A multi-walled carbon nanotube biosensor and its preparation method and application

Abstract

The present invention discloses a biosensor based on multi-walled carbon nanotubes, its preparation method, and application. The sensor includes a phospholipid bilayer and three sizes of carbon nanotubes. The outer carbon nanotubes are injected into a solution via a syringe. As ion channels form, the middle carbon nanotubes and inner carbon nanotubes are injected in sequence. The smaller diameter carbon nanotubes are captured by the larger diameter carbon nanotubes due to van der Waals energy, and finally a multi-walled carbon nanotube biosensor is assembled. By adding a polypeptide to the solution, the inner carbon nanotubes can be pulled to different lengths through the van der Waals interaction between different amino acids and the inner carbon nanotubes, thereby identifying amino acid sites on the polypeptide and amino acids at specific sites. The present invention has strong physical sensitivity to subtle changes in single molecules, does not require any chemical modification, and has high redesign flexibility for specific applications, and can provide sufficient resolution to identify and distinguish amino acids in real time.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a multi-walled carbon nanotube biosensor and a preparation method and application thereof. Background Art

[0002] Proteins are structural elements in all living cells, responsible for biological structure and the balance of internal systems. They play a key role in biological processes such as basic metabolism, DNA replication, and cell motility. The unique function of a particular protein is often determined by the amino acid sequence that constitutes its primary structure, which is naturally synthesized from the information encoded in genes. Protein sequencing will provide important information for disease diagnosis and treatment.

[0003] In proteomics, Edman degradation and mass spectrometry are two commonly used methods for protein detection and sequencing. However, these techniques still have limitations because they require sufficiently pure samples, have low throughput, cannot analyze multiple proteins simultaneously, are time-consuming, and are difficult to integrate into portable devices. Therefore, people have to use other technologies such as tunneling current and fluorescent labeling. Although tunneling current technology was proposed earlier, the combination of each amino acid at the junction can be oriented in multiple conformations, thereby inducing different levels of current signal. Therefore, the passage of proteins through the gap between the two electrodes requires strict control to successfully measure effective tunneling current. Moreover, the subtle differences between more than twenty amino acids also increase the difficulty of distinguishing between them. Optical imaging has the characteristics of high-throughput detection, but fluorescent labeling may affect the inherent properties of protein molecules, hindering the natural characterization of molecules, and there is a lack of fluorophores that can label more than twenty amino acids.

[0004] In contrast, nanopores, as versatile molecular sensors, offer great potential for low-cost, high-throughput applications and have been widely used in molecular detection and sequencing. Carbon nanotubes (CNTs) possess an ideal tubular structure, high strength, thermal and electrical conductivity, and unique electronic properties, making them suitable for a wide range of applications. Studies have shown that short CNTs, with lengths comparable to the thickness of a lipid bilayer, can spontaneously insert into living cell membranes and lipid bilayers. These CNTs inserted into membranes can form channels for use as biosensors. When molecules are transported through CNTs, they may interact non-covalently with the inner wall of the CNTs, reducing translocation rates and generating distinguishable modulations of ionic currents. However, ionic currents alone are currently unable to distinguish between multiple amino acids, accurately read the sites of polypeptide residues, or identify amino acids at specific sites. Therefore, there is an urgent need to develop new technologies to enable rapid and accurate gene sequencing. Summary of the Invention

[0005] To solve the above problems, the present invention discloses a multi-walled carbon nanotube biosensor. By adding a polypeptide to the solution, the inner carbon nanotube can be pulled to different lengths through the van der Waals interaction between different amino acids and the inner carbon nanotube, thereby identifying the amino acid sites on the polypeptide and the amino acids at specific sites.

[0006] The biosensor includes a substrate, a lipid bilayer, an outer carbon nanotube, an intermediate carbon nanotube disposed in the outer carbon nanotube and capable of reciprocating along the outer carbon nanotube, an inner carbon nanotube disposed in the intermediate carbon nanotube and capable of reciprocating along the intermediate carbon nanotube, a cis side, a trans side, a working electrode, a ground electrode, a probe wire, and a fluorescence microscope;

[0007] The same electrolyte solution is distributed in the cis side and the trans side. Here, monovalent cations are selected as the main ion species, and the monovalent cations include but are not limited to K + 、L i + and Na + ; The cis side and the trans side are connected through a micron channel of the substrate.

