阅读说明：本技术 一种固态纳米管及其制备方法和应用 (Solid nanotube and preparation method and application thereof ) 是由 金永东 周亚 于 2021-02-03 设计创作，主要内容包括：本发明涉及生物分析技术领域,公开了一种固态纳米管及其制备方法和应用。本发明所述固态纳米管在其内部管口处,围绕固态纳米管内壁修饰有PDA膜。本发明采用一种简便的方法在固态纳米管上原位聚合多巴胺,修饰后的纳米管的直径从90-120纳米左右降低到10纳米以下,由于PDA与寡核苷酸存在π-π共轭和氢键作用,其穿孔速率会大大减缓,同时通过PDA与碱基之间的相互作用区分出相同长度的不同碱基以及通过停留时间来区分不同长度的寡核苷酸,从而达到修饰后固态纳米管分析单个寡聚核苷酸链的长度、碱基、以及修饰基团的目的。(The invention relates to the technical field of biological analysis and discloses a solid-state nanotube and a preparation method and application thereof. The PDA film is modified around the inner wall of the solid nano tube at the inner tube opening of the solid nano tube. The invention adopts a simple method to polymerize dopamine in situ on the solid nanotube, the diameter of the modified nanotube is reduced to below 10nm from about 90-120nm, the perforation rate can be greatly slowed down due to pi-pi conjugation and hydrogen bond action between PDA and oligonucleotide, different bases with the same length can be distinguished through the interaction between PDA and bases, and oligonucleotides with different lengths can be distinguished through retention time, thus achieving the purpose of analyzing the length, the bases and the modified groups of a single oligonucleotide chain by the modified solid nanotube.)

1. A solid nanotube is characterized in that a PDA film is modified around the inner wall of the solid nanotube at the opening of the inner part of the solid nanotube, and the diameter of the modified solid nanotube is below 10 nanometers.

2. The solid-state nanotube of claim 1, wherein the solid-state nanotube is a glass nanotube.

3. Use of the solid-state nanotubes of claim 1 or 2 for the detection of individual oligonucleotides or for the preparation of a product for the detection of individual oligonucleotides.

4. Use of the solid-state nanotubes of claim 1 or 2 for detecting proteins or for preparing products for detecting proteins.

5. The method for preparing solid nanotubes of claim 1, wherein the nanotube opening of the solid nanotubes injected with dopamine aqueous solution is placed in copper sulfate solution to polymerize until the diameter of the solid nanotubes is below 10nm, and the solid nanotubes are polymerized in situ on the inner wall of the nanotube opening by non-covalent bond to form PDA membrane, thus obtaining the solid nanotubes.

6. The method of claim 5, wherein the concentration of said aqueous dopamine solution is 4-8 mg/mL.

7. The method according to claim 5 or 6, wherein the dopamine aqueous solution is a dopamine hydrochloride aqueous solution.

8. The method according to claim 5, wherein the concentration of copper sulfate is 30 mM/L.

9. A method for detecting a single nucleotide, characterized in that a single nucleotide to be detected is detected using the solid-state nanotube according to claim 1 or 2, and the length, base species, and functional group information of the oligonucleotide are analyzed by the difference in the puncture time in the original current.

Technical Field

The invention relates to the technical field of biological analysis, in particular to a solid-state nanotube and a preparation method and application thereof.

Background

Over the past two decades, scientists have developed a large number of DNA detection and analysis methods. Recently, nanopore technologies have been rapidly developed and widely used in bioassays and related assays due to their superior single molecule confinement sensing capabilities. The movement of a single nanoparticle within a nanopore or the passage of a single molecule may cause a significant change in ionic current across the pore. Generally, information on the volume, length, etc. of individual substances and molecules can be analyzed from the magnitude and residence time of the ion current. The nano-pore has wide application in the fields of nano-particle dynamic transmission process research, protein detection, DNA single nucleotide polymorphism detection and the like. However, identification of oligonucleotides based on nanopore technology currently presents several challenges due to their extremely short length and fast translocation speed. A few reports exist for the detection of oligonucleotides using biological nanopores. This is because the biological nanopore is smaller in size, matching that of an oligonucleotide.

