A two-dimensional sub-nanopore detection system, a nucleic acid molecule detection device and their applications

Abstract

The present invention provides a two-dimensional sub-nanopore detection system, a nucleic acid molecule detection device and their applications. The angstrom-level two-dimensional sub-nanopore detection system constructed in the present invention prepares a graphene oxide film by the method of layer-by-layer self-assembly. The interlayer spacing between multiple adjacent two single-layer graphene oxides constitutes a two-dimensional sub-nanopore. By modifying probe molecules on the outer surface of the two-dimensional sub-nanopore, biological target molecules are detected. Since the interlayer spacing size of the two-dimensional sub-nanopore is at the angstrom level, the background of its sensing signal is low, it can distinguish solutes in electrolytes of different sizes, obtain more analyzable signals, overcome the defect of high background signal of one-dimensional nanochannels, thus solving the problem of low detection sensitivity of target molecules and improving the detection limit to 1 aM. The preparation method of the detection system of the present invention is simple and its performance is stable, and the method for detecting nucleic acid molecules is simple.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical molecule detection, and particularly relates to a two-dimensional sub-nanopore detection system, a nucleic acid molecule detection device and their applications. Background Art

[0002] With the development and progress of nanotechnology, pores with nano- and sub-nano sizes have gradually attracted people's attention. Nanoanalysis technology has been widely studied in the discipline of analytical chemistry, and biosensing and molecular detection have become one of the important research fields of nanoanalysis technology. For traditional one-dimensional nanochannels, due to the limitations of nanomanufacturing, it is difficult to achieve channels with an angstrom-level size. Therefore, during the process of ion signal sensing, high background signals are likely to be generated, thereby reducing the detection sensitivity. In addition, due to the size limitations of one-dimensional nanochannels, it is difficult to distinguish the effects of different electrolytes on sensing detection, and there are fewer analyzable sensing signals, further reducing the detection sensitivity. Therefore, how to construct a detection system with angstrom-level channels and improve the sensitivity of biosensing and molecular detection is an important problem currently faced. Summary of the Invention

[0003] The purpose of the present invention is to provide, in view of the above deficiencies of the prior art, a two-dimensional sub-nanopore detection system, a nucleic acid molecule detection device and their applications.

[0004] To achieve the above purpose, the present invention adopts the following technical solutions:

[0005] The first object of the present invention provides a two-dimensional sub-nanopore detection system. The two-dimensional sub-nanopore detection system includes a porous support and a functionalized graphene oxide film disposed on the porous support. The porous support includes any one of anodic aluminum oxide film or porous polymer film; the functionalized graphene oxide film is formed by layer-by-layer assembly of multiple sheet-like single-layer graphene oxides, and the layer spacing between two adjacent single-layer graphene oxides is at the angstrom level. The layer spacings between all adjacent two single-layer graphene oxides constitute sub-nanopores; the outer surface of the functionalized graphene oxide film is modified with probe molecules for capturing target molecules; by detecting the interaction between the probe molecules and the target molecules to be detected, the target molecules to be detected carry their own electric fields, so that the ion current signal of the sub-nanopores of the two-dimensional sub-nanopore detection system is amplified, and high-sensitivity target molecules are identified.

[0006] Further, the functionalized graphene oxide film is obtained by the following method:

[0007] S1. Prepare the functionalized graphene oxide film

[0008] Step S11: Disperse sheet-like single-layer graphene oxide and cross-linking agent dimethylsiloxane in deionized water to obtain a mixed solution A;

[0009] Step S12: After the mixed solution A obtained in step S21 is heated and stirred at a constant temperature for a period of time, stop stirring, and then cool the solution to room temperature to obtain a casting solution B.

[0010] Step S13: Drop the casting solution B obtained in step S22 onto the porous support polyethylene terephthalate, and naturally deposit it at room temperature to obtain a layer-by-layer self-assembled functionalized graphene oxide film.

