Preparation method of dot-matrix biochip

Abstract

The invention provides a preparation method of a dot-matrix biochip. According to the method, a nanopore dot matrix is formed on a chip substrate through nanoimprinting, a functional modification layer is uniformly formed on the surface of the dot matrix through hydrogel, and then a deflected ion beam is used for etching the substrate; the included angle between the incident direction of the ion beam and the surface of the slide holder is theta during etching, so that the functional modification layer is selectively etched; the functional modification layer at the bottom and part of the side wall of the nanopore can be reserved through etching with an included angle, and the other part of the functional modification layer is completely etched by the ion beam; an ion source of IBE is argon, the chemical property is stable, the chemical components of the material are not obviously influenced, and the damage of chemical residues to the performance of the functional material is avoided; iBE does not generate chemical reaction byproducts, so that surface residual particles and pollution risks are reduced; the IBE ion beam energy is adjustable, relatively mild etching avoids damage to the substrate, and the influence on crystal lattices and the surface state of a surface layer material is small.

Description

Technical Field

[0001] The 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 matrix biochip is a micro device that performs high-throughput analysis of a large number of biological samples in a miniaturized dot matrix format. Each dot represents a "detection site" or "array point" that can specifically bind to target molecules such as DNA, RNA or protein. Dot matrix biochips are widely used in gene analysis, disease detection, protein interaction research, drug screening and environmental monitoring.

[0003] The general structure of a dot matrix biochip includes: substrate, dot matrix structure, surface chemical modification layer, and probe molecules; selective modification is an important step in the production of dot matrix biochips. Its purpose is to allow functional materials to modify specific areas. Selective modification technology can fix probe molecules in the modified area, and probe molecules will not be fixed in the unmodified area, which helps to reduce background noise, improve signal-to-noise ratio, reduce nonspecific adsorption, etc., to ensure the independence and accuracy of the dot matrix chip. The surface functionalized material is evenly coated on the substrate by spin coating, spray coating, etc., and then a selective surface is formed by technical means, that is, the functionalized material is retained at the bottom and side walls of the dot matrix, and the material needs to be removed on the upper surface, so that the probe molecules are finally fixed in the dot matrix, and the area outside the dot matrix is ​​removed by other methods such as ultrasound. There are two main technical methods for forming a selectively modified surface. One is the dispensing process, that is, after preparing a surface-treated substrate, using a spotting device to set the volume and spacing of the spotting droplets, the reagent loaded in the spotting device is periodically dotted on the substrate through a dispensing needle to form 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, leaving 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, and 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 the protruding part of the substrate surface, thereby achieving the purpose of removing the surface and retaining the material inside the lattice, and finally achieving selective modification.

[0004] The dispensing process has the following defects: Although the dispensing process can quickly form a dot matrix structure with selective modification, it also has significant disadvantages. On the one hand, it is difficult to achieve a high-density dot matrix due to the limitation of the sampling accuracy, and high-throughput detection is impossible. On the other hand, the sample droplets are easily affected by the surface properties of the substrate, the ambient temperature and humidity, etc., resulting in poor dot matrix uniformity, which affects the 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 material surface 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] Prepare arrayed nanopores 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 modification layer on a wafer stage of an ion etcher and performing etching treatment to obtain a dot matrix biochip;

[0013] Among them, 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 5-10sccm, the screen grid voltage is 550-560eV, the ion beam current is 110-120mA, the wafer stage speed is 10-20rpm, and the etching time is 10-20s.

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

[0016] Providing a master, wherein the surface of the master 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, demoulding, and obtaining a pattern on the flexible substrate opposite to the master to form a daughter plate;

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

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

[0020] Preferably, the material used for the functional 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, epoxy, polysaccharide material, biotin, hydroxylated PEG, and amino PEG.