[0008] The outer carbon nanotubes are inserted into the lipid bilayer; the middle carbon nanotubes are confined in the outer carbon nanotubes due to the van der Waals force between them and the outer carbon nanotubes; the inner carbon nanotubes are confined in the middle carbon nanotubes due to the van der Waals force between them, the outer carbon nanotubes and the middle carbon nanotubes.

[0009] In the application of the multi-walled carbon nanotube biosensor in carbon nanotube pore detection technology, the analyte to be detected is one or more of a polymer, a protein, a polypeptide, a peptide, a drug or a diagnostic agent; the analyte is detected through the carbon nanotube pore by electrophoresis or electroosmosis;

[0010] In the application of the multi-walled carbon nanotube biosensor in carbon nanotube pore detection technology, when the analyte is a polypeptide, the specific steps are as follows:

[0011] (1) contacting the polypeptide with the carbon nanotube nanopore, so that the polypeptide moves relative to the carbon nanotube nanopore;

[0012] (2) When the polypeptide moves relative to the nanopore of the carbon nanotube, the van der Waals energy between the polypeptide and the inner carbon nanotube drives the inner carbon nanotube to move. When the inner carbon nanotube is pulled to the point where it is about to leave the middle carbon nanotube, the inner carbon nanotube drives the middle carbon nanotube to move, and the displacement of the middle carbon nanotube is measured, wherein the measurement indicates one or more characteristics of the polypeptide, and thereby characterizes the amino acid sites of the polypeptide and the amino acids at specific sites.

[0013] (3) When the polypeptide leaves the carbon nanotube pore, the carbon nanotube will return to its original position due to the van der Waals energy between the carbon nanotubes. When the next polypeptide moves through the carbon nanotube nanopore, the carbon nanotube will perform the next measurement.

[0014] The present invention also discloses a method for preparing a multi-walled carbon nanotube biosensor, comprising the following steps:

[0015] (4) Preparation of outer carbon nanotubes: The outer nanotubes are prepared by chemical vapor deposition or electrochemical method, with a length of 5-6 nm and a diameter of

[0016] 1.2-1.4nm;

[0017] (5) Preparation of intermediate carbon nanotubes: The intermediate nanotubes are prepared by chemical vapor deposition or electrochemical methods for preparing nanotubes, and the length of the intermediate nanotubes is 5-6 nm and the diameter is 1.8-2.2 nm;

[0018] (6) Preparation of inner carbon nanotubes: inner nanotubes are prepared by chemical vapor deposition or electrochemical methods for preparing nanotubes, with a length of 0.8-1.2 nm and a diameter of 3.2-3.6 nm;

[0019] (7) performing fluorescence treatment on the outer carbon nanotubes, the middle carbon nanotubes, and the inner carbon nanotubes, respectively, by first treating the carbon nanotubes with a surfactant;

[0020] After functional modification, a suitable fluorescent dye is selected to react with the surface of the carbon nanotube; the reaction product is purified and separated, and it is confirmed that the fluorescent marker has been successfully bound to the surface of the carbon nanotube;

[0021] (8) Construction of phospholipid membrane system: The construction method of phospholipid membrane can refer to

[0022] Montal-Mueller method, thereby forming a stable lipid bilayer membrane on the micropores of the substrate;

[0023] (9) Constructing multi-walled carbon nanotube nanochannels: insert the outer carbon nanotubes into the lipid bilayer through a micro syringe. When a current step occurs, it indicates that the carbon nanotubes have been successfully inserted into the lipid layer. When the current changes steadily, inject the middle carbon nanotubes into the substrate channel through a micro syringe. When the middle carbon nanotubes move around the outer carbon nanotubes, they will be captured by the outer carbon nanotubes to form double-walled carbon nanotubes. When the current becomes smaller, it means that double-walled carbon nanotubes have been formed. When the current changes steadily, inject the inner carbon nanotubes into the substrate channel through a micro syringe. When the inner carbon nanotubes move around the middle carbon nanotubes, they will be captured by the middle carbon nanotubes to form triple-walled carbon nanotubes. When the current becomes smaller, it indicates that the biosensor is successfully assembled.

[0024] (10) dissolving the obtained detection object in an electrolyte solution in the cis side;

[0025] (11) applying a voltage to the electrolyte solution through electrodes a and b, so that the detection object can enter the multi-walled carbon nanotube by electrophoresis or electroosmosis and produce relative movement in the pores of the sensing area of ​​the carbon nanotube, generating a corresponding specific blocking current by applying voltage, and observing the intensity of fluorescence under a fluorescence microscope to characterize the displacement of the middle layer carbon nanotube and the inner layer carbon nanotube pulled by the polypeptide;

[0026] Among them, the characteristics obtained during the detection process, such as blocking current and fluorescence intensity, include indicating whether the peptide has passed through, different amino acid sites of the peptide, and the type of amino acid at a specific site. The data on the change of fluorescence intensity over time will vary depending on the type of amino acid passing through.

