Method for modulating pore size of a solid-state nanopore array template

Abstract

The application discloses a method for regulating the aperture of a solid-state nanopore array template, comprising the following steps: taking a metal target and a ceramic target as co-sputtering targets, magnetron sputtering co-depositing a metal nanowire array-ceramic composite material on a pretreated substrate, immersing in chemical etching liquid A for etching, then washing, immersing in chemical etching liquid B for etching at 15-35 DEG C for 1-15 min for aperture regulation, and washing to obtain the solid-state nanopore array template after aperture regulation; the diameter of the nanopore can be finely regulated to 0.1-1.2 nm. In the application, the solid-state nanopore array template is prepared by co-sputtering of the target material, and then the nanopore diameter is effectively and controllably regulated by using a Lewis acid or a Lewis acid base, so that the sub-nanometer size regulation is realized, and the method can be applied to the detection field of various biological macromolecules with similar sizes.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of nanomaterials, and particularly relates to a method for regulating the aperture of a solid-state nanopore array template. BACKGROUND

[0002] Solid-state nanopores have great application in biomolecule detection, especially in DNA and RNA sequencing, and also have broad prospects in potential applications such as nanofiltration, supercapacitors, energy conversion and storage. In the process of biomolecule detection, nanopores are embedded in a thin film and immersed in an electrolyte solution, and the electric field on both sides will drive the molecules to pass through the nanopores and produce corresponding ion current signal fluctuations, and the size, structure and other information of the biomolecules can be determined by analyzing the current changes. For biomolecule detection, when the diameter of the nanopore matches the size of the target molecule (consistent with the size of the molecule or slightly larger), the detection result will have the characteristics of high sensitivity and high resolution.

[0003] For common biomolecules such as DNA duplexes (diameter about 2 nm), protein complexes (diameter 1.5-2.5 nm), amino acid polymers (diameter 5.5±1.1-8 nm) and the like, the size difference between them is very small (sub-nanometer level). Considering the diversity and size characteristics of biomolecules, obtaining size-controllable and sub-nanometer scale continuously adjustable nanopores is of great significance for biomolecule detection, single molecule detection and gene sequencing.

[0004] In recent years, the preparation of solid-state nanopores mainly uses local high-energy microprocessing technology, such as laser beam irradiation, high-energy particle (ion) beam irradiation, high-voltage discharge, electroporation, etc. Yuan et al. (Controllable fabrication of solid-state nanopores array by electron beam shrinking. Int. J. Mach. Tool Manufact., 2020, 159:103623) used focused ion beam technology to prepare nanopores with a pore size range of 41-166 nm on a Si3N4 film, and then used high-energy electron beam irradiation to make hydrocarbon compounds deposit on the edge of the nanopore to reduce the diameter of the nanopore. The smallest diameter of the nanopore prepared by this technology is 5.8 nm.

[0005] Liu et al. (Direct fabrication of sub-10 nm nanopores in WO3 nanosheets using single swift heavy ions. Nano Lett., 2023, 23:4502) used rapid heavy ions to directly fabricate sub-10 nm random nanopores on WO3 nanosheets. By selecting ions of different types and energies, the pore size and density of the nanopores can be controlled. However, preparation techniques such as electron beam and rapid heavy ion breakdown have problems such as the need for high-energy beams, the limited number of nanopores prepared in a single run, and high equipment costs. Furthermore, the use of single solid-state nanopores for detection is plagued by more complex and cumbersome procedures, high preparation costs, and low sequencing efficiency, all of which significantly limit the efficiency of molecular detection.

[0006] One of the methods to improve the detection efficiency of solid-state nanopores is to use solid-state nanopore array devices to achieve high-throughput physical sequencing. Researchers have made many new attempts in the preparation of solid-state nanopore arrays, and large-scale self-ordered solid-state nanopore films have been attempted to be prepared. The Chinese patent document with publication number CN115093948A discloses a method for resolving biomolecules and its solid-state nanopore four-hole array device. The patent uses a helium ion microscope to etch a four-nanopore array structure with a nanopore diameter of 5-10 nm and a distance between the pore centers of 30-200 nm. Although this method produces a highly regular solid-state nanopore array, its molecular detection results are limited to the detection of smaller biomolecules due to the limitations of the solid-state nanopore pore size range. In addition, the invention only focuses on further reducing the diameter of the nanopore, and does not consider the importance of continuously adjustable diameter for improving the resolution of molecular detection.