[0011] Furthermore, the modification of the probe molecules on the outer surface of the functionalized graphene oxide film is obtained by the following method:

[0012] S2. Modify probe molecules on the outer surface of the functionalized graphene oxide film

[0013] Step S21: Sputter metal chromium and metal gold successively on the graphene oxide film to obtain a functionalized graphene oxide film with an activated outer surface. The thickness of the metal chromium is 5 - 10 nm, preferably 10 nm, and the thickness of the metal gold is 5 - 10 nm, preferably 10 nm.

[0014] Step S22: Dissolve tris(2 - carboxyethyl)phosphine hydrochloride in tris(hydroxymethyl)aminomethane buffer solution to obtain a solution C for opening the disulfide bond of the probe, with a concentration of 30 mM.

[0015] Step S23: Take the solution C obtained in step S22, add a certain amount of probe molecule solution and tris(hydroxymethyl)aminomethane buffer solution, and oscillate and mix evenly for 3 - 5 min to obtain a probe molecule solution D with a concentration of 0.5 - 2 M.

[0016] Step S24: Take the solution D obtained in step S23, drop it onto the functionalized graphene oxide with an activated outer surface obtained in S21, let it stand at room temperature for at least 1 h, wash it with deionized water for 3 - 5 min, and dry it with nitrogen to obtain a probe - modified functionalized graphene oxide film.

[0017] Furthermore, in step S11, the size of the flaky single - layer graphene oxide is 0.5 - 1 μm; the mass ratio of the flaky single - layer graphene oxide, cross - linker dimethylsiloxane, and deionized water is 1:(40 - 60):285.

[0018] Furthermore, in step S12, the reaction temperature is 90 - 110 °C, and the reaction time is 5 - 7 h.

[0019] Furthermore, in step S13, the area of the porous support is 5 mm × 5 mm, the pore diameter is 30 nm, and the pore density is 5×10 7 / cm 2 。

[0020] The second object of the present invention is to provide the above two-dimensional sub-nanopore detection system for qualitative and quantitative analysis of nucleic acid molecules.

[0021] The third object of the present invention is to provide a nucleic acid molecule detection device, which includes a symmetric electrolytic cell and the two-dimensional sub-nanopore detection system according to any one of claims 1-6. The functionalized graphene oxide film of the two-dimensional sub-nanopore detection system is clamped between the symmetric electrolytic cells. The symmetric electrolytic cell is filled with an electrolyte, and the electrolyte includes any one of LiCl and LiTf2N.

[0022] Furthermore, the concentration of LiCl or LiTf2N is 0.01M.

[0023] The fourth object of the present invention is to provide a method for detecting nucleic acid molecules using the above nucleic acid molecule detection device, including the following steps:

[0024] Step S1, preparing a sample

[0025] Drop 30 μL of the target nucleic acid molecule solution onto the probe-modified functionalized graphene oxide film obtained in step S24. After standing at room temperature for at least 12 h, wash it with deionized water for 3-5 min and dry it with nitrogen to obtain the functionalized graphene oxide film captured with the target nucleic acid molecule;

[0026] Samples for the standard curve: Prepare hybridization buffer solutions of nucleic acid molecules with different standard concentrations; Prepare functionalized graphene oxide films captured with nucleic acid molecules of different standard concentrations;

[0027] Samples of nucleic acid molecules to be detected: Prepare functionalized graphene oxide films captured with nucleic acid molecules to be detected;

[0028] Step S2, detecting the ion current signal

[0029] Detect the ion current signal of the probe-modified functionalized graphene oxide film, denoted as I0;

[0030] Detect the ion current signals of the functionalized graphene oxide films capturing nucleic acid molecules with different standard concentrations, denoted as I i , the current increment of the ion current signal where i represents the target nucleic acid of different concentrations;

[0031] Detect the ion current signal of the functionalized graphene oxide film capturing the nucleic acid molecule to be detected, denoted as I 样 , the current increment of the ion current signal

[0032] Step S3, drawing a standard curve

[0033] Taking the nucleic acid molecule concentration as the abscissa and the current increment as the ordinate, perform curve fitting according to the logistic regression model to draw a standard curve;

[0034] S4. Implement sample detection

[0035] Put the β of the nucleic acid molecule to be detected 样 into the standard curve drawn in step S3, and calculate the concentration of the nucleic acid molecule in the sample to be detected.