However, the biological nanopore is extremely sensitive to conditions such as pH and temperature, which prevents practical application of the biological nanopore under severe conditions, and the existing biological nanopore is basically difficult to directly distinguish single-stranded oligonucleotides. Although solid state nanotubes have the advantages of easy modification, thermal stability, and acid and alkali resistance, the naked solid state nanopore is generally not considered to be well used for detecting single oligonucleotides due to its generally large pore size, mismatch with the size of a single oligonucleotide, and the naked nanotube does not have the ability to specifically recognize a single base.

Disclosure of Invention

In view of the above, the present invention provides a simple method for modifying a solid nanotube and the obtained solid nanotube, which can prolong the perforation time of a single oligonucleotide and interact with the oligonucleotide, so as to analyze information such as the length, the base, and the modification group of the single oligonucleotide chain.

Another object of the present invention is to provide the use of the above-mentioned solid nanotubes for detecting single oligonucleotides or for preparing products for detecting single oligonucleotides.

In order to achieve the above purpose, the invention provides the following technical scheme:

a PDA (poly dopamine) film is modified around the inner wall of a solid nano tube at the inner tube opening of the solid nano tube.

The previous literature reports that the single-stranded DNA perforation time is mostly in microsecond or millisecond time scale, but the single-stranded DNA perforation time is delayed by several orders of magnitude to reach the level of second by in-situ polymerization of PDA at the orifice of the solid nanotube. The reduced perforation rate and the multifunctional properties of PDA enable the detection and base recognition of oligonucleotide chains. The modified solid-state nanotube can be used for analyzing information such as the length, the base, the modifying group and the like of a single oligonucleotide chain.

The solid nanotube prepared by the pipe orifice in-situ polymerization PDA method can reduce the aperture of the bare solid nanotube to below 10 nm. In the specific embodiment of the invention, the solid-state nanotube is a glass nanotube with an initial pore size of 90-120 nm.

The aperture of the solid-state nanotube modified by the method is estimated by an empirical formula according to an I-V curve, the aperture can be calculated to be reduced to below 10nm, the surface of the modified nanopore carries more negative charges, and the rectification shows a more negative trend. Conversely, unmodified nanopores are larger in size or exhibit a less pronounced negative propensity to rectify, and oligonucleotides that pass through the nanopore will fail to detect any perforation events.

Meanwhile, the invention also takes oligonucleotide consisting of 20bp C base as a detection object, and passes through the naked glass nanopore and the modified glass nanopore provided by the invention to detect the original current traces of the naked glass nanopore and the modified glass nanopore. The results show that bare glass nanopores fail to detect any perforation events due to the large pore size and low signal-to-noise ratio. The polydopamine-modified glass nano-pore provided by the invention has small pore diameter, interacts with oligonucleotide, and the perforation time reaches the order of seconds.

Based on the test results, the invention provides the application of the solid nanotube in detecting single oligonucleotide or preparing a product for detecting single oligonucleotide. Meanwhile, the modified solid nanotube can also be used for detecting protein due to the surface multifunctional property thereof, so the invention also provides the application of the solid nanotube in detecting protein or the application in preparing a product for detecting protein.

In addition, the invention also provides a preparation method of the solid-state nanotube, wherein the pipe orifice of the solid-state nanotube injected with dopamine aqueous solution is placed in copper sulfate solution to be polymerized until the diameter of the solid-state nanotube is below 10 nanometers, and the solid-state nanotube is obtained by polymerizing the PDA film on the inner wall of the pipe orifice of the nanotube through non-covalent bond action. Wherein, the non-covalent bond includes metal coordination or chelation, hydrogen bond, accumulation, quinoline charge transfer compound, and the like, dopamine is combined on the substrate to diffuse through the non-covalent bond to form an effective adsorption layer, and then polymerized on the inner surface of the nanopore orifice, when the oligonucleotide passes through the PDA modified nanopore, the perforation rate can be greatly reduced due to pi-pi conjugation and hydrogen bond action between the PDA and the oligonucleotide, and the flow diagram is shown in figure 1.

The concentration of the dopamine aqueous solution and the polymerization time can be determined through tests, a preferable reference is given in the invention, the concentration of the dopamine aqueous solution is 4-8mg/mL, and the polymerization time is more than or equal to 48 h; in a specific embodiment of the invention, the invention polymerizes for 48h at a concentration of 4mg/mL to achieve a reduction in orifice diameter to less than 10 nm.

