Solid-state nanopores, their preparation methods, small peptide detection methods and applications

Abstract

The present invention belongs to the technical field of molecular detection, and particularly relates to a solid-state nanopore, a preparation method thereof, a small peptide detection method and applications. Based on the single-molecule solid-state nanopore detection technology, the WS2 / MoS2 vertical heterojunction structure grown by the CVD method is transferred onto a silicon nitride chip by means of PMMA wet-assisted transfer as a nanopore carrier material, and a solid-state nanopore is prepared by dielectric breakdown. Finally, the formed solid-state nanopore is used to detect the electrical signal dimension of GSH. The present invention provides a novel carrier material for fabricating solid-state nanopores, which promotes the development of solid-state nanopores to a certain extent and has broad application prospects in the fields of biophysics and biomedicine.

Description

Technical Field

[0001] The present invention belongs to the technical field of molecular detection, and particularly relates to a solid-state nanopore, a preparation method thereof, a small peptide detection method and an application thereof. Background Art

[0002] Due to its good mechanical stability, thermal stability, chemical stability, easy modification and other characteristics, solid-state nanopores have been greatly developed in recent years. Traditional solid-state nanopores have problems such as it being difficult to prepare nanopores with a pore diameter less than 2 nm and a thickness less than 10 nm, and it being difficult to precisely control the chemical environment of the inner surface of the nanopore. The emergence of single-atom-layer two-dimensional materials has well solved the above problems and also significantly improved the spatial resolution of nanopores. Currently, single-atom-layer two-dimensional materials are mainly divided into graphene, boron nitride (BN), and transition metal dichalcogenides (TMD). TMD has excellent optoelectronic properties, chemical stability, and mechanical properties. Among them, molybdenum disulfide (MoS2) and tungsten disulfide (WS2) are typical representatives. Using the sub-nanometer single-layer films of these two materials to fabricate solid-state nanopores for single-molecule analysis can provide a high spatial resolution and achieve excellent sensing sensitivity.

[0003] Currently, the preparation of solid-state nanopores from monolayer or few-layer WS2 and MoS2 grown by CVD and the single-molecule analysis of DNA have been reported in the literature. However, the preparation of solid-state nanopore devices from the composite vertical films formed by the two metal sulfides has not been reported in current patents and literature. At the same time, there is no literature or patent reporting the detection of GSH using such solid-state nanopores. Summary of the Invention

[0004] In view of this, the present invention intends to grow a WS2 / MoS2 vertical heterojunction structure at a relatively low temperature by chemical vapor deposition (CVD) method, use this vertical heterojunction film to fabricate a solid-state nanopore, and then use this solid-state nanopore to detect the small molecule glutathione (GSH).

[0005] One of the purposes of the present invention is to provide a novel solid-state nanopore, which uses the WS2 / MoS2 vertical heterojunction structure as a carrier material, providing technical support for further detection of GSH.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] A solid-state nanopore, characterized in that the solid-state nanopore uses the WS2 / MoS2 vertical heterojunction structure as a carrier material.

[0008] Furthermore, the pore diameter of the solid-state nanopore is 2 - 3 nanometers, and the thickness is within 10 nanometers.

[0009] The second object of the present invention is to provide a method for preparing a solid-state nanopore.

[0010] To achieve the above object, the present invention adopts the following technical solutions:

[0011] The method for preparing the solid-state nanopore described in Object 1 includes the following steps:

[0012] S1: Grow a WS2 / MoS2 vertical heterojunction structure on a P-doped SiO2 / Si substrate by chemical vapor deposition;

[0013] S2: Use a PMMA wet-assisted transfer method to transfer the WS2 / MoS2 vertical heterojunction structure obtained in S1 from the substrate surface to a silicon nitride chip as a nanopore carrier material;

[0014] S3: Prepare a solid-state nanopore on the nanopore carrier material obtained in S2 by dielectric breakdown.

[0015] Further, in S1, the P-doped SiO2 / Si substrate is sequentially placed in deionized water, acetone, ethanol, and deionized water for ultrasonic cleaning, and the ultrasonically cleaned SiO2 / Si substrate is gently blown with nitrogen to remove organic and inorganic contaminants on the surface.

