow rate is 5sccm, the screen voltage is 550eV, the ion beam current is 110mA, the wafer stage speed is 10rpm, and the etching time is 20s.

[0102] Comparative Example 1

[0103] This comparative example provides a method for preparing a biochip, comprising the following steps:

[0104] S1. Nanopores arranged in an array are prepared on the surface of a silicon wafer substrate with a thickness of 675 μm. The specific preparation method is as follows:

[0105] S11. Providing a master having an array of nanopores on its surface; performing a cleaning process on the master: using an SPM solution, namely, mixing 98% concentrated sulfuric acid and 30% hydrogen peroxide in a volume ratio of 7:3 to obtain an SPM solution; soaking the master in the SPM solution for 15 minutes, then rinsing with deionized water, drying, and setting aside;

[0106] S12, coating the first imprinting glue on the master after cleaning in S11, specifically using spin coating method to spin coat the first imprinting glue; the first imprinting glue spin coating process: dropping the first imprinting glue on the master, spin coating at a speed of 4000 r / min for 60 seconds; using nano-imprinting equipment to imprint the flexible substrate PET film on the first imprinting glue, ultraviolet curing, demolding, and the PET film replicates the pattern opposite to the master, that is, the sub-master is obtained; curing using ultraviolet light with a wavelength of 365 nanometers, the ultraviolet light intensity is 300 mj / cm 2 ;

[0107] S13, dropping the adhesion promoter on the substrate surface and spin coating at a speed of 3000 r / min for 30 s, then baking at 115°C for 3 min; after baking and waiting for the substrate to cool, spin coating the second imprinting glue, dropping the second imprinting glue on the adhesion promoter and spin coating at a speed of 2000 r / min for 60 s, and baking at 115°C for 2 min;

[0108] S14, using nano-imprinting equipment to imprint the sub-master on the second imprinting glue, ultraviolet curing, demolding, and obtaining the same arrayed nanoholes as the master pattern on the second imprinting glue, that is, arrayed nanoholes are prepared on the substrate surface; curing using ultraviolet light with a wavelength of 365 nanometers, the ultraviolet light intensity is 300 mj / cm 2 ; the nanohole diameter is 200 nm, the height (i.e. depth) is 100 nm, and the distance between any two adjacent nanoholes is 500 nm;

[0109] S2, coating the functional modification material on the substrate surface, the second imprinting glue surface and the nanohole to form a functional modification layer, to obtain a biochip;

[0110] The material used for the functional modification layer is acrylamide hydrogel containing carboxyl groups, and the preparation method of the functional modification layer comprises the following steps:

[0111] S21, mixing 1 mL of acrylamide hydrogel containing carboxyl groups with 5 mg of fluorescein isothiocyanate to obtain a functional modification material; the preparation method of the acrylamide hydrogel containing carboxyl groups is: adding 1 g of poly(acrylamide-acrylic acid) partial sodium salt to 100 mL of deionized water, stirring, and obtaining the acrylamide hydrogel containing carboxyl groups;

[0112] S22, dropping the functional modification material on the substrate surface, the second imprinting glue surface and the nanohole, and spin coating at a speed of 3000 r / min for 60 s, baking at 80°C for 10 min to form a functional modification layer; the thickness of the functional modification layer is 30 nm.

[0113] Performance test

[0114] The biochip was prepared according to the method in the Example 1, and periodic fluorescent dot array signal was presented under the fluorescence microscope using light with 488 nm excitation wavelength, and the result is shown in Figure 7

[0115] The biochip was prepared according to the method in the Comparative Example 1, and whole surface fluorescent signal was presented under the fluorescence microscope using light with 488 nm excitation wavelength, and the result is shown in Figure 8

[0116] IBE was used to selectively etch the functionalized modification layer, and the result is shown in Figures 7-8 It can be seen that, before the IBE selective etching, the functionalized modification material coated the whole surface of the nanopore array, that is, the whole surface fluorescent signal was detected; after the IBE selective etching of the functionalized modification layer, the fluorescent signal was presented in the nanopore, that is, there was functionalized material in the nanopore, and the functionalized material outside the nanopore was removed, that is, the selective modification was realized, and then the periodic fluorescent dot array signal was presented.