[0027] Beneficial effects of this hair

[0028] 1. The sensor in the biosensor of the present invention is the inner carbon nanotube. The size of the sensing area can be controlled by adjusting parameters such as the length or diameter of the carbon nanotube. There is no need to redesign the experimental equipment, only the sample preparation of the carbon nanotube needs to be changed, which has high design flexibility.

[0029] 2. The nanopores of the present invention are composed of carbon nanotubes. Due to the strong van der Waals interaction energy between the protein molecules to be tested and the middle layer of carbon nanotubes, this reduces the speed of the protein molecules passing through the pores to a certain extent, thereby improving the detection accuracy of protein molecules.

[0030] 3. The present invention detects protein molecules based on translocations caused by protein molecules pulling the carbon nanotubes along the pore axis. This is primarily due to the varying van der Waals forces between the side chains of different amino acids and the inner wall of the carbon nanotubes. Therefore, when the protein molecules to be tested move into the sensing region, different amino acids pull the carbon nanotubes to different displacements, thereby enabling sequencing. Thanks to the ideal, smooth tubular structure of the carbon nanotubes, amino acids of different sizes create steric hindrance when moving through the sensing region, further enhancing the discrimination between different amino acids and improving the accuracy of protein detection.

[0031] 4. The sequencing method of the present invention can not only distinguish different amino acids, but also identify different amino acid sites on protein molecules and the types of amino acids at specific sites, especially the distinction between isomeric amino acids.

[0032] 5. The method of the present invention continues the characteristics of nanopore sequencing, namely, low cost and strong physical sensitivity to subtle changes in single molecules, without the need for any other operations on the protein molecules to be tested or the biosensor, such as chemical modification. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic structural diagram of the multi-walled carbon nanotube biosensor device proposed in Example 1 of the present invention;

[0034] Figure 2 The electrostatic potential distribution diagram of the nanopore of the multi-walled carbon nanotube biosensor in buffer a / buffer b of 2M KCl / 2MKCl and voltage of 1V;

[0035] Figure 3 The displacement map of the inner carbon nanotube when the multi-walled carbon nanotube biosensor is pulled when sequencing a homogeneous peptide (sequence is N'-TTTTTTTTTTTT-'C);

[0036] Figure 4 This is the displacement diagram of the inner carbon nanotube being pulled when the multi-walled carbon nanotube biosensor sequences the peptide (sequence is N'-TTTTTLLTTTTT-'C);

[0037] Figure 5 This is the displacement map of the inner carbon nanotube being pulled when the multi-walled carbon nanotube biosensor sequences the peptide (sequence is N'-TTTTT II TTTTT-'C);

[0038] Figure 6 This is the displacement diagram of the inner carbon nanotube being pulled when the multi-walled carbon nanotube biosensor sequences the peptide (sequence is N'-TLLTTTTTTTTT-'C);

[0039] Figure 7This is the displacement diagram of the inner carbon nanotube being pulled when the multi-walled carbon nanotube biosensor sequences the peptide (sequence is N'-TTTTTTTTTLLT-'C);

[0040] Figure 8 This is the displacement diagram of the inner carbon nanotube being pulled when the multi-walled carbon nanotube biosensor sequences the peptide (sequence is N'-TII TTTTTTLLT-'C). DETAILED DESCRIPTION

[0041] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following specific embodiments are intended only to illustrate the present invention and are not intended to limit the scope of the present invention. It should be noted that the terms "front," "rear," "left," "right," "up," and "down" used in the following description refer to directions in the accompanying drawings, and the terms "inward" and "outward" refer to directions toward or away from the geometric center of a particular component, respectively.