[0016] Further, in S1, the reactant preparation method is as follows: Dissolve 4 mg of molybdenum trioxide, 4 mg of tungsten trioxide, and 4 mg of sodium chloride in 1 mL of absolute ethanol to prepare a reactant suspension.

[0017] Further, in S1, set the CVD device growth program as follows:

[0018] The first step is to heat up to 200 °C within 10 min;

[0019] The second step is to heat up to 700 °C within 20 min to reach the MoS2 growth temperature;

[0020] The third step is to continue for 8 min at 700 °C to grow the MoS2 structure;

[0021] The fourth step is to heat up to 875 °C within 7 min to reach the WS2 growth temperature;

[0022] The fifth step is to continue for 6 min at 875 °C to grow the WS2 structure.

[0023] Further, in S2, before transfer, the silicon nitride chip is cleaned with plasma for 2 min to increase the hydrophilicity of the silicon nitride chip.

[0024] Further, in S2, the transferred silicon nitride chip is left standing overnight to air dry the moisture.

[0025] Further, place the air-dried silicon nitride chip in acetone to remove the PMMA on the surface.

[0026] Further, in S3, by measuring the I-V curve and fitting, the conductance G is obtained.

[0027] Further still, if the conductance G is within 0.5, it indicates that the WS2 / MoS2 vertical heterojunction structure has been successfully transferred to the silicon nitride chip.

[0028] Further, S3 specifically includes:

[0029] 1) Assemble the transferred silicon nitride chip with the flow cell;

[0030] 2) Inject the buffer solution into both ends of the flow cell, and connect the patch clamp to measure the I-V curve;

[0031] 3) Fit the obtained I-V curve to obtain the conductance G;

[0032] 4) Provide a current pulse to form the solid-state nanopore through dielectric breakdown.

[0033] A current pulse is provided by a power meter, and the transferred thin film will be exposed to an electric field with an intensity equivalent to the dielectric strength of the film. The charge accumulation and thermal effect of the strong electric field will generate leakage current at the structural defects of the thin film, resulting in dielectric breakdown of the film and the formation of nanopores.

[0034] Further, in 2), the buffer solution is a mixed solution of KCl, Tris, and EDTA, and the pH value of the buffer solution is 8; the concentration of KCl is 1M, the concentration of Tris is 10mM; the concentration of EDTA is 1mM.

[0035] The third object of the present invention is to provide a method for detecting the electrical signal of small molecule glutathione using a solid-state nanopore.

[0036] To achieve the above object, the present invention adopts the following technical solutions:

[0037] The method for detecting small molecule glutathione using the solid-state nanopore described in object one includes the following steps:

[0038] (1) Prepare a glutathione test sample solution;

[0039] (2) Add the glutathione sample test solution obtained in (1) to the cis end of the flow cell, and add buffer 1 to the trans end; detect the ion current blockage signal generated by glutathione passing through the solid-state nanopore;

[0040] (3) Analyze the ion current blockage signal of glutathione obtained in (2).

[0041] Further, in step (2), the buffer 1 is a mixed solution of KCl and 1×PBS; the concentration of KCl is 1 M; the pH value of the mixed solution is 7.4.

[0042] Further, in step (1), a glutathione test sample solution is prepared using the buffer 1 as a diluent.

[0043] Further, in step (3), the analysis software is Clampfit software.

[0044] Based on the single-molecule solid-state nanopore detection technology, the WS2 / MoS2 vertical heterojunction structure grown by the CVD method is transferred onto a silicon nitride chip as a nanopore carrier material through a wet-assisted transfer method using polymethyl methacrylate (PMMA), and a solid-state nanopore is prepared by dielectric breakdown. Finally, the formed solid-state nanopore is used to detect the electrical signal of GSH in the electrical dimension. Different from the currently reported WS2 nanopores and MoS2 nanopores, the present invention prepares solid-state nanopores from a composite vertical thin film formed by two metal sulfides, integrating the characteristics of the two materials. The two materials grow vertically and overlap with obvious boundaries, and have different interactions with biomolecules. By regulating the interaction behavior between the material and biomolecules at different sites on different thin films, the sensing performance of the thin film nanopore for small peptides can be significantly improved. The detection of the electrical signal of the small peptide GSH using the composite metal sulfide vertical thin film prepared solid-state nanopore of the present invention is a novel nanopore analysis platform, which has not been reported in current literature and patents.