[0007] Therefore, it is necessary to explore a method for preparing a solid-state nanopore array structure with continuously adjustable diameter to further improve the resolution, sensitivity and testing efficiency of solid-state nanopores for biological molecules, so as to realize the widespread application of solid-state nanopores in biological detection (high-throughput detection), gene sequencing (high-throughput sequencing) and other fields. Summary of the Invention

[0008] The present invention addresses the problem that the diameter of nanopores is difficult to micro-control and is not suitable for the detection of biomolecules with small size differences. It provides a method for preparing a solid-state nanopore template that can adjust the pore size at the sub-nanometer scale. It can achieve nanopore diameter control in a small range of 0.1-1.2 nm. The preparation method is simple and can be prepared on a large area without the need for complex process preparation conditions such as photolithography or high-energy electron beam.

[0009] To achieve the above object, the technical solution adopted by the present invention is:

[0010] A method for regulating the pore size of a solid nanopore array template, comprising the steps of:

[0011] Step 1, ultrasonic cleaning and plasma treatment of the substrate;

[0012] Step 2, co-sputtering of a metal nanowire array-ceramic composite film on the substrate using a metal target and a ceramic target as co-sputtering targets;

[0013] Step 3, etching the composite material in chemical etching solution A and then cleaning to obtain a solid nanopore array template;

[0014] Step 4, etching the solid nanopore array template in chemical etching solution B at 15-35 DEG C for 1-15 min to regulate the pore size, and then cleaning to obtain a solid nanopore array template with regulated pore size;

[0015] The pore size regulation refers to a change of 0.1-1.2 nm in the diameter of the nanopores in the solid nanopore array template;

[0016] The chemical etching solution A is an aqueous solution of NH3H2O and / or H2O2, and the molar fraction of the solute in the aqueous solution is 0.3-1 mol / L;

[0017] The chemical etching solution B is an aqueous solution of one or more of NaOH, KOH, HCl and H2SO4, and the molar fraction of the solute in the aqueous solution is 0.01-1 mol / L.

[0018] In the present application, a solid nanopore array can be prepared in a large area by the method of magnetron sputtering, and then the diameter of the solid nanopore is regulated by controllable wet etching, i.e. the metal phase is completely removed by using chemical etching solution A, and then the diameter of the solid nanopore is regulated by using Lewis acid or Lewis base in chemical etching solution B. Since the diameter of the nanopore is small, there is a strong capillary force, and the etching solution in the nanopore is firmly adsorbed, and cannot flow naturally with the etching solution in the beaker. With the progress of the reaction, the concentration of the etching solution in the nanopore is lower than that in the beaker, and the change in the pore diameter caused by single etching can be effectively realized by controlling the etching time. Meanwhile, after the speed control element reaction occurs, with the progress of the reaction, the content of silanol and siloxane hydrolyzed in the solid nanopore increases, which slows down the reaction process to a certain extent, until the reaction is in a balanced state, i.e. the amount of consumed compounds and the amount of corresponding generated compounds are consistent, and a reversible equilibrium state is maintained, so that fine regulation in a very small diameter range can be realized.

[0019] The substrate comprises one or more of single crystal silicon wafer, quartz glass sheet, sapphire, flexible PET and Al foil;

[0020] The ultrasonic treatment in step 1 includes treating with one or more of acetone, ethanol, deionized water, and ultrasonic treatment for 10-30 min; the plasma treatment is 5-20 x 10 -5 Pa for 5-20 min.

[0021] The diameter of the nanopores in the solid nanopore array template is 1-10 nm, the average spacing of the nanopores is 3-20 nm, and the array period is 6-40 nm.