[0036] Compared with the prior art, the beneficial effects brought by the technical solution provided by the present invention are:

[0037] The biomolecule detection system of the two-dimensional sub-nanometer channel at the angstrom level constructed by the present invention uses the layer-by-layer self-assembly method to prepare a graphene oxide film. The layer spacing between multiple adjacent two single-layer graphene oxides constitutes a two-dimensional sub-nanometer channel. The biological target molecule is detected by modifying the probe molecule on the outer surface of the two-dimensional sub-nanometer channel. Since the layer spacing size of the two-dimensional sub-nanometer channel is at the angstrom level, the background of its sensing signal is low, and it can distinguish the solutes in electrolytes of different sizes, obtaining more analyzable signals, thus solving the problem of low detection sensitivity. Modifying the probe molecule on the outer surface to detect the target overcomes the defect of high background signal of the one-dimensional nanochannel, improves the detection sensitivity, and raises the detection limit to 1 aM. The preparation method and detection method of the present invention are simple and have good performance stability. Description of the drawings

[0038] Figure 1 It is a comparison chart of the stability in water of the graphene oxide film prepared in Example 1 and the graphene oxide film prepared in Comparative Example 1;

[0039] Figure 2 It is the AFM diagram and height analysis diagram of single-layer graphene oxide in the graphene oxide films prepared in Example 1 and Comparative Example 1;

[0040] Figure 3 It is the XRD diagram of the graphene oxide films and the support prepared in Example 1 and Comparative Example 1;

[0041] Figure 4a It is the SEM diagram of the cross section of the graphene oxide film prepared in Example 1;

[0042] Figure 4b It is the SEM diagram of the plane of the graphene oxide film prepared in Example 1;

[0043] Figure 4c It is the SEM diagram of the plane of the graphene oxide film modified with the probe mercapto-PNA molecule;

[0044] Figure 4dSEM image of the surface of the graphene oxide thin film modified with the probe thiol-DNA molecule;

[0045] Figure 5 Gel electrophoresis diagram of Example 1;

[0046] Figure 6 Gel electrophoresis diagram of Example 2;

[0047] Figure 7 Current increment diagram for detecting target DNA molecules at different concentrations under the electrolyte LiCl condition in Example 1;

[0048] Figure 8 Current increment diagram for detecting target DNA molecules at different concentrations under the electrolyte LiTf2N condition in Example 2;

[0049] Figure 9 Current increment diagram for detecting target DNA molecules at different concentrations under the electrolyte LiCl condition in Example 3;

[0050] Figure 10 Current increment diagram for detecting target DNA molecules at different concentrations under the electrolyte LiTf2N condition in Example 4.

[0051] Figure 11 Logistic regression linear fitting diagram of target molecule DNA concentration - current increment. Detailed implementation manners

[0052] To make the objectives, technical solutions and advantages of the present invention clearer, the following further describes in detail the specific implementation manners of the present invention with reference to specific embodiments and the accompanying drawings. For those not specified in the embodiments regarding specific technologies or conditions, they shall be carried out according to the technologies or conditions described in the literature in the field or according to the product specifications. For reagents or instruments not specified for the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0053] The base sequences of the probes and target molecules used in this embodiment are shown in Table 1 below.