[0045] The fourth object of the present invention is to provide an application of the solid-state nanopore described in the first object in constructing a nanopore analysis platform for ion transport, DNA sequencing, and / or biomolecule detection.

[0046] Further, the application of the solid-state nanopore in detecting single molecules such as nucleic acids and proteins.

[0047] The fifth object of the present invention is to provide an application of the WS2 / MoS2 vertical heterojunction structure in preparing solid-state nanopores.

[0048] The beneficial effects of the present invention are as follows:

[0049] 1. The present invention discloses a novel solid-state nanopore based on a WS2 / MoS2 composite metal sulfide vertical heterojunction thin film, and uses this solid-state nanopore to achieve the detection of small peptide GSH at the single-molecule level.

[0050] 2. The present invention provides a novel carrier material for fabricating solid-state nanopores, which promotes the development of solid-state nanopores to a certain extent and has broad application prospects in the fields of biophysics and biomedicine; WS2 / MoS2 solid-state nanopores can be used to detect different single molecules such as nucleic acids and proteins in the future and have great development potential; this method provides a novel two-dimensional material solid-state nanopore, which greatly promotes the application and research of two-dimensional material solid-state nanopores in aspects such as ion transport, DNA sequencing, and biomolecule detection.

[0051] 3. This patent uses the WS2 / MoS2 vertical heterojunction composite structure as the nanopore carrier material. Compared with a single structure, the control of its growth conditions is more precise than that of a single material; in terms of membrane transfer, it has high mechanical strength and is easier to transfer successfully; in terms of the sensing performance of nanopores, the stress and flicker noise of the composite material are both small, and the composite material can have an obvious difference in the interaction with the detected biomolecules, thereby significantly improving the time resolution and sensitivity of sensing. Therefore, the composite material nanopore of the present invention has advantages in terms of membrane transfer to the sensing performance of nanopore devices. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 It is a schematic flow diagram of the transfer of WS2 / MoS2 vertical heterojunction thin film, nanopore preparation, and the detection current signal of small peptides;

[0053] Figure 2 It is an optical microscope picture of the WS2 / MoS2 vertical heterojunction structure grown by CVD;

[0054] Figure 3 It is an optical microscope picture of the transferred WS2 / MoS2 vertical heterojunction thin film transferred on a silicon nitride chip;

[0055] Figure 4 It is a statistical result graph of the through-pore signal of 20nM GSH passing through the WS2 / MoS2 nanopore under a 300mV bias voltage in buffer1; among them, Figure 4 (a) is a scatter plot of the blocking time and the blocking current amplitude, Figure 4 (b) is a bar graph of the blocking time, Figure 4 (c) is a bar graph of the blocking current amplitude, Figure 4 (d) is a bar graph of the blocking rate;

[0056] Figure 5 It is an optical characterization graph of 20nM GSH passing through the WS2 / MoS2 nanopore under a 300mV bias voltage in buffer1. Among them, Figure 5 (a) is an optical microscope picture of the WS2 / MoS2 vertical heterojunction structure, indicating the selected area for Raman analysis and testing; Figure 5(b) is for Figure 5 the Raman scattering diagrams of points 1, 2, and 3 shown in (a). Specific embodiments

[0057] The technical solutions of the present invention will be further clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all embodiments. Therefore, based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative efforts belong to the protection scope of the present invention.

[0058] In the embodiments of the present invention, first, a WS2 / MoS2 vertical heterojunction structure is grown on a P-doped SiO2 / Si substrate by chemical vapor deposition (CVD). Then, the WS2 / MoS2 heterojunction structure is transferred from the surface of the SiO2 / Si growth substrate to a silicon nitride chip by means of wet-assisted transfer of polymethyl methacrylate (PMMA). Next, nanopores are prepared on this thin film by dielectric breakdown. Finally, the electrical signals of glutathione (GSH) are detected using the nanopores formed in this thin film.