[0022] The filling rate of the metal phase in the composite film is 5-30%, and the volume fraction of the ceramic phase is 70-95%.

[0023] The metal target material includes one or more of Ag target, Cu target, and Al target; and the ceramic target material includes one or more of Al2O3, AlN, and SiO2.

[0024] Preferably, the ceramic target material is selected from SiO2 or AlN, because the Al2O3 target material has a serious slagging phenomenon during preparation.

[0025] In the magnetron sputtering in step 2, the power of the metal target sputtering is 10-20 W, the power of the ceramic target sputtering is 60-100 W, and the pre-deposition pressure is 5-10 x 10 -5 Pa.

[0026] In step 3, the etching temperature is 15-35℃, and the etching time is 1-6 h, preferably 1-2 h. The purpose of step 3 is to etch away the metal phase completely. Therefore, a long etching time is required to completely remove the metal phase. Since the etching liquid A does not react with the ceramic matrix, this process does not affect the diameter of the nanopores.

[0027] Preferably, the etching time in step 4 is 1-5 min, preferably 3-4 min, which can achieve the regulation of the nanopore diameter of about 0.1-0.5 nm, and the regulation is more accurate.

[0028] Preferably, the solute molar concentration of the chemical etching liquid A is 0.5-1 mol / L.

[0029] Preferably, the solute molar concentration of the chemical etching liquid B is 0.1-1 mol / L.

[0030] The thickness of the nanopore layer in the solid nanopore array film is 10-300 nm.

[0031] The array template structure has superhydrophilicity, and the immersion solution can include any one of deionized water, NH3·H2O, NaOH solution, KOH solution, HCl solution, H2SO4 solution, and HNO3 solution.

[0032] The porosity of the solid-state nanopore array template is 7%-64%.

[0033] In some embodiments, the step 4 is repeated to perform pore diameter regulation of the nanopore with a diameter change greater than 1.2 nm, and the sample etching time is a cumulative etching time.

[0034] Compared with the prior art, the present application has the following beneficial effects:

[0035] (1) In the present application, the solid-state nanopore array template is prepared by target co-sputtering of metal phase and ceramic phase, and then the Lewis acid or Lewis base is used to effectively adjust or control the nanopore diameter for precise regulation, so as to realize sub-nanometer size regulation, which can be applied to the detection field of various similar biological macromolecules.

[0036] (2) In the present application, the preparation method of the solid-state nanopore array template is simple, compared with the traditional solid-state nanopore preparation methods such as electron beam lithography and high-energy particle bombardment, which is conducive to integration and can be prepared in a large area, and can further improve the material preparation efficiency.

[0037] (3) The solid-state nanopore array template with adjustable pore diameter prepared in the present application is uniform in size and pore diameter, which is beneficial for the passage of biological molecules and improves the detection efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 It is a flowchart of the preparation method of the solid-state nanopore array of Example 1.

[0039] Figure 2 It is a surface SEM image of the solid-state nanopore array structure after etching to remove the metal phase in step 5 of Example 1.

[0040] Figure 3 It is a surface SEM image of the solid-state nanopore array structure after increasing the nanopore diameter by two-step etching in step 7 of Example 1.

[0041] Figure 4 It is a real object image of the metal nanowire array-ceramic composite metamaterial large-area sample prepared in step 3 of Example 2, and b is a real object image of the solid-state nanopore array structure after etching to remove the metal phase in step 5.

[0042] Figure 5 It is a film thickness and filling rate curve corresponding to different etching times in Example 3.

[0043] Figure 6 It is a contact angle test image of the sample with a cumulative etching time of 18 min in Example 3. DETAILED DESCRIPTION

[0044] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below with examples. It should be understood that the specific examples described herein are only used to explain the present application and not to limit the present application. Those skilled in the art can modify or replace equivalently without departing from the spirit and scope of the present application, which should be covered within the protection scope of the present application.

[0045] The raw materials used in the following specific embodiments are all purchased from the market.