[0054] Table 1 Sequences of probe thiol-PNA, thiol-DNA and target DNA

[0055]

[0056] Example 1

[0057] Construct a two-dimensional sub-nanopore detection system with a probe of thiol-PNA molecule to detect target DNA molecules

[0058] S1. Prepare a functionalized graphene oxide thin film

[0059] S11. Disperse 0.5 - 1μm flaky single - layer graphene oxide and cross - linker dimethyl silicone in 5 mL of deionized water to obtain a mixed solution A;

[0060] S12. After heating and stirring the mixed solution A obtained in step S11 at a constant temperature of 90 - 110 °C for 5 - 7 h, stop stirring, and then cool the solution to room temperature to obtain a casting solution B;

[0061] S13. Take about 10 μL of the casting solution B obtained in step S12 and drop it on a porous support polyethylene terephthalate. The area of the porous support is 5 mm × 5 mm, the pore diameter is 30 nm, and the pore density is 5×10 7 / cm 2 Naturally deposit at room temperature to obtain a layer - by - layer self - assembled functionalized graphene oxide film.

[0062] S2. Modify the outer surface of the functionalized graphene oxide film with probe molecules

[0063] S21. Sputter metal chromium and metal gold on the graphene oxide film in sequence. The average sputtering rate of the metal is 0.05 nm / s to obtain a functionalized graphene oxide film with an activated outer surface. The thickness of the metal chromium is 10 nm, and the thickness of the metal gold is 10 nm;

[0064] S22. Weigh a certain mass of tris(2 - carboxyethyl)phosphine hydrochloride and dissolve it in 100 μL of tris(hydroxymethyl)aminomethane buffer solution (20 mM, pH = 7.4) to obtain a solution C with a concentration of 30 mM for opening the disulfide bond of the probe;

[0065] S23. Take 10 μL of the solution C obtained in step S22, add 1 μL of 100 μM probe thiol - PNA molecular solution and 89 μL of tris(hydroxymethyl)aminomethane buffer solution, and oscillate and mix well for 3 - 5 min to obtain a probe thiol - PNA molecular solution D with a concentration of 1 μM;

[0066] S24. Take 30 μL of the solution D obtained in step S23 and drop it on the outer - surface - activated functionalized graphene oxide obtained in step (1). After standing at room temperature for 1 h, wash it with deionized water for 3 - 5 min and dry it with nitrogen to obtain a probe - modified functionalized graphene oxide film.

[0067] Use the two - dimensional sub - nanometer pore detection system of this embodiment to detect nucleic acid molecules. In this embodiment, probe thiol - PNA molecules are used to detect target DNA molecules, and the concentrations of the targets are 1 μM, 10 nM, 100 pM, 1 pM, 10 fM, 100 aM, and 1 aM.

[0068] Device for detecting nucleic acid molecules: A self-made symmetric electrolytic cell is used. The functionalized graphene oxide film captured by the target is sandwiched between the electrolytic cells. Approximately 0.5 mL of electrolyte is added to the two-sided electrolytic cells. The electrolyte is LiCl with a concentration of 0.01 M. Two self-made Ag / AgCl electrodes are respectively placed in the two electrolytic cells and connected to an electrochemical workstation with the instrument model CHI650D for I-V curve testing.

[0069] The steps for detecting the target DNA molecule to be detected are as follows:

[0070] (1) Preparation of samples

[0071] Drop 30 μL of target DNA molecule solutions with different concentrations (1 μM, 10 nM, 100 pM, 1 pM, 10 fM, 100 aM, and 1 aM) onto the probe-modified functionalized graphene oxide film obtained in step S24. After standing at room temperature for about 12 h, wash with deionized water for 3 - 5 min and dry with nitrogen to obtain the functionalized graphene oxide film captured by the target.

[0072] (2) Detection of ion current signal

[0073] Detect the ion current signal of the functionalized graphene oxide film, denoted as I0;

[0074] Detect the ion current signal of the functionalized graphene oxide film capturing nucleic acid molecules with different concentrations, denoted as I i , the current increment of the ion current signal where i represents the target nucleic acid with different concentrations;

[0075] (3) Plotting of the standard curve

[0076] Taking the target DNA molecule concentration as the abscissa and the current increment as the ordinate, perform curve fitting according to the logistic regression model to plot the standard curve;

[0077] Example 2

[0078] The implementation operation is the same as that of Example 1, except that the electrolyte is LiTf2N with a concentration of 0.01 M.