Preferably, the dopamine aqueous solution is dopamine hydrochloride aqueous solution; the concentration of the copper sulfate is 30 mM/L. The present inventors tried to initiate polymerization of dopamine using oxygen according to the previously reported process, but since the tip of the nanopore is small and it is not easy to inject oxygen, a copper sulfate solution was selected to allow slow polymerization and no oxygen injection was required.

According to the application, the invention provides a method for detecting single nucleotide, which uses the solid-state nanotube as described in claim 1 or 2 to detect the single nucleotide to be detected, and analyzes the length, base type and functional group information of the oligonucleotide through the difference of the perforation time in the original current.

According to the technical scheme, dopamine is polymerized on the solid-state nanotube in situ by a simple method, the diameter of the modified nanotube is reduced to below 10 nanometers from about 90-120 nanometers, the perforation rate of the modified nanotube is greatly reduced due to pi-pi conjugation and hydrogen bond action between PDA and oligonucleotide, different basic groups with the same length are distinguished through the interaction between PDA and the basic groups, and oligonucleotides with different lengths are distinguished through retention time, so that the purpose of analyzing the length, the basic groups and the modified groups of a single oligonucleotide chain by the modified solid-state nanotube is achieved.

Drawings

FIG. 1 is a schematic diagram of the preparation process of polydopamine modified solid nanotubes and the detection of single oligonucleotides according to the present invention;

FIG. 2 is a graph showing current-voltage curves before and after modification of glass nanotubes after dopamine polymerization; a represents the dopamine modified nanotube result, B represents the bare tube result;

FIG. 3 shows the original current traces of the same oligonucleotide passing through a bare glass nanopore (a) and a polydopamine-modified glass nanopore (b), respectively; the ordinate refers to the magnitude of the current change and the abscissa is a time scale.

Detailed Description

The invention discloses a solid-state nanotube modification method, an obtained solid-state nanotube and application, and can be realized by appropriately improving process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the solid state nanotubes and methods of modification and use of the same have been described in the examples, it will be apparent to those skilled in the art that the techniques of the present invention may be practiced and applied by modifying or appropriately combining the solid state nanotubes and methods of preparation and use of the same without departing from the spirit, scope and spirit of the invention.

The solid nanotube provided by the present invention, the preparation method and the application thereof are further described below.

Example 1: preparation of solid nanotubes modified according to the invention

The pipe orifice of the glass solid nanotube (with the diameter of 90-120 nm) injected with 4mg/mL dopamine hydrochloride aqueous solution is placed in 30mM/L copper sulfate solution for 48 hours, and is polymerized into a PDA membrane on the inner wall of the pipe orifice of the nanotube through non-covalent bond action, so that the solid nanotube with the diameter of below 10nm is obtained.

Example 2: comparison of the Nano-solid tube of the invention with bare Nano-solid tube

1. Polydopamine-modified glass nanopore electrochemical test

The diameter of a single base and the current value were calculated from the I-V curve using an oligonucleotide consisting of 20bp bases of C bases to cross the bare glass nanopore and the polydopamine modified glass nanopore (solid state nanotube modified in example 1) as follows:

R＝U/I；

R＝ρl/S；S＝πd²/4；

from the above formula

The size of the modified nanopore can be calculated to obtain the pore diameter of about 1.8nm according to the diameter of 1nm of the single-stranded oligonucleotide and the ratio of the initial current value to the current change value of 3.3.

Therefore, the pore diameter of the nano solid tube is reduced to be less than 10nm after the nano solid tube is modified, the surface of the modified nano hole has more negative charges, and rectification shows a more negative trend. On the contrary, in the bare tube, the current is larger under the same voltage, and the size of the nanopore is correspondingly larger, or the rectification shows a positive tendency, and when the oligonucleotide passes through the nanopore, any perforation event cannot be detected (fig. 2).

2. Detection contrast of Single oligonucleotides

The raw current trace results using an oligonucleotide consisting of a C base of 20bp base through a bare glass nanopore (a in fig. 3) and a polydopamine modified glass nanopore (b in fig. 3) show that the bare glass nanopore fails to detect any perforation event due to the large pore size and low signal-to-noise ratio. The hole diameter of the polydopamine modified glass nano-hole is small, the polydopamine modified glass nano-hole interacts with oligonucleotide, and the perforation time reaches the order of seconds.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.