Functionalized two-dimensional nanopore membrane containing probe modification as well as preparation method and application of functionalized two-dimensional nanopore membrane

Abstract

The invention belongs to the technical field of nano material detection and application, and particularly relates to a probe-modified functionalized two-dimensional nanopore membrane and a preparation method and application thereof.The probe-modified functionalized two-dimensional nanopore membrane comprises a graphene oxide two-dimensional nanopore membrane and a crown ether probe on the graphene oxide two-dimensional nanopore membrane, according to the graphene oxide two-dimensional nanopore membrane, after flaky graphene oxide non-methylated polypeptide with carboxyl on the surface penetrates through the prepared probe-modified two-dimensional functionalized nanopore membrane and is combined with a probe through a hydrogen bond, a pore channel is blocked to cause current change, but methylated polypeptide is not combined with the probe and passes through the pore channel; therefore, the purpose of enriching methylated polypeptide is achieved. The driven solution is collected, the eluent of the functionalized two-dimensional nano-pore membrane is subjected to mass spectrum testing, methylated polypeptide is found in the driven solution, non-methylated polypeptide is found in the eluent of the functionalized two-dimensional nano-pore membrane, and therefore the enrichment effect of the functionalized two-dimensional nano-pore membrane is verified.

Description

Technical Field

[0001] The present invention belongs to the technical field of nanomaterial detection and application, and in particular relates to a functional two-dimensional nanoporous membrane modified with a probe, and a preparation method and application thereof. Background Art

[0002] Protein methylation, one of the most important post-translational modifications, has attracted considerable attention due to its crucial role in numerous biological processes, including DNA repair, RNA processing, transcriptional regulation, and protein phase separation. Recent research evidence suggests that imbalanced histone methylation can contribute to tumor development and progression, with altered methylation levels of certain histones also associated with increased cancer recurrence and poor survival. Therefore, monitoring the status and levels of histone methylation in tumor tissue is crucial for tumor diagnosis and prognosis.

[0003] Currently, there are two main analytical methods for methylation modification. The first is immunoaffinity (antibody-based), which is not only costly but also requires the development of specific antibodies for various methylation sites and modification forms, lacking pan-specificity. The other is chromatography, but this is limited to the analysis of histone samples and is still a long way from being applicable to complex biological samples.

[0004] In recent years, biomimetic nanopores have attracted the attention of many scientists with their advantages such as controllable pore size and strong stability. In addition, the surface of nanopores has different active functional groups, which can be easily connected with probe molecules to achieve various functions, such as single-molecule DNA sequencing, protein conformational characterization, post-translational modification recognition, ligand binding allosteric recognition, and certain molecular mechanism research and applications. Summary of the Invention

[0005] Based on this, it is necessary to provide a functional two-dimensional nanoporous membrane modified with probes and its preparation method and application, which can realize the distinction and enrichment of methylated post-translationally modified peptides and the indirect detection of methylases.

[0006] To achieve the above objectives, the present invention provides a probe-modified functionalized two-dimensional nanoporous membrane, wherein the probe-modified functionalized two-dimensional nanoporous membrane comprises a graphene oxide two-dimensional nanoporous membrane and a crown ether probe on the graphene oxide two-dimensional nanoporous membrane.

[0007] Furthermore, the graphene oxide two-dimensional nanoporous membrane is a sheet of graphene oxide with carboxyl groups on the surface.

[0008] The present invention also provides a method for preparing the probe-modified functionalized two-dimensional nanoporous membrane, comprising the following steps:

[0009] (1) preparing a graphene oxide substrate material into a graphene oxide dispersion;

[0010] (2) modifying the crown ether probe on the graphene oxide surface of the graphene oxide dispersion by an amide reaction to obtain a functionalized graphene oxide dispersion;

[0011] (3) filtering the functionalized graphene oxide dispersion into a solid film by vacuum filtration to obtain a solid film.

[0012] Furthermore, in step (1), the graphene oxide dispersion is prepared by dispersing flaky graphene oxide powder in pure water and stirring the mixture by magnetic force.

[0013] Furthermore, in step (2), the amide reaction is to activate the carboxyl groups on the surface of graphene oxide through EDC and NHS and combine with the crown ether probe containing amino group.