[0042] See also Figure 1 The present invention provides a multi-walled carbon nanotube biosensor, comprising a ground electrode 10, a working electrode 1 8, a working electrode 2 9, a cis-side 6, a trans-side 7, an outer carbon nanotube 3, an intermediate carbon nanotube 4 disposed within the outer carbon nanotube 3 and capable of reciprocating along the outer carbon nanotube 3, an inner carbon nanotube 5 disposed within the intermediate carbon nanotube 3 and capable of reciprocating along the intermediate carbon nanotube 3, a fluorescence microscope 13, a lipid bilayer 2, and a substrate 1. The cis-side 6 and trans-side 7 are each filled with an electrolyte solution. The ground electrode 10 is connected to the electrolyte solution on the cis-side 6 and connected to the ground via a wire. The working electrode 2 9 is connected to the electrolyte solution on the trans-side 7 and is connected to a power supply and an electrical signal acquisition device via a probe 11 and a wire 12. The cis-side 6 and trans-side 7 are connected via a micron channel in the substrate 1. These micron channels are typically used to support the lipid bilayer membrane and construct the carbon nanotube pores. The cis side 6 and the trans side 7 together form a detection cell, which can hold the analyte and the electrolyte solution and integrate a signal acquisition device required for carbon nanotube nanopore detection, wherein the signal acquisition device includes an analog-to-digital converter and a signal amplifier.

[0043] The same electrolyte solution is distributed in the cis side and the trans side of the present invention, wherein monovalent cations are selected as the main ion species, including but not limited to K + 、L i + and Na + .

[0044] The present invention selects a polypeptide as the object to be detected. When the polypeptide moves relative to the carbon nanotube nanopore, the van der Waals energy between the polypeptide and the inner carbon nanotube drives the inner carbon nanotube to move. When the inner carbon nanotube is pulled to the point where it is about to leave the middle carbon nanotube, the inner carbon nanotube drives the middle carbon nanotube to move. The displacement of the middle carbon nanotube is measured, wherein the measurement indicates one or more characteristics of the polypeptide, and thereby characterizes the amino acid sites of the polypeptide and the amino acids at specific sites.

[0045] When the polypeptide leaves the carbon nanotube, the carbon nanotube will return to its original position due to the van der Waals energy between the carbon nanotubes. When the next polypeptide moves through the nanopore of the carbon nanotube, the carbon nanotube will perform the next measurement.

[0046] Under the influence of an external electric field, cations and water molecules on the cis and trans sides will undergo directional movement. Driven by electroosmosis or electrophoresis, the relative movement of peptides dissolved in the electrolyte solution and the carbon nanotube pores can be controlled. A fluorescence microscope located above the trans side will monitor the changes in the fluorescence intensity of the fluorescent carbon nanotubes in real time.

[0047] The present invention provides a multi-walled carbon nanotube biosensor as described above, and also provides a method for detecting polypeptides based on the device, the method comprising the following steps:

[0048] (1) Preparation of outer carbon nanotubes: The outer carbon nanotubes are prepared by chemical vapor deposition or electrochemical method. The length of the outer carbon nanotubes is

[0049] 5-6nm, diameter 1.2-1.4nm;

[0050] (2) Preparation of intermediate carbon nanotubes: The intermediate carbon nanotubes are prepared by chemical vapor deposition or electrochemical methods for preparing carbon nanotubes, and the length of the intermediate carbon nanotubes is 5-6 nm and the diameter is 1.8-2.2 nm;

[0051] (3) Preparation of inner carbon nanotubes: The inner carbon nanotubes are prepared by chemical vapor deposition or electrochemical method. The length of the inner carbon nanotubes is

[0052] 0.8-1.2nm, diameter 3.2-3.6nm;

[0053] (4) performing fluorescence treatment on the outer carbon nanotubes, the middle carbon nanotubes, and the inner carbon nanotubes, respectively, by: first treating the carbon nanotubes with a surfactant; then performing functional modification on the carbon nanotubes; selecting a suitable fluorescent dye to react with the surface of the carbon nanotubes; purifying and separating the reaction products, and confirming that the fluorescent marker has been successfully bound to the surface of the carbon nanotubes;

[0054] (5) Construction of phospholipid membrane system: The construction method of phospholipid membrane can refer to

[0055] Montal-Mueller method, thereby forming a stable lipid bilayer membrane on the micropores of the substrate;

[0056] (6) Constructing multi-walled carbon nanotube nanopores: insert the outer carbon nanotubes into the lipid bilayer through a micro syringe. When a current step occurs, it means that the carbon nanotubes have been successfully inserted into the lipid layer. When the current changes steadily, the middle carbon nanotubes are injected into the substrate pores through a micro syringe. When the middle carbon nanotubes move around the outer carbon nanotubes, they are captured by the outer carbon nanotubes to form double-walled carbon nanotubes. When the current becomes smaller, it means that double-walled carbon nanotubes are formed. When the current changes steadily, the inner carbon nanotubes are injected into the substrate pores through a micro syringe. When the inner carbon nanotubes move around the middle carbon nanotubes, they are captured by the middle carbon nanotubes to form triple-walled carbon nanotubes. When the current becomes smaller, it means that the biosensor is successfully assembled.