[0059] Example 1

[0060] The first step: growing the WS2 / MoS2 vertical heterojunction structure using CVD

[0061] First, the P-doped SiO2 / Si substrate is successively placed in deionized water, acetone, ethanol, and deionized water for ultrasonic cleaning, and the ultrasonically cleaned SiO2 / Si substrate is gently blown with nitrogen to remove organic and inorganic contaminants on the surface. At the same time, 4 mg of molybdenum trioxide (MoO3), 4 mg of tungsten trioxide (WO3), and 4 mg of sodium chloride (NaCl) are dissolved in 1 mL of absolute ethanol to prepare a reactant suspension. Then, 10 μL of the reactant suspension is dropped onto the cleaned SiO2 / Si substrate and placed on a heating stage, and heated and dried at 50 °C to remove the solvent. Then, a growth program is set on the CVD device: the first step, heating to 200 °C within 10 min; the second step, heating to 700 °C within 20 min to reach the MoS2 growth temperature; the third step, maintaining at 700 °C for 8 min to grow the MoS2 structure; the fourth step, heating to 875 °C within 7 min to reach the WS2 growth temperature; the fifth step, maintaining at 875 °C for 6 min to grow the WS2 structure. Finally, the heated and dried SiO2 / Si substrate is placed in the CVD device to grow the WS2 / MoS2 vertical heterojunction structure.

[0062] As Figures 1 - 3 shown, Figure 1Schematic diagram of the transfer of WS2 / MoS2 vertical heterojunction thin film, preparation of nanopores, and detection of current signals of small peptides. The whole process undergoes four steps: CVD growth of WS2 / MoS2 vertical heterojunction structure, transfer, pore preparation, and testing. Figure 2 Figure shows the CVD-grown WS2 / MoS2 vertical heterojunction structure observed under an optical microscope.

[0063] Figure 3 Figure shows the state of the transferred WS2 / MoS2 vertical heterojunction thin film on a silicon nitride chip observed through an optical microscope. The black square in the figure is the silicon nitride suspension window.

[0064] Step 2: Transfer the WS2 / MoS2 vertical heterojunction structure onto a silicon nitride chip

[0065] Drop 20 μL of PMMA solution onto the grown SiO2 / Si substrate, and spin-coat it on a spin coater at 500 rpm for 5 s and 3000 rpm for 40 s to uniformly spin-coat the PMMA solution on the wafer. Then, heat the spin-coated wafer at 120 °C for 10 min to dry the PMMA. Meanwhile, prepare 1 M sodium hydroxide (NaOH) solution. Then, gently scrape the periphery of the dried wafer with a knife to make it easier for the NaOH solution to penetrate between the PMMA and the Si substrate to etch SiO2. Place the scraped wafer in 1 M NaOH solution and heat it at 37 °C for two hours to remove SiO2. Lift the film floating in the NaOH solution with a glass slide and rinse it in deionized water. Meanwhile, clean the silicon nitride chip with plasma for 2 min to increase the hydrophilicity of the silicon nitride chip. Then, transfer the film onto the silicon nitride chip under a microscope (as shown in Figure 1 and Figure 3 ). Let the transferred silicon nitride chip stand overnight to air-dry the moisture. Finally, place the air-dried silicon nitride chip in acetone to remove the PMMA.

[0066] Step 3: Preparation of nanopores and detection of electrical signals of GSH

[0067] 1. Preparation of nanopores

[0068] The transferred silicon nitride chip is assembled with the flow cell. A buffer solution of 1M KCl, 10mM Tris, 1mM EDTA, pH8 is injected into both ends of the flow cell, and the patch clamp is connected to measure the I-V curve. The obtained I-V curve is fitted to obtain the conductance G. If the conductance G is very small, it indicates the successful transfer of the WS2 / MoS2 heterojunction structure. Then, a current pulse is provided by a power meter, and the transferred thin film is exposed to an electric field with an intensity equivalent to the dielectric strength of the film. Charge accumulation and thermal effects in the strong electric field will generate leakage current at the structural defects of the thin film, resulting in dielectric breakdown of the film and the formation of nanopores.