[0021] Preferably, the material used for the functional modification layer is an acrylamide-based hydrogel containing a carboxyl group, and the method for preparing the functional 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) into 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 glue 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 500-1000 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 the dot matrix biochip of the present invention has the following beneficial effects compared with the prior art:

[0033] The preparation method of the dot matrix biochip of the present invention forms a nanopore dot matrix on a chip substrate by nanoimprinting, uniformly forms a functional modification layer on the surface of the dot matrix by hydrogel, and then etches the substrate by using 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 functional modification layer; 0°<θ<90°; the functional modification layer at the bottom and part of the side wall of the nanopore can be retained by etching with the angle, while the functional modification layer of 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, is easy to realize highly anisotropic etching, and is convenient for realizing 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 gas, which has stable chemical properties and is a purely physical etching process. It 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 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] In order 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 prior art descriptions. Obviously, the drawings described below are only some embodiments of the present invention, and for ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0035] Figure 1 It is a schematic diagram of placing a substrate having a functionalized modified layer on a wafer stage of an ion etcher for etching according to the present invention;

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

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

[0038] Figure 4 A schematic diagram of the structure of a 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 It is a schematic diagram of the structure of nanopores with the same array arrangement as the master plate obtained on the second imprinting glue 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 described embodiments 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 creative work 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, further definition and explanation thereof is not required in subsequent drawings.

[0045] The following are described in detail. 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 the present application, the term "including" means "including but not limited to". Various embodiments of the present invention may exist in the form of a range; it should be understood that the description in the form of a range is only for convenience and simplicity, and should not be understood as a rigid limitation on the scope of the present invention; therefore, it should be considered that the range description has specifically disclosed all possible sub-ranges and single values ​​within the range. For example, it should be considered that the range description from 1 to 6 has 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 quoted 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 arrayed 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 modification layer on a wafer stage of an ion etcher, and performing etching treatment to obtain a dot matrix biochip;

[0050] Among them, 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 functional modification material on the surface of the substrate 10 and inside the nanopores 11 to form a functional modification layer 12; placing the substrate formed with the functional modification layer 12 on a wafer stage 1 of an ion etcher for etching; the present invention adopts ion beam etching (IBE) to selectively etch the functional modification layer 12, and ion beam etching (IBE) is an etching process that uses ion beams 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, has good directionality, and is 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 obvious effect on the chemical composition of the material, thus avoiding the damage of chemical residues to the performance of the functionalized modified layer material. And IBE does not produce chemical reaction by-products, 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, which does not affect 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, the substrate 10 formed with the functionalized modified layer 12 is placed on the stage 1 of the 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°, and 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, d is the etching depth of the side wall of the nanopore 11, and the bottom and part of the side wall of the nanopore 11 can be retained by etching with an angle, while the functional modification layer 12 of the other part is cleaned by ion beam etching, refer to 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 stage of an ion etcher, and in the step of etching, the etching process parameters are: the ion source is argon (Ar), the argon flow rate is 5-10 sccm, the screen voltage is 550-560 eV, the ion beam current is 110-120 mA, the stage speed is 10-20 rpm, and the etching time is 10-20 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, the rotation speed of the wafer stage is 10 to 20 rpm, and 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°, or 89°. Furthermore, 3°≤θ≤12°.

[0056] In some embodiments, the array of nanopores is prepared on the surface of the substrate, specifically comprising the following steps:

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

[0058] S12, coating a first embossing adhesive on the surface of the master, embossing the flexible substrate on the first embossing adhesive, UV curing, demoulding, obtaining a pattern on the flexible substrate opposite to the master, and 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, demoulding, and obtaining 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 a high-precision mass-production ultraviolet nanoimprint lithography device of Qingdao Tianren Micro-Nano Technology Co., Ltd. to obtain nanopores with the same array arrangement as the master plate on the second imprint glue; wherein the preparation method of the daughter plate is specifically as follows:

[0062] S111, providing a master, wherein the master surface has arrayed nanopores; 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, and then rinsing with deionized water and drying for standby use;

[0063] S112, coating the first embossing glue on the master after cleaning in S111, specifically using a spin coating method to spin coat the first embossing glue; the first embossing glue spin coating process: dripping the first embossing glue 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 imprint glue, UV-cured, demolded, and a 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 intensity of the ultraviolet light is 300-350 mj / cm 2 .

[0065] like Figure 5 As shown, it is a schematic diagram of the structure of the sub-plate 2, including a flexible substrate 21, and a first embossing adhesive is cured to form an array of bosses 22. It can be understood that the bosses 22 are compatible with the nanopores on the mother plate and also with the nanopores 11 on the second embossing adhesive 13.