[0079] Example 3

[0080] Construct a two-dimensional sub-nanopore detection system with a probe of thiol-DNA molecule to detect the target DNA molecule.

[0081] The implementation operation is the same as that of Example 1, except that the probe molecule is thiol-DNA.

[0082] Example 4

[0083] The implementation operation is the same as that of Example 1, except that the probe molecule is thiol-DNA, the film after modification with the probe molecule is named AuDNA-GO, and the electrolyte is LiTf2N with a concentration of 0.01 M.

[0084] Comparative Example 1

[0085] The implementation operation is the same as that of Example 1, except that the crosslinking agent dimethyl silicone is not added.

[0086] In order to better elaborate that the two-dimensional sub-nanopore detection system of the present invention has low background interference and high detection sensitivity for detecting nucleic acid molecules, the applicant has carried out the following research content:

[0087] As Figure 1 shown, it is a comparison diagram of the stability in water of the graphene oxide film prepared in Example 1 and the graphene oxide film prepared in Comparative Example 1. It can be seen from the figure that when water was just added, the film of Comparative Example 1 was slightly dispersed in water; after half an hour, the film of Comparative Example 1 was significantly dispersed in water, while the film of Example 1 was not dispersed. After 6 hours, most of the film of Comparative Example 1 was dispersed in water and a small part remained on the support; after 48 hours, almost all of the film of Comparative Example 1 was dispersed in water, while the film of Example 1 was still not dispersed. This shows that the functionalized graphene oxide film prepared in Example 1 has good stability in aqueous solution.

[0088] As Figure 2 shown, it is the AFM diagram and height analysis diagram of single-layer graphene oxide in the graphene oxide films prepared in Example 1 and Comparative Example 1. It can be seen from the figure that the thickness of the single-layer graphene oxide functionalized with the crosslinking agent in Example 1 is relatively uniform. According to the height analysis diagram, the thickness of the single-layer functionalized graphene oxide is about 2.5 nm; the thickness of the single-layer graphene oxide in the comparative example is also relatively uniform. From the height analysis diagram, the thickness of the single-layer graphene oxide is about 1.0 nm. This shows that the surface of graphene oxide was successfully modified with the crosslinking agent dimethyl silicone, and the thickness of the crosslinking agent is about 1.5 nm.

[0089] As Figure 3 shown, it is the XRD diagram of the graphene oxide films and the support prepared in Example 1 and Comparative Example 1. It can be seen from the figure that the film of Example 1 has a diffraction characteristic peak only at 10.96°, corresponding to the diffraction peak of the (d 002 ) crystal plane. The interlayer spacing of the functionalized graphene oxide was calculated by the Bragg equation to be The thin film of Comparative Example 1 had diffraction peaks at 10.65° and 12.25°, and the support had a diffraction peak at 12.28°. This indicated that the diffraction peak at 12.25° in the thin film of Comparative Example 1 was the diffraction peak of the support. The XRD diffraction peak of the thin film of Example 1 in the present invention was shifted 0.31° to the right relative to the thin film of Comparative Example 1, which indicated that the layer spacing of the thin film of Example 1 was smaller than that of the thin film of Comparative Example 1. It was illustrated that after the graphene oxide film was functionalized with the crosslinking agent, the layers were closer to each other and not easily dispersed.