[0046] Example 1

[0047] In this embodiment, single crystal silicon wafer is selected as the substrate, and the array template prepared after regulation and control is a solid nanopore array. The film layer thickness is 108.2 nm, the nanopore diameter is 4.6 nm, and the nanopore filling rate is 35.1%. The flowchart of the preparation method of this embodiment is shown in Figure 1 .

[0048] In this embodiment, the solid nanopore array template is first deposited with a metal nanowire array-ceramic composite metamaterial on the substrate by magnetron sputtering, wherein the metal is Ag and the ceramic phase is Al2O3. The metal phase in the composite layer is first etched and removed by a continuous chemical etching method to obtain a basic solid nanopore array template. The target size solid nanopore array is obtained by further chemical etching. The specific preparation method is as follows:

[0049] Step 1, the substrate is sequentially placed in acetone, ethanol and deionized water, ultrasonic cleaning for 15 min and dried with nitrogen, and the silicon wafer is fixed on the substrate tray;

[0050] Step 2, the tray is loaded into the deposition chamber of the magnetron sputtering equipment, the pre-deposition pressure is 5-10×10 -5 Pa, the substrate surface is bombarded by plasma for 10 min to further clean the substrate;

[0051] Step 3, under the condition of room temperature, control the co-sputtering of metal target and ceramic target under argon atmosphere, the sputtering power of metal Ag target is 12 W, the sputtering power of ceramic Al2O3 target is 100 W, and the plasma bombardment power is 20 W. After 0.8 h of deposition, the target and plasma bombardment power are turned off, and a metal nanowire array-ceramic composite metamaterial with a metal filling rate of 21.2% is obtained;

[0052] Step 4, under the condition of room temperature, the substrate coated with metal nanowire array-ceramic composite metamaterial is placed in 1 mol / L ammonia water for 2 h etching to obtain a solid nanopore array template;

[0053] Step 5, the ammonia-etched substrate is cleaned with 0.5 mol / L nitric acid solution, deionized water and ethanol for 5 min respectively at room temperature, and dried with nitrogen;

[0054] Step 6, the prepared solid-state nanopore array template after cleaning and drying is placed in 1 mol / L NaOH etching solution at room temperature, and the etching time is 10 min;

[0055] Step 7, the etched silicon wafer is cleaned with 0.4 mol / L NaOH solution, deionized water and ethanol at room temperature, and the cleaning time is 5 min, and dried with nitrogen, to obtain a solid-state nanopore array template with a nanopore diameter of 4.6 nm;

[0056] The surface morphology of the above-mentioned thin film sample is observed and analyzed by scanning electron microscopy (SEM), Figure 2 The SEM image of the solid-state nanopore array structure obtained in step 4 in Example 1 is given, Figure 3 The solid-state nanopore array template with expanded pore diameter obtained in step 7 in Example 1 is given.

[0057] Through optical ellipsometer measurement, effective medium theory fitting and microstructure characterization analysis, the thickness of the above-mentioned solid-state nanopore array structure changes from 110 nm to 108.2 nm, the nanopore diameter changes from 3.6±0.1 nm to 4.6±0.1 nm, and the nanopore filling rate changes from 21.2% to 35.1%, realizing the sub-nanometer scale adjustable and controllable preparation of solid-state nanopore array.

[0058] Example 2

[0059] In this example, Al foil is selected as the substrate, the substrate size is 10 cm×10 cm, the array template prepared after regulation is a solid-state nanopore array, the film thickness is 107.3 nm, the nanopore diameter is 4.7 nm, and the nanopore hole filling rate is 35.1%.