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

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

[0068] S13, firstly perform a viscosity enhancement treatment on the substrate, drip a viscosity enhancer on the surface of the substrate, and spin-coat it at a speed of 3000 r / min for 30 seconds, and 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 glue, drip the second embossing glue on the viscosity enhancer, 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, using nanoimprint equipment, imprinting the daughter plate on the second imprint glue, UV curing, demolding, and obtaining the array of nanopores arranged in the same pattern as the mother plate on the second imprint glue, that is, preparing the array of nanopores on the surface of the substrate; specifically, using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300-350 mj / cm 2 .

[0070] After the above method realizes the nanopore array on the substrate by nanoimprinting, in practice, the arrayed nanopores can also be prepared on the substrate by ultraviolet lithography, inkjet printing and other methods. The nanopore array is realized on the substrate by the nanoimprinting method, which is lower in cost and is conducive to 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 are 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, IPNR-PC2000, etc. from Imprint, and German Micro Resist nano-embossing adhesives such as mr-NIL6000 series, mr-UVCur06, and mr-UVCur21 can also be used.

[0075] In some embodiments, the tackifier is a conventional commercially available tackifier, for example, the micro-nano lithography tackifier SurPass, model SurPass 3000, 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 a carboxyl group, 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 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.; it 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 functional 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 sub-plate is embossed on the second embossing adhesive, UV-cured, and demolded, and the nanopores 11 with the same array arrangement as the master plate are obtained on the second embossing adhesive 13, as shown in FIG. Figure 6 Then the functional modification material is coated on the surface of the substrate 10, the surface of the second imprint glue 13 and the nanopore 11 to form a functional modification layer 12, such as Figure 3 As shown, the substrate 10 with the functionalized modified layer 12 is finally placed on the wafer stage of an ion etching machine, and the functionalized modified layer is selectively etched to finally obtain Figure 4 The bio-array chip shown.

[0087] The following further describes the preparation method of the dot matrix biochip of the present application with specific examples. This section further describes the content of the present invention in conjunction with specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0088] In the following examples, the first embossing adhesive is mr-UVCur06, the second embossing adhesive is mr-UVCur21, the tackifier is SurPass 4000; the partial sodium salt of poly(acrylamide-acrylic acid), i.e., poly(acrylamide-co-acrylic acid), 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. Arrayed nanopores 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, the surface of which has arrayed nanopores; performing a cleaning process on the master: using an SPM solution, that is, mixing concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass fraction of 30% 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 and drying, and setting aside;

[0093] S12, coating the first imprint glue on the motherboard after cleaning in S11, specifically using spin coating to spin coat the first imprint glue; the first imprint glue spin coating process: dripping the first imprint glue on the motherboard, and spin coating at a speed of 4000r / min for 60 seconds; using nanoimprint equipment, imprinting the flexible substrate PET film on the first imprint glue, UV curing, demolding, and replicating the pattern opposite to the motherboard on the PET film to obtain a daughter board; using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300mj / 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, and then baking at 115° C. for 3 minutes; after baking, wait for the substrate to cool down, and then spin coating the second embossing glue, adding the second embossing glue 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, using nanoimprint equipment, imprinting the daughter plate on the second imprint glue, UV curing, demoulding, and obtaining the same array of nanopores as the mother plate pattern on the second imprint glue, that is, preparing the array of nanopores on the substrate surface; using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300mj / cm 2The diameter of the nanopore is 200 nm, the height (i.e., depth) is 100 nm, and the distance between any two adjacent nanopores is 500 nm.

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

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

[0098] S21, mixing 1 mL of acrylamide hydrogel containing carboxyl groups with 5 mg of fluorescein isothiocyanate to obtain a functionalized modified material; the preparation method of acrylamide hydrogel containing carboxyl groups is as follows: adding 1 g of partial sodium salt of poly(acrylamide-acrylic acid) to 100 mL of deionized water, stirring, and obtaining acrylamide hydrogel containing carboxyl groups;

[0099] S22, dropping the functional modification material on the surface of the substrate, the surface of the second imprinting glue and the nanopores, and spin coating at a speed of 3000 r / min 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 modification layer on a wafer stage of an ion etcher, and performing etching treatment 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 rotation 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. Arrayed nanopores 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, the surface of which has arrayed nanopores; performing a cleaning process on the master: using an SPM solution, that is, mixing concentrated sulfuric acid with a mass concentration of 98% and hydrogen peroxide with a mass fraction of 30% 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 and drying, and setting aside;