[0014] Furthermore, in step (3), the vacuum filtration is to discharge the liquid in the functionalized graphene oxide dispersion under a negative pressure environment to achieve solid-liquid separation.

[0015] The present invention also provides an application of the functionalized two-dimensional nanopore membrane modified with a probe in distinguishing methylated post-translationally modified polypeptides, wherein the methylated post-translationally modified polypeptides pass through the functionalized two-dimensional nanopore membrane modified with the probe without binding to the probe, while the non-methylated post-translationally modified polypeptides bind to the probe through hydrogen bonds when passing through the two-dimensional functionalized nanopore membrane modified with the probe.

[0016] Furthermore, the method of distinguishing methylated post-translationally modified polypeptides includes adding a methylated modified polypeptide solution to an electrolytic cell, driving the polypeptide through the layer where the crown ether probe is located on the functional two-dimensional nanopore membrane containing the probe modification under the simultaneous current and concentration difference, and then detecting the current change ratio and / or collecting the solution after the driving action for mass spectrometry characterization.

[0017] The present invention also provides an application of the functionalized two-dimensional nanoporous membrane modified with a probe in detecting the efficiency of a methylation enzymatic reaction.

[0018] Furthermore, during the methylation enzymatic reaction, the substrate non-methylated polypeptide is partially converted into a methylated polypeptide. The mixed solution after the enzymatic reaction is added to an electrolytic cell containing the functionalized two-dimensional nanopore membrane modified with the probe. Driven by the current and concentration difference, the incompletely reacted substrate non-methylated polypeptide binds to the probe, blocking the pores and reducing the electrochemical signal. The smaller the degree of reduction in the electrochemical signal, the higher the efficiency of the methylation enzymatic reaction.

[0019] Compared with the prior art, the present invention has the following advantages and technical effects:

[0020] Under actuation, unmethylated polypeptides, when passing through the probe-modified two-dimensional functionalized nanopore membrane prepared by the present invention, hydrogen bond with the probes, blocking the pores and causing a change in current. Methylated polypeptides, however, pass through the pores without binding to the probes, thereby enriching the methylated polypeptides. The post-actuation solution and the eluate from the functionalized two-dimensional nanopore membrane were collected and subjected to mass spectrometry. Methylated polypeptides were detected in the post-actuation solution, while unmethylated polypeptides were detected in the eluate from the functionalized two-dimensional nanopore membrane, thus verifying the enrichment effect.

[0021] By utilizing the difference in the response of the probe-modified two-dimensional functionalized nanopore membrane to non-methylated and methylated peptides, the methylase is indirectly detected by measuring the methylation degree of the substrate peptide before and after the methylase-catalyzed reaction. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] 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. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0023] Figure 1 Schematic diagram of the functionalized two-dimensional nanopore modified with probes prepared in the present invention;

[0024] Figure 2 The plan view and cross-sectional view of the functionalized two-dimensional nanochannel modified with probes of the present invention;

[0025] Figure 3 Schematic diagram of the electrolytic cell used in the present invention;

[0026] Figure 4 is the current change rate (Ratio, R) after the functionalized two-dimensional nanopore containing the crown ether probe prepared in the experimental example of the present invention is combined with the non-methylated polypeptide;

[0027] Figure 5 The R values ​​of the functionalized two-dimensional nanopore containing crown ether probes (GO-Crown) prepared in the experimental example of the present invention after binding to different methylated peptides (non: Un, mono: Mono, di: Di, tri: Tri), and the R values ​​of the two-dimensional nanopore without probes (GO) in the control group after binding to different methylated peptides;

[0028] Figure 6 The mass spectrometry results of the functionalized two-dimensional nanopore containing the crown ether probe prepared in the experimental example of the present invention after binding to the methylated polypeptide and the non-methylated polypeptide;

[0029] Figure 7This is the response curve of the functionalized two-dimensional nanopore containing the crown ether probe prepared in the experimental example of the present invention to the non-methylated polypeptide substrate that was not completely reacted after the enzymatic reaction. DETAILED DESCRIPTION

[0030] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0031] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0032] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0033] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0034] As used herein, "include", "comprising", "having", "containing", etc. are all open-ended terms, meaning including but not limited to. The room temperature described below is 25°C.