[0060] The array template with solid-state nanopores in this example is first deposited with a metal nanowire array-ceramic composite metamaterial on the substrate by magnetron sputtering, wherein the metal is Cu and the ceramic phase is SiO2. By continuous chemical etching method, the metal phase in the composite layer is etched and removed to obtain a basic solid-state nanopore array template, and the target size solid-state nanopore array is obtained by further chemical etching. The specific steps are as follows:

[0061] Step 1, after the substrate is cleaned with ethanol and deionized water for 5 min, the substrate is fixed on the substrate tray;

[0062] Step 2, the tray is loaded into the deposition chamber of the magnetron sputtering device, and the pre-deposition pressure is 5-10x10 -5 Pa, the substrate surface is further cleaned by plasma bombardment for 20 min;

[0063] Step 3, under the condition of room temperature, the metal target and the ceramic target are co-sputtered in an argon atmosphere, the sputtering power of the metal Cu target is 10 W, the sputtering power of the ceramic SiO2 target is 70 W, and the plasma bombardment power is 30 W. The argon gas flow during co-sputtering is 10 sccm, the substrate rotates at a constant speed, and after 1.5 h of deposition, the target and the plasma bombardment power are turned off, and a metal nanowire array-ceramic composite metamaterial with a metal filling rate of 21.2% is obtained;

[0064] Step 4, under the condition of room temperature, the substrate coated with the metal nanowire array-ceramic composite metamaterial is placed in 1 mol / L H2O2, and after 1.8 h of etching, a solid-state nanopore array template is prepared;

[0065] Step 5, under the condition of room temperature, the silicon wafer etched by nitric acid is sequentially cleaned with deionized water and ethanol for 5 min, and dried with nitrogen;

[0066] Step 6, under the condition of room temperature, the solid-state nanopore array template prepared after cleaning and drying is placed in 0.1 mol / L HCl solution, and the etching time is 10 min;

[0067] Step 7, under the condition of room temperature, the silicon wafer etched by HCl is sequentially cleaned with 0.05 mol / L NaOH solution, deionized water and ethanol, and the cleaning time is 5 min, and dried with nitrogen, to obtain a solid-state nanopore array template with a pore diameter of 4.7 nm;

[0068] The obtained sample is measured by optical ellipsometer, fitted by effective medium theory, and analyzed by microstructure characterization, Figure 4 a shows the actual picture of the large-area prepared sample of the metal nanowire array-ceramic composite metamaterial on the flexible substrate prepared in step 3 of Example 2, Figure 4 b is the actual picture of the corresponding solid-state nanopore array template in step 5.

[0069] Through optical ellipsometer measurement and effective medium theory fitting, the thickness of the above-mentioned solid-state nanopore array structure changes from 100 nm to 107.3 nm, the nanopore diameter changes from 4±0.2 nm to 4.7±0.1 nm, and the nanopore filling rate changes from 25.9% to 35.1%, realizing the controllable preparation of large-area adjustable solid-state nanopore array.

[0070] Compared with the sample of Example 1, the diameter of the nanopore can be regulated in sub-nanometer scale, the substrate of the solid nanopore can be applied on a flexible substrate and can be prepared in a large area.

[0071] Example 3

[0072] The metal nanowire array-ceramic composite metamaterial is plated according to the sputtering parameters and preparation process of Example 1, except that different etching times are recorded under the same experimental conditions to obtain different thicknesses and filling rates of the solid nanopore array.

[0073] Steps 1-3 are the same as in Example 1, and step 4 is adjusted to place the six metal nanowire array-ceramic composite metamaterial plated samples in 1 mol / L ammonia water at room temperature, and after 2 h of etching, six solid nanopore array templates are prepared;

[0074] Step 5, under room temperature conditions, the silicon wafer etched with nitric acid is sequentially cleaned with deionized water and ethanol for 5 min, and dried with nitrogen;

[0075] Step 6, under room temperature conditions, the six solid nanopore array templates prepared after cleaning and drying are placed in 0.3 mol / L HCl solution, and the etching time of the solid nanopore is 3 min each time, which experiences three stages of rapid chemical reaction, reduced chemical reaction speed, and chemical reaction into equilibrium state within 3 min, and then repeats etching in the newly configured 0.3 mol / L HCl solution for 3 min. The cumulative etching time of the six samples is 3 min, 6 min, 9 min, 12 min, 15 min, and 18 min, respectively;