[0090] As Figure 4a shown, it was the SEM image of the cross section of the graphene oxide film prepared in Example 1. It could be seen from the figure that the graphene oxide film was stacked neatly and had an ordered structure; as Figure 4b shown, it was the SEM image of the plane of the graphene oxide film prepared in Example 1. It could be seen from the figure that the plane of the graphene oxide film was flat and no large cracks were found, indicating that the graphene oxide film was successfully prepared; as Figure 4c shown, it was the SEM image of the plane of the graphene oxide film modified with the probe mercapto-PNA molecule; as Figure 4d shown, it was the SEM image of the plane of the graphene oxide film modified with the probe mercapto-DNA molecule. It could be seen from the figure that after the probe molecule was modified, the change in the film surface was not obvious compared with the graphene oxide film and it was still relatively flat. This was because the probe molecule was relatively small, only about 4 nm, so it would not change the outer surface structure of the graphene oxide.

[0091] As Figure 5 shown, it was the gel electrophoresis image of Example 1. It could be seen from the figure that there was no band for the neutral probe mercapto-PNA in lane 2; there was only 1 band for the negatively charged target DNA in lane 3. Compared with the indicator band, it was found that the number of bases of the target DNA was about 20 base pairs, which was similar to the number of bases of the sequence listed in Table 1; there were 3 bands for the probe-target binding in lane 4. Compared with the indicator band, it was found that the number of base pairs after the probe-target binding was less than 40 base pairs. The bottom band was the target DNA band, the middle band was between 20 and 40 base pairs, that is, the probe-target binding product, and the top band was due to the fact that a small amount of the mercapto groups in the probe mercapto-PNA were not reduced by TCEP and still formed dimers of PNA with disulfide bonds. At this time, the probe PNA was complementary to the target DNA, that is, the product of the complementary dimer PNA and the target DNA. From the above results, the probe and the target were successfully bound in the present invention.

[0092] As Figure 6As shown, it is the gel electrophoresis diagram of Example 2. It can be seen from the figure that the negatively charged probe thiol-DNA with only 13 bases in No. 4 does not show a band, which is due to the small number of bases and small mass, and under the action of the electric field, it runs out of the gel plate. The bands of No. 1, No. 2, and No. 3 are normal, and the gel electrophoresis results are the same as Figure 5 the results in

[0093] As Figure 7 shown, it is the current increment diagram of detecting target DNA molecules with different concentrations under the condition of electrolyte LiCl in Example 1. It can be seen from the figure that after modifying target DNA molecules with different concentrations, the ionic current has a large increase compared with only modifying the probe thiol-PNA molecules. This is because the negatively charged target DNA molecules provide another self-built electric field for the ions in addition to the externally applied electric field, accelerating the movement rate of the ions in the thin film, that is, the ionic current increases. In addition, through differential analysis, it is found that when detecting high-concentration (greater than 10 nM) target DNA molecules, the difference is obvious, and the P value is less than 0.001, showing 3 stars; when detecting medium and low concentrations (100 aM to 100 pM), the difference is lower than that of high concentrations, and the P value is between 0.001 and 0.01, showing 2 stars. When detecting a concentration of 10 fM, due to the small difference between samples, the error bar is very small. Therefore, when performing differential analysis, the P value is less than 0.001, showing 3 stars; when detecting a low concentration (1 aM), the difference is the lowest, and the P value is between 0.01 and 0.05, showing 1 star. These results are mainly attributed to the decrease in the charge amount on the outer surface of the functionalized graphene oxide thin film and the decrease in the self-built electric field strength after the DNA concentration decreases, and the effect of ion acceleration also decreases accordingly, resulting in a decrease in the ionic current increment. From the results of differential analysis, the detection limit for detecting target DNA molecules is 1 aM.