[0106] S12, coating the first imprint glue on the motherboard after cleaning in S11, specifically using spin coating to spin coat the first imprint glue; the first imprint glue spin coating process: dripping the first imprint glue on the motherboard, and spin coating at a speed of 4000r / min for 60 seconds; using nanoimprint equipment, imprinting the flexible substrate PET film on the first imprint glue, UV curing, demolding, and replicating the pattern opposite to the motherboard on the PET film to obtain a daughter board; using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300mj / cm 2 ;

[0107] S13, adding a tackifier to the surface of the substrate, and spin coating at a speed of 3000 r / min for 30 seconds, and then baking at 115° C. for 3 minutes; after baking, wait for the substrate to cool down, and then spin coating the second embossing glue, adding the second embossing glue to the tackifier, spin coating at a speed of 2000 r / min for 60 seconds, and baking at 115° C. for 2 minutes;

[0108] S14, using nanoimprint equipment, imprinting the daughter plate on the second imprint glue, UV curing, demoulding, and obtaining the same array of nanopores as the mother plate pattern on the second imprint glue, that is, preparing the array of nanopores on the substrate surface; using ultraviolet light with a wavelength of 365 nanometers for curing, and the ultraviolet light intensity is 300mj / cm 2 The diameter of the nanopore is 200 nm, the height (i.e., depth) is 100 nm, and the distance between any two adjacent nanopores is 500 nm.

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

[0110] The material used for the functional modification layer is an acrylamide-based hydrogel containing a carboxyl group. The preparation method of the functional modification layer includes the following steps:

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

[0112] S22, adding the functional modification material dropwise onto the substrate surface, the second imprinting adhesive surface and the nanopores, and spin coating at a speed of 3000 r / min 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.

[0113] Performance Testing

[0114] According to the method in Example 1, a dot matrix biochip was prepared. Using light with an excitation wavelength of 488 nanometers under a fluorescence microscope, a periodic fluorescence dot matrix signal was displayed. The results are as follows: Figure 7 shown.

[0115] A biochip was prepared according to the method in Comparative Example 1. Using light with an excitation wavelength of 488 nanometers under a fluorescence microscope, a fluorescence signal of the entire surface can be displayed under a fluorescence microscope. The results are as follows: Figure 8 shown.

[0116] Depend on Figures 7-8 By comparison, it can be seen that before IBE selective etching, the functionalized modification material coated the entire surface of the nanopore array, that is, the fluorescence signal was detected on the entire surface; after the functionalized modification layer was selectively etched by IBE, a fluorescence signal appeared inside the nanopore, that is, there was functionalized material inside the nanopore, and the functionalized material in the area outside the nanopore was removed, that is, selective modification, thereby realizing a periodic fluorescence dot matrix signal.

[0117] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a dot matrix biochip, characterized in that: The following steps are involved: Prepare arrayed nanopores 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 modification layer on a wafer stage of an ion etcher and performing etching treatment to obtain a dot matrix biochip; Among them, 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, characterized in that: 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 5-10sccm, the screen voltage is 550-560eV, the ion beam current is 110-120mA, the wafer stage rotation speed is 10-20rpm, and the etching time is 10-20s.

3. The method for preparing a dot matrix biochip according to claim 1, characterized in that: The nanopores arranged in an array are prepared on the surface of the substrate, specifically comprising the following steps: Providing a master, wherein the surface of the master 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, demoulding, and obtaining a pattern on the flexible substrate opposite to the master to form a daughter plate; Coating a tackifier on the surface of the substrate, heating and baking, then coating a second embossing adhesive, heating and baking; The daughter plate is printed on the second printing glue, UV-cured, and demoulded to obtain the same array of nanopores as the mother plate on the second printing glue, 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, characterized in that: The material used for the functionalized modification layer includes, but is not limited to, an acrylamide-based hydrogel containing a functionalized group; the functionalized group includes, but is not limited to, any one of a carboxyl group, an epoxy group, a polysaccharide material, biotin, a hydroxylated PEG, and an aminated PEG.

5. The method for preparing a dot matrix biochip according to claim 1, characterized in that: The material used for the functional modification layer is an acrylamide-based hydrogel containing a carboxyl group, and the preparation method of the functional 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, characterized in that: 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) into 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, characterized in that: 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 glue 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, characterized in that: 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, characterized in that: The diameter of the nanopore is 200-300 nm and the height is 100-150 nm; And / or, the thickness of the substrate is 500-1000 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, characterized in that: 3°≤θ≤12°.

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