[0057] (7) selecting the polypeptide to be tested, wherein the tested polypeptide includes different homogeneous peptides, amino acid mutations at different sites, different amino acid mutations at a specific site, and different sites and different amino acid mutations on the same polypeptide;

[0058] (8) dissolving the detection object obtained in step S37 in the electrolyte solution in the cis side;

[0059] (9) applying a voltage to the electrolyte solution through the working electrode, so that the detection object can enter the multi-walled carbon nanotube by electrophoresis or electroosmosis and produce relative movement in the pores of the sensing area of ​​the carbon nanotube, generating a corresponding specific blocking current by applying a voltage, and observing the intensity of fluorescence under a fluorescence microscope to characterize the displacement of the middle layer carbon nanotube and the inner layer carbon nanotube pulled by the polypeptide;

[0060] Among them, for the characteristics obtained during the detection process, such as blocking current and fluorescence intensity; characterization includes indicating whether the polypeptide passes through, different amino acid sites of the polypeptide, and the amino acid type at a specific site. Figure 8 As shown, the data on the temporal change of fluorescence intensity show differences depending on the type of amino acid passing through.

[0061] Next, the effects of the present invention will be further described with reference to specific examples:

[0062] Example 1:

[0063] Multi-walled carbon nanotube biosensor for detection of homogeneous peptides.

[0064] A homogeneous peptide (sequence: N'-TTTTTTTTTTTT-'C) was detected using a multi-walled carbon nanotube biosensor using 2M KCl as both the cis- and trans-side electrolyte solutions at a voltage of 140mV. Based on the displacement and statistics of the middle-layer carbon nanotubes, it was found that when the homogeneous peptide moved relative to the carbon nanotube sensing channel, the middle-layer carbon nanotubes were significantly pulled. Since the amino acids on the homogeneous peptides were identical, the distances they were pulled were similar. When the homogeneous peptide left the carbon nanotube channel, the middle-layer carbon nanotubes returned to their original position. Figure 3 .

[0065] Example 2:

[0066] Multi-walled carbon nanotube biosensors detect different amino acid mutations at the same site.

[0067] The cis-side and trans-side electrolyte solutions were both 2MKCl, the voltage was 140mV, and the multi-walled carbon nanotube biosensor was used to detect peptides with different mutation sites (sequences are N'-TTTTTLLTTTTT-'C and N'-TTTTT II TTTTT-'C). According to the displacement and statistics of the middle layer carbon nanotubes, when the peptide moves relatively through the carbon nanotube sensing channel, the middle layer carbon nanotubes are significantly pulled. When the mutation site moves to the inner layer carbon nanotube sensing area, the displacement of the middle layer carbon nanotubes becomes larger. Figure 4 、 5 As shown, the carbon nanotubes exhibit different displacement levels for Thr and Leu on the same peptide, and also for different amino acid Ile mutations at the same site, demonstrating that multi-walled carbon nanotube biosensors can distinguish different amino acids at specific sites. When the peptide leaves the carbon nanotube pore, the middle carbon nanotube returns to its original position.

[0068] Example 3:

[0069] Multi-walled carbon nanotube biosensors detect amino acid mutations at different sites.

[0070] The cis-side and trans-side electrolyte solutions were both 2MKCl and the voltage was 1V. The multi-walled carbon nanotube biosensor was used to detect peptides with different mutation sites (sequences are N'-TLLTTTTTTTTT-'C and N'-TTTTTTTTTLLT-'C). According to the displacement and statistics of the middle layer carbon nanotubes, when the peptide moves relatively through the carbon nanotube sensing channel, the middle layer carbon nanotubes are significantly pulled. When different mutation sites move to the inner layer carbon nanotube sensing area, the displacement of the middle layer carbon nanotubes becomes larger. Figure 6 、 7As shown, the carbon nanotubes exhibit different displacement levels for Thr and Leu on the same peptide, and the displacement changes at different times for amino acid mutations at different sites, indicating that the multi-walled carbon nanotube biosensor can distinguish amino acids at different sites. When the peptide leaves the carbon nanotube channel, the middle carbon nanotube returns to its original position.

[0071] Example 4:

[0072] Multi-walled carbon nanotube biosensors detect mutations at different sites and in different categories of amino acids.