[0094] As Figure 8 shown, it is the current increment diagram of detecting target DNA molecules with different concentrations under the condition of electrolyte LiTf2N in Example 2. The results of the ionic current increment in the figure are the same as Figure 7 the results in Figure 7 and as the concentration of the detected target DNA molecules decreases, the ionic current increment also decreases, and the principle is the same as Figure 7 . However, the ionic current increment at each concentration is about an order of magnitude larger than that in - which is attributed to the fact that Tf2N - ions are more likely to migrate on organic molecules, so it is easier to pass through the graphene oxide thin film functionalized by the cross-linking agent compared with Cl - . Through differential analysis, it is found that when using LiTf2N electrolyte, the lowest concentration for detecting target DNA molecules is also 1 aM, and the P value at this time is between 0.01 and 0.05, showing 2 stars, which is more obvious than the difference with LiCl.

[0095] As shown Figure 9 in the figure, it is the current increment diagram for detecting target DNA molecules with different concentrations under the condition of electrolyte LiCl in Example 3. Similar to Application Example 1, as the concentration of the detected target DNA molecule decreases, the ion current increment decreases. In addition, through differential analysis, it is found that when the concentration of the target DNA molecule decreases to 10 fM, the current signals of the probe thiol-DNA molecule and the captured target DNA molecule cannot be distinguished, and the detection limit at this time is 1 pM. This phenomenon is mainly attributed to the fact that the probe thiol-DNA molecule carries a small amount of negative charge, which causes an increase in the ion current and cannot be distinguished from the ion current signal after capturing low-concentration target molecules.

[0096] As shown Figure 10 in the figure, it is the current increment diagram for detecting target DNA molecules with different concentrations under the condition of electrolyte LiTf2N in Example 4. Similar to Application Example 1, after capturing the target molecule, the ion current signal increases and decreases as the concentration of the target DNA molecule decreases. According to the results of differential analysis, the lowest concentration for detecting the target DNA molecule is 1 aM.

[0097] As shown Figure 10 in the figure, under the test condition of 0.01 M LiCl, with the nucleic acid molecule concentration as the abscissa and the current increment as the ordinate, a standard curve of concentration-current increment is plotted by curve fitting according to the logistic regression model. It can be seen from the figure that except for the point at 10 nM, the rest of the points are distributed near the curve, indicating that the fitted curve of the present invention has a good linear relationship.

[0098] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A two-dimensional sub-nanopore detection system, characterized in that The two-dimensional sub-nanopore detection system includes a porous support and a functionalized graphene oxide film disposed on the porous support. The porous support is polyethylene terephthalate; the functionalized graphene oxide film is formed by layer-by-layer assembly of multiple sheet-like single-layer graphene oxides. The layer spacing between two adjacent single-layer graphene oxides is at the angstrom level, and the layer spacings between all adjacent two single-layer graphene oxides constitute sub-nanopores; the outer surface of the functionalized graphene oxide film is modified with probe molecules for capturing target molecules, and the probe molecules are thiol-PNA molecules; by detecting the interaction between the probe molecules and the target molecules to be detected, the target molecules to be detected carry their own electric fields, so that the ion current signal of the sub-nanopores of the two-dimensional sub-nanopore detection system is amplified, realizing high-sensitivity detection of the target molecules. The functionalized graphene oxide film is obtained by the following method: S1. Prepare the functionalized graphene oxide film S11. Disperse sheet-like single-layer graphene oxide and cross-linking agent dimethyl silicone in deionized water to obtain a mixed solution A; S12. After the mixed solution A obtained in step S11 is heated and stirred at a constant temperature for a period of time, stop stirring, and then cool the solution to room temperature to obtain a casting solution B; S13. Drop the casting solution B obtained in step S12 onto the porous support polyethylene terephthalate, and naturally deposit it at room temperature to obtain a layer-by-layer self-assembled functionalized graphene oxide film.