[0069] 2. Detection of GSH electrical signals

[0070] (1) Preparation of test samples: Buffer1: A buffer solution of 1M KCl, 1×PBS, pH7.4. The GSH stock solution is diluted with Buffer1 to obtain a 20nM GSH test sample solution.

[0071] (2) Add 20nM GSH sample test solution to the cis end of the flow cell and Buffer1 to the trans end. Then, connect the flow cell to the patch clamp and apply a 300mV bias voltage to measure the ion current blockage signal generated by GSH passing through the nanopores of about 2nm.

[0072] (3) The collected GSH electrical signals are analyzed using Clampfit software.

[0073] The data characteristic distribution is as Figure 4 and Figure 5 shown. Figure 4 It is a graph of the blocking time (T = 0.12ms), blocking current amplitude (A = 169pA), and blocking rate (I b / I o = 0.027) obtained by applying a 300mV bias voltage to 20nM GSH in an electrolyte of 1M KCl, 1×PBS, pH7.4 using the prepared WS2 / MoS2 nanopores with an aperture of about 2nm. From the preliminary detection results, it can be seen that this new type of nanopore can detect GSH and shows a good capture rate and signal-to-noise ratio. It proves that the constructed new nanopore analysis platform is successful, providing a new direction for subsequent detection and analysis of single molecules using this platform. Figure 5 It is the optical microscope structure diagram of the WS2 / MoS2 vertical heterojunction structure and the Raman scattering characterization diagram of the thin film material. Raman scattering analysis was performed on different regions of the composite metal sulfide vertical thin film, including WS2, MoS2, and the boundary between the two materials. Each region shows obvious material characteristic scattering, confirming that the grown material is a vertical heterojunction structure.

Claims

1. A method for detecting small molecule glutathione based on solid-state nanopores, characterized in that It includes the following steps: (1) Prepare a glutathione test sample solution; (2) Add the glutathione sample test solution obtained in (1) to the cis end of the flow cell, and add buffer 1 to the trans end; Detect the ion current blockage signal generated by glutathione passing through the solid-state nanopore; (3) Analyze the ion current blockage signal of glutathione obtained in (2); The solid-state nanopore uses a WS2 / MoS2 vertical heterojunction structure as the carrier material, the pore diameter of the solid-state nanopore is 2-3 nanometers, and the thickness is within 10 nanometers.

2. The method according to claim 1, wherein In step (2), the buffer 1 is a mixed solution of KCl and 1×PBS, and the pH value of the mixed solution is 7.

4.

3. The method according to claim 1, characterized in that The preparation method of the solid-state nanopore includes the following steps: S1: Grow a vertical heterojunction structure on a P-doped SiO2 / Si substrate by chemical vapor deposition; S2: Use a PMMA wet-assisted transfer method to transfer the vertical heterojunction structure obtained in S1 from the substrate surface to a silicon nitride chip as the nanopore carrier material; S3: Prepare a solid-state nanopore on the nanopore carrier material obtained in S2 by dielectric breakdown.

4. The method according to claim 3, wherein In S1, the P-doped SiO2 / Si substrate is sequentially placed in deionized water, acetone, ethanol, and deionized water for ultrasonic cleaning.

5. The method according to claim 3, characterized in that, In S1, set the CVD device growth program as follows: The first step is to heat up to 200°C within 10 minutes; The second step is to heat up to 700°C within 20 minutes; The third step is to maintain at 700°C for 8 minutes; The fourth step is to heat up to 875°C within 7 minutes; The fifth step is to maintain at 875°C for 6 minutes.

6. The method according to claim 3, characterized in that, In S3, measure the I-V curve and fit to obtain the conductance G.

7. The method according to claim 6, characterized in that, If the conductance G is within 0.5, it indicates the successful transfer of the WS2 / MoS2 vertical heterojunction structure to the silicon nitride chip.

8. Application of a solid-state nanopore in detecting small molecule glutathione, characterized in that, The solid-state nanopore uses a WS2 / MoS2 vertical heterojunction structure as the carrier material, the pore diameter of the solid-state nanopore is 2-3 nanometers, and the thickness is within 10 nanometers; The application is for non-disease diagnosis and treatment purposes.

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