Nanopore probe utilizing host-guest interaction and preparation method and application thereof

Abstract

The invention discloses a nanopore probe utilizing interaction of a subject and an object as well as a preparation method and application of the nanopore probe. The nanopore probe is composed of a guest molecule, a recognition site and a traction sequence, wherein the guest molecule is used for being combined with the host molecule, so that the displacement time of the probe in the nanopore is slowed down while the interaction between the probe and the nanopore is enhanced; the traction sequence provides power for the whole probe molecule to enter a nanopore detection area under the driving of electrophoretic force; the recognition sites can be used for placing various micromolecular to-be-detected objects (such as amino acids, side chain modified amino acids, nucleotides, neurotransmitters and the like). The probe is simple in structure, small in sample quantity demand, high in detection sensitivity, simple in operation method, stable and uniform in output signal, and extremely high in distinguishing capability on 20 amino acids and post-translational modifications. By taking the probe # imgabs0 # host molecule compound as an example, twenty different amino acids can be distinguished by the method.

Description

Technical Field

[0001] The present invention belongs to the fields of amino acid recognition, protein sequencing, proteomics research and disease diagnosis, and specifically relates to a nanopore probe utilizing host-guest interaction and a preparation method and application thereof. Background Art

[0002] Proteins play a large number of functions in organisms, including DNA replication, catalyzing metabolic reactions in organisms, acting as messengers between cells, maintaining acid-base balance and fluid balance in organisms, and providing stable structures for cells and organisms. They are composed of long amino acid chains, the sequence of which is determined by the nucleotide sequence of the gene. The amino acid chains fold into specific three-dimensional structures to determine their physiological activity. However, gene sequences cannot directly encode information about protein abundance, post-translational modifications or shearing. Even slight changes in the main protein sequence may have relevant effects. Therefore, proteomics research is in great need of an efficient and economical protein sequencing and post-translational modification detection strategy. Nanopore technology, due to its successful application in DNA sequencing, also has great potential in the field of protein sequencing. In the field of protein sequencing, there are three main challenges: protein unfolding, controllable perforation of polypeptide chains, and recognition of single amino acids. Currently, the academic community usually uses reverse electrophoretic force and electroosmotic flow to control the displacement speed of the analyte in the nanopore. At the same time, the nanopore is site-directed mutated or modified to enhance the interaction between the nanopore and the analyte, thereby slowing down the penetration time of the analyte, ultimately obtaining a recognition signal with longer duration and more obvious characteristics.

[0003] In supramolecular chemistry, host-guest chemistry describes complexes composed of two or more molecules or ions that are held together by forces other than covalent bonds in a unique structural relationship. Such non-covalent interactions include ionic bonds, hydrogen bonds, van der Waals forces, and hydrophobic interactions. Under such interactions, host-guest molecules can maintain a relatively moderate interaction strength. When applied to the nanopore detection process, they can not only produce a distinctive and long-lasting recognition signal, but also release their interaction under the action of external forces, which allows the simultaneous detection of multiple probes during the nanopore detection process. Therefore, applying host-guest interactions to the design of nanopore probes has great detection potential. Summary of the invention

[0004] The purpose of the present invention is to provide a nanopore probe that utilizes host-guest interaction, which not only enhances the interaction between the probe and the nanopore, but also slows down the displacement time of the probe in the nanopore, thereby increasing the detection capability of the nanopore and further performing qualitative and / or quantitative analysis of target molecules.

[0005] In the first aspect, the present invention provides a nanopore probe utilizing host-guest interaction, wherein the nanopore probe is composed of a guest molecule, a recognition site, and a traction sequence; wherein the guest molecule is used to bind to the host molecule, thereby enhancing the interaction between the probe and the nanopore while slowing down the displacement time of the probe in the nanopore; the traction sequence provides the power for the entire probe molecule to be driven by the electrophoretic force into the nanopore detection area; the recognition site can be used to place various small molecule analytes (such as amino acids, side-chain modified amino acids, nucleotides, neurotransmitters, etc.).

[0006] Furthermore, the guest molecule on the probe is selected from at least one of ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives.

[0007] Furthermore, the traction sequence on the probe is selected from: polynucleotides (such as PolyT, PolyA), random nucleotide sequences (length can be any length, such as 10mer, 20mer, 30mer), charged polypeptides (such as PolyR, PolyD, PolyK, PolyE, PolyH, length can be any number of amino acids, such as 7, 8, 9), polymers (such as (PEG) n ) at least one of.

[0008] Furthermore, the main molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other types of cyclodextrin derivatives, cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], cucurbituril

[10] , and other types of cucurbituril derivatives.

[0009] According to one embodiment of the present invention, the polypeptide probe for amino acid recognition may be FGXD n , X represents any one of the 20 natural amino acids to be tested, n represents the degree of polymerization of polyaspartic acid (Poly D). The guest molecule in the probe is phenylalanine (F), and the pulling sequence is polyaspartic acid.

[0010] In a second aspect, the present invention provides a method for preparing the above-mentioned probe.

[0011] The preparation method of the probe provided by the present invention comprises the following steps: connecting the recognition site on the probe to the traction sequence through a coupling reaction such as an amide reaction; connecting the guest molecule to the recognition site-traction sequence through a coupling reaction such as an amide reaction and a click chemistry reaction.

[0012] The charged polypeptide in the pulling sequence of the probe can be synthesized by a polypeptide synthesizer, the polynucleotide and the random sequence of nucleotide can be synthesized by a DNA synthesizer, and the polymer can be purchased directly.

[0013] In a third aspect, the present invention provides a probe composition.

[0014] The probe composition provided by the present invention comprises the above-mentioned probe of the present invention and a main molecule.

[0015] In a fourth aspect, the present invention also provides a probe Host molecular complex.

[0016] The probe provided by the present invention The host molecule complex is a probe formed by the above-mentioned probe of the present invention and the host molecule through host-guest interaction, so that the guest molecule in the probe is combined with the host molecule The host molecule complex. When used, the probe The host molecule complex temporarily suspends the recognition site of the probe in the sensing area of ​​the nanopore to improve the nanopore's recognition ability for the analyte molecules.

[0017] In a fifth aspect, the present invention