[0073] Using 2M KCl as the electrolyte solution on both the cis and trans sides, and a voltage of 140mV, the multi-walled carbon nanotube biosensor was used to detect peptides with different mutation sites (sequence: N'-TII TTTTTTLLT-'C). Based on the displacement and statistics of the middle carbon nanotube, it can be seen that when the peptide moves relative to the carbon nanotube sensing channel, the middle carbon nanotube is significantly pulled. When different mutation sites move to the inner carbon nanotube sensing area, the displacement of the middle carbon nanotube becomes larger. Figure 8 As shown, the carbon nanotubes exhibit different displacement levels for Thr, Leu, and Ile on the same peptide, and the displacement changes at different times for amino acid mutations at different sites, demonstrating that the multi-walled carbon nanotube biosensor can distinguish different amino acids at different sites on the same peptide. When the peptide leaves the carbon nanotube pore, the middle carbon nanotube returns to its original position.

[0074] Those skilled in the art will understand that the foregoing descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will be able to modify the technical solutions described in the foregoing embodiments or substitute equivalents for some of the technical features. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included within the scope of protection of the present invention.

[0075] The technical means disclosed in the solution of the present invention are not limited to the technical means disclosed in the above-mentioned embodiment, but also include technical solutions composed of any combination of the above technical features.

Claims

1. A multi-walled carbon nanotube biosensor, characterized by: The invention comprises a substrate (1), a lipid bilayer (2), an outer carbon nanotube (3), an intermediate carbon nanotube (4) arranged in the outer carbon nanotube (3) and reciprocating along the outer carbon nanotube (3), an inner carbon nanotube (5) arranged in the intermediate carbon nanotube (4) and reciprocating along the intermediate carbon nanotube (4), a cis side (6), a trans side (7), a working electrode 1 (8), a working electrode 2 (9), a ground electrode (10), a probe (11), a wire (12) and a fluorescence microscope (13); the cis side (6) is connected to the trans side (7) through a nanochannel of the carbon nanotube; the ground electrode (10) is connected to the electrolyte solution of the cis side (6); and the fluorescence microscope located above the trans side (7) detects changes in the fluorescence intensity of the fluorescent carbon nanotube in real time.

2. The multi-walled carbon nanotube biosensor according to claim 1, characterized in that: The same electrolyte solution is distributed in the cis side (6) and the trans side (7).

3. The multi-walled carbon nanotube biosensor according to claim 2, characterized in that: Monovalent cations were selected as the main ion species in the electrolyte solution.

4. The multi-walled carbon nanotube biosensor according to claim 1, characterized in that: The intermediate layer carbon nanotubes (4) are confined in the outer layer carbon nanotubes (3) due to the van der Waals force between the intermediate layer carbon nanotubes (4) and the outer layer carbon nanotubes (3).

5. The multi-walled carbon nanotube biosensor according to claim 1, characterized in that: The inner layer carbon nanotubes (5) are confined in the middle layer carbon nanotubes (4) due to the van der Waals force between the inner layer carbon nanotubes (5), the outer layer carbon nanotubes (3) and the middle layer carbon nanotubes (4).

6. Application of the multi-walled carbon nanotube biosensor according to claim 1 in carbon nanotube pore detection technology, characterized in that: The analyte to be detected is one or more of a polymer, a protein, a polypeptide, a drug or a diagnostic agent; the analyte is detected by passing through the pores of the carbon nanotubes through electrophoresis or electroosmosis.

7. Application of the multi-walled carbon nanotube biosensor according to claim 6 in carbon nanotube pore detection technology, characterized in that: When the analyte is a polypeptide, the specific steps are as follows: bringing the polypeptide into contact with the carbon nanotube nanopore and causing it to move relative to the carbon nanotube nanopore; When the polypeptide moves relative to the carbon nanotube nanopore, the van der Waals energy between the polypeptide and the inner carbon nanotube drives the inner carbon nanotube to move. When the inner carbon nanotube is pulled to the point where it is about to leave the middle carbon nanotube, the inner carbon nanotube drives the middle carbon nanotube to move. The displacement of the middle carbon nanotube is measured, wherein the measurement indicates one or more characteristics of the polypeptide and thereby characterizes the amino acid sites of the polypeptide and the amino acids at specific sites. When the polypeptide leaves the carbon nanotube pore, the carbon nanotube will return to its original position due to the van der Waals energy between the carbon nanotubes. When the next polypeptide moves through the carbon nanotube pore, the carbon nanotube will perform the next measurement.

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