[0035] The technical concept of the present invention is to provide a functionalized two-dimensional nanoporous membrane modified with probes and a preparation method thereof, so as to specifically distinguish methylated post-translationally modified polypeptides, thereby achieving the enrichment of methylated polypeptides and the indirect detection of methylases.

[0036] Specifically, the functionalized two-dimensional nanopore membrane containing probe modification of the present invention is a two-dimensional graphene oxide nanopore membrane in which the crown ether probe and the non-methylated polypeptide are combined through hydrogen bond interaction, thereby blocking the pores and causing the electrochemical signals before and after actuation to change, thereby achieving the distinction between methylated post-translationally modified polypeptides.

[0037] The method for preparing a probe-modified functionalized nanoporous membrane of the present invention comprises the following steps:

[0038] (1) Under magnetic stirring, pure water is added to the provided graphene oxide base material to prepare a graphene oxide dispersion of 2 to 4 mg / mL.

[0039] (2) Under magnetic stirring, 40-50 mg / mL EDC (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) and 20-30 mg / mL NHS (N-hydroxysuccinimide) were added to the dispersion of step S1 to activate the carboxyl groups on the surface of graphene oxide.

[0040] (3) Under magnetic stirring, 8-10 mg / mL of a crown ether probe is added to the dispersion of step S2. The amino groups of the crown ether probe react with the carboxyl groups on the surface of the graphene oxide to modify the probe on the graphene oxide sheet.

[0041] (4) Through vacuum filtration technology, with the support of water filter membrane, pure water is discharged under negative pressure environment to achieve solid-liquid separation and obtain a solid membrane.

[0042] The schematic diagram of the functionalized two-dimensional nanopore modified with probe prepared in the present invention is as follows: Figure 1 As shown, the plan view and cross-section view are as follows Figure 2 shown.

[0043] Example 1

[0044] The structure of the crown ether probe used in this embodiment is shown in Figure 1:

[0045]

[0046] The method for preparing a functionalized two-dimensional nanoporous membrane modified with a crown ether probe in this embodiment comprises the following steps:

[0047] S1, magnetic stirring for 12 hours to obtain a 2.5 mg / mL graphene oxide dispersion, followed by the addition of 50 mg / mL EDC and 30 mg / mL NHS to activate the carboxyl groups on the graphene oxide surface;

[0048] S2, adding 10 mg / mL crown ether probe to the dispersion;

[0049] S3, vacuum filtration to form a solid membrane, wherein the pore size of the water filter membrane is 0.45μm and the diameter is 25mm. The pressure of vacuum filtration is less than -0.1MPa. In order to obtain a stable membrane, after the filtration is completed, the membrane is placed in a drying oven at 60℃ and dried for 2h, and then the prepared membrane is peeled off from the water filter membrane.

[0050] This embodiment also provides a method for indirectly detecting methylase using a two-dimensional nanoporous membrane modified with a crown ether probe, comprising the following steps:

[0051] Under the enzymatic reaction, the substrate non-methylated polypeptide is partially converted into methylated polypeptide. The mixture after the enzymatic reaction is added to Figure 3 In the electrolytic cell shown, after actuation, unreacted substrate (unmethylated peptide) binds to the probe, blocking the pores and reducing the electrochemical signal. The smaller the reduction in electrochemical signal, the more efficient the methylation enzymatic reaction.

[0052] Detect the changes in transmembrane ionic current of the two-dimensional nanopore membrane. The detection results of ionic current changes are shown in Figure 4 ,Depend on Figure 4 It can be seen that as the concentration of non-methylated peptide increases, the current change ratio also increases.

[0053] The solution after driving was collected and tested by mass spectrometry. The results are shown in Figure 6 ,Depend on Figure 6 It can be seen that the non-methylated polypeptide binds to the probe and cannot pass through the two-dimensional nanopore membrane, while the methylated polypeptide does not bind to the probe and passes through the two-dimensional nanopore membrane to the other side of the electrolytic cell.

[0054] Comparative Example 1

[0055] The present invention also provides a control test for detecting whether a methylated polypeptide is present using a two-dimensional nanoporous membrane without probe modification. The test method and steps are the same as the detection of polypeptides using the two-dimensional functionalized nanoporous membrane containing probes.

[0056] The results of the control experiment are shown in Figure 5 ,Depend on Figure 5 It can be seen that in the two-dimensional nanopore membrane without probes, neither methylated nor unmethylated peptides will cause changes in the current signal.