[0117] The above only describes the preferred embodiments of the present application and is not intended to limit the present application, and any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.​​

Claims

1. A method for preparing a dot matrix biochip, characterized in that: The following steps are involved: Nanopores arranged in an array are prepared on the surface of the substrate; Coating a functional modification material on the surface of the substrate and in the nanopores to form a functional modification layer; Placing the substrate with the functionalized modified layer on a slide stage of an ion etching machine and performing etching to obtain a dot matrix biochip; During etching, the angle between the incident direction of the ion beam and the surface of the wafer stage is θ, so as to selectively etch the functionalized modification layer; 0°<θ<90°.

2. The method for preparing a dot matrix biochip according to claim 1, wherein: The substrate with the functionalized modified layer is placed on the wafer stage of an ion etcher for etching. The etching process parameters are as follows: the ion source is argon, the argon flow rate is 5-10 sccm, the screen voltage is 550-560 eV, the ion beam current is 110-120 mA, the wafer stage speed is 10-20 rpm, and the etching time is 10-20 s.

3. The method for preparing a dot matrix biochip according to claim 1, wherein: The nanopores arranged in an array are prepared on the surface of the substrate, specifically comprising the following steps: Providing a master, wherein the master surface has arrayed nanopores; Coating a first embossing adhesive on the surface of the master, embossing the flexible substrate on the first embossing adhesive, UV curing, and demoulding to obtain a pattern on the flexible substrate opposite to that of the master, thereby forming a daughter plate; Coating a tackifier on the surface of the substrate, heating and baking, then coating a second embossing adhesive, and heating and baking; The daughter plate is printed on the second imprint adhesive, UV-cured, and demoulded to obtain the same array of nanopores as the mother plate on the second imprint adhesive, that is, the array of nanopores is prepared on the substrate surface.

4. The method for preparing a dot matrix biochip according to claim 1, wherein: The material used for the functionalized modification layer includes but is not limited to acrylamide-based hydrogel containing functional groups; the functional groups include but are not limited to any one of carboxyl groups, epoxy groups, polysaccharide materials, biotin, hydroxylated PEG, and amino PEG.

5. The method for preparing a dot matrix biochip according to claim 1, wherein: The material used for the functionalized modification layer is an acrylamide-based hydrogel containing a carboxyl group, and the preparation method of the functionalized modification layer comprises the following steps: The acrylamide-based hydrogel containing carboxyl groups is mixed with fluorescein isothiocyanate to obtain a functionalized modified material; The functional modification material is spin-coated on the substrate surface and in the nanopores, and baked at 80-100° C. for 10-15 minutes to form a functional modification layer.

6. The method for preparing a dot matrix biochip according to claim 5, wherein: The volume mass ratio of the acrylamide hydrogel containing carboxyl groups to fluorescein isothiocyanate is (1-3) mL: (5-10) mg; The method for preparing the acrylamide-based hydrogel containing carboxyl groups is as follows: adding partial sodium salt of poly(acrylamide-acrylic acid) to deionized water and stirring to obtain the acrylamide-based hydrogel containing carboxyl groups; the mass volume ratio of partial sodium salt of poly(acrylamide-acrylic acid) to water is (0.5-2) g: (100-150) mL.

7. The method for preparing a dot matrix biochip according to claim 3, wherein: A tackifier is coated on the surface of the substrate, and the substrate is heated and baked at 115-120° C. for 3-4 minutes, and then a second embossing adhesive is coated on the surface of the substrate, and the substrate is heated and baked at 115-120° C. for 2-3 minutes.

8. The method for preparing a dot matrix biochip according to claim 1, wherein: The substrate includes any one of a sapphire substrate, a silicon substrate, a quartz glass substrate, a polyimide substrate, a polyethylene terephthalate substrate, and a polyethylene naphthalate substrate.

9. The method for preparing a dot matrix biochip according to claim 1, wherein: The nanopore has a diameter of 200 to 300 nm and a height of 100 to 150 nm; and / or, the thickness of the substrate is 500 to 1000 μm; And / or, the thickness of the functional modification layer is 30-50 nm.

10. The method for preparing a dot matrix biochip according to claim 1, wherein: 3°≤θ≤12°.

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