[0076] Step 7, under room temperature conditions, the silicon wafer etched with HCl is sequentially cleaned with 0.05 mol / L NaOH solution, deionized water, and ethanol, and the cleaning time is 5 min, and dried with nitrogen to prepare solid nanopore array templates with different pore diameters and filling rates;

[0077] The obtained samples are measured by optical ellipsometer, fitted by effective medium theory, and analyzed by microstructure characterization, and the experimental results are shown in Figure 5 The thickness of the above solid nanopore array structure changes from 109.7 nm to 107.7 nm, and the nanopore filling rate changes from 21.2% to 46.8% as the etching time increases, realizing the controllable preparation of the solid nanopore array. The measurement results of the solid nanopore array sample with a cumulative etching time of 18 min are shown in Figure 6As shown, the contact angle is obviously less than 30°, having superhydrophilicity. Compared with the sample of Example 1, the obtained solid-state nanopore sample can have a nanopore diameter controllable in sub-nanometer scale in a single etching by regulating the etching time, and can realize fine-tuning of pores in a larger size through repeated etching, which has important application value for detection of biological molecules with similar sizes.

[0078] The above detailed description of the specific embodiments of the present application has explained the technical solutions and beneficial effects of the present application, and it should be understood that the above description is only the preferred embodiments of the present application and is not used to limit the present application. Any modification, supplement and equivalent replacement, etc. made on the principle of the present application shall be included in the protection scope of the present application, and the protection scope of the present application is defined by the scope of the claims.

Claims

1. A method for regulating pore size of a solid-state nanopore array template, characterized in that: Including steps: Step 1, ultrasonic cleaning and plasma treatment of the substrate; Step 2, using a metal target and a ceramic target as co-sputtering targets, magnetron sputtering co-deposit a metal nanowire array-ceramic composite film on a substrate; the metal target includes one or more of an Ag target, a Cu target, and an Al target; the ceramic target includes one or more of Al2O3, AlN, and SiO2; Step 3, immersing the composite material in a chemical etching solution A, etching, and then cleaning to obtain a solid nanopore array template; Step 4, immersing the solid nanopore array template in chemical etching solution B at 15-35° C. for 1-15 minutes to adjust the pore size, and washing to obtain the solid nanopore array template after pore size adjustment; The pore size control refers to the change of the diameter of the nanopores in the solid-state nanopore array template to 0.1-1.2nm; The chemical etching solution A is an aqueous solution of NH3·H2O and / or H2O2, and the solute molar fraction in the aqueous solution is 0.3-1 mol / L; The chemical etching solution B is an aqueous solution of one or more of NaOH, KOH, HCl, and H2SO4, and the solute molar fraction in the aqueous solution is 0.01-1 mol / L.

2. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The substrate includes one or more of a single crystal silicon wafer, a quartz glass wafer, a sapphire, a flexible PET and an Al foil; In step 1, the ultrasonic treatment includes using one or more solutions of acetone, ethanol, and deionized water for 10-30 minutes; the plasma treatment is carried out at 5-20×10 -5 Pa deposition pressure for 5-20 minutes.

3. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The diameter of the nanopores in the solid-state nanopore array template is 1-10 nm, the average spacing between the nanopores is 3-20 nm, and the array period is 6-40 nm.

4. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The metal phase filling rate in the composite film is 5-30%, and the ceramic phase volume accounts for 70-95%.

5. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: In step 2, during magnetron sputtering, the power of metal target sputtering is 10-20W, the power of ceramic target sputtering is 60-100W, and the pre-deposition pressure is 5-10×10 -5 Pa.

6. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: In step 3, the etching temperature is 15-35° C., and the etching time is 1-6 hours.

7. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The thickness of the nanopore layer in the solid-state nanopore array film is 10-300 nm.

8. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The porosity of the solid-state nanopore array template is 7%-64%.

9. The method for regulating pore size of a solid-state nanopore array template according to claim 1, wherein: The method also includes repeating step 4 to adjust the pore size so that the diameter of the nanopore changes by more than 1.2 nm.

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