[0057] In summary, the probe-modified two-dimensional nanopore membrane provided by the present invention can be used to distinguish and detect methylated polypeptides and indirectly detect methyltransferases. Specifically, the present invention modifies the crown ether probe into the two-dimensional nanopore sheet by covalent action. Due to the non-covalent binding of the target and the crown ether probe molecule, the nanopore is blocked, resulting in a change in the nanopore transmembrane current. The functionalized two-dimensional nanopore membrane modified with the crown ether probe of the present invention can distinguish methylated modified polypeptides, and the enrichment effect of the functionalized two-dimensional nanopore membrane can be verified by auxiliary mass spectrometry results. In addition, by measuring the degree of methylation of the substrate non-methylated polypeptide before and after the enzymatic reaction, indirect detection of the methyltransferase is achieved.

[0058] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. A functionalized two-dimensional nanoporous membrane modified with a probe, characterized in that: The functionalized two-dimensional nanoporous membrane modified with probes comprises a graphene oxide two-dimensional nanoporous membrane and a crown ether probe on the graphene oxide two-dimensional nanoporous membrane.

2. The probe-modified functionalized two-dimensional nanoporous membrane according to claim 1, wherein: The graphene oxide two-dimensional nanoporous membrane is a sheet of graphene oxide with carboxyl groups on the surface.

3. The method for preparing a probe-modified functionalized two-dimensional nanoporous membrane according to any one of claims 1 to 2, wherein: The steps include: (1) preparing a graphene oxide substrate material into a graphene oxide dispersion; (2) modifying the crown ether probe on the graphene oxide surface of the graphene oxide dispersion by an amide reaction to obtain a functionalized graphene oxide dispersion; (3) filtering the functionalized graphene oxide dispersion into a solid film by vacuum filtration to obtain a solid film.

4. The method for preparing a probe-modified functionalized two-dimensional nanoporous membrane according to claim 3, wherein: In step (1), the graphene oxide dispersion is prepared by dispersing flaky graphene oxide powder in pure water and stirring the mixture by magnetic force.

5. The method for preparing a probe-modified functionalized two-dimensional nanoporous membrane according to claim 3, wherein: In step (2), the amide reaction is to activate the carboxyl groups on the surface of graphene oxide by EDC and NHS and combine with the crown ether probe containing amino group.

6. The method for preparing a probe-modified functionalized two-dimensional nanoporous membrane according to claim 3, wherein: In step (3), the vacuum filtration is to discharge the liquid in the functionalized graphene oxide dispersion under a negative pressure environment to achieve solid-liquid separation.

7. Use of the probe-modified functionalized two-dimensional nanoporous membrane according to any one of claims 1 to 2 in distinguishing and enriching methylated post-translationally modified polypeptides, characterized in that: The methylated post-translationally modified polypeptide passes through the probe-modified functional two-dimensional nanopore membrane without binding to the probe, and the non-methylated post-translationally modified polypeptide binds to the probe through hydrogen bonds when passing through the probe-modified two-dimensional functional nanopore membrane.

8. The use according to claim 7, characterized in that The differentiation comprises adding a methylated modified polypeptide solution to an electrolytic cell, driving the polypeptide through the layer where the crown ether probe is located on the probe-modified functional two-dimensional nanopore membrane according to any one of claims 1 to 2 under the simultaneous current and concentration difference, and then detecting the current change ratio and / or collecting the solution after the driving action for mass spectrometry characterization.

9. Use of the probe-modified functionalized two-dimensional nanoporous membrane according to any one of claims 1 to 2 in detecting the efficiency of a methylation enzymatic reaction.

10. The use according to claim 7, characterized in that During the methylation enzymatic reaction, the substrate non-methylated polypeptide is partially converted into a methylated polypeptide. The mixed solution after the enzymatic reaction is added to an electrolytic cell containing the probe-modified functionalized two-dimensional nanoporous membrane according to any one of claims 1-2. Under the simultaneous drive of current and concentration difference, the incompletely reacted substrate non-methylated polypeptide binds to the probe, blocking the pores and reducing the electrochemical signal. The smaller the degree of reduction in the electrochemical signal, the higher the efficiency of the methylation enzymatic reaction.

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