2. The two-dimensional sub-nanopore detection system according to claim 1, wherein The modification of the probe molecules on the outer surface of the functionalized graphene oxide film is obtained by the following method: S2. Modify the probe molecules on the outer surface of the sub-nanopores S21. Sputter metal chromium and metal gold on the graphene oxide film in sequence to obtain a functionalized graphene oxide film with an activated outer surface. The thickness of the metal chromium is 5-10 nm, and the thickness of the metal gold is 5-10 nm; S22. Dissolve tris(2-carboxyethyl)phosphine hydrochloride in tris(hydroxymethyl)aminomethane buffer solution to obtain a solution C for opening the disulfide bond of the probe, with a concentration of 30 mM; S23. Take the solution C obtained in step S22, add a certain amount of probe molecule solution and tris(hydroxymethyl)aminomethane buffer solution, and oscillate and mix well for 3-5 min to obtain a probe molecule solution D with a concentration of 0.5-2 M; S24. Take the solution D obtained in step S23, drop it onto the functionalized graphene oxide with an activated outer surface obtained in S21, let it stand at room temperature for at least 1 h, wash it with deionized water for 3-5 min, and dry it with nitrogen to obtain a probe-modified functionalized graphene oxide film.

3. The two-dimensional sub-nanopore detection system according to claim 1, wherein In step S11, the size of the sheet-like single-layer graphene oxide is 0.5-1 μm.

4. The two-dimensional sub-nanopore detection system according to claim 3, characterized in that, The mass ratio of the sheet-like single-layer graphene oxide, cross-linking agent dimethyl silicone and deionized water is 1:(40-60):

285.

5. The two-dimensional sub-nanopore detection system according to claim 1, characterized in that In step S12, the reaction temperature is 90-110 °C, and the reaction time is 5-7 h.

6. The two-dimensional sub-nanopore detection system according to claim 1, characterized in that, In step S13, the area of the porous support is 5 mm × 5 mm, the pore diameter is 30 nm, and the pore density is 5×10 7 / cm 2 .

7. A two-dimensional sub-nanopore detection system according to any one of claims 1-6 is used for qualitative and quantitative analysis of nucleic acid molecules.

8. A nucleic acid molecule detection device, characterized in that, It includes a symmetric electrolytic cell and a two-dimensional sub-nanopore detection system as described in any one of claims 1-6. The functionalized graphene oxide film of the two-dimensional sub-nanopore detection system is clamped between the symmetric electrolytic cells. The symmetric electrolytic cells are filled with an electrolyte, and the electrolyte includes any one of LiCl and LiTf2N.

9. The nucleic acid molecule detection device according to claim 8, wherein, The concentration of the LiCl or LiTf2N is 0.01 M.

10. A method for detecting nucleic acid molecules using the device according to any one of claims 8-9, characterized in that, It includes the following steps: S1. Prepare samples Drop 30 μL of the target nucleic acid molecule solution onto the probe-modified functionalized graphene oxide film obtained in step S24. After standing and reacting at room temperature for at least 12 h, wash it with deionized water for 3-5 min and dry it with nitrogen to obtain the functionalized graphene oxide film capturing the target nucleic acid molecule; Samples for the standard curve: Prepare hybridization buffer solutions of nucleic acid molecules with different standard concentrations; Prepare functionalized graphene oxide films capturing nucleic acid molecules with different standard concentrations; Samples of nucleic acid molecules to be detected: Prepare functionalized graphene oxide films capturing nucleic acid molecules to be detected; S2. Detect the ion current signal Detect the ion current signal of the probe-modified functionalized graphene oxide film, denoted as I0; Detect the ionic current signals of functionalized graphene oxide films that capture nucleic acid molecules of different standard concentrations, denoted as I i , the current increment of the ionic current signal where i represents target nucleic acids of different concentrations; Detect the ionic current signal of the functionalized graphene oxide film that captures the nucleic acid molecule to be measured, denoted as I 样 , the current increment of the ionic current signal S3. Draw the standard curve With the nucleic acid molecule concentration as the abscissa and the current increment as the ordinate, perform curve fitting according to the logistic regression model to draw the standard curve; S4. Perform sample detection Bring the β of the nucleic acid molecule to be tested 样 into the standard curve drawn in step S3, and calculate the concentration of the nucleic acid molecule in the sample to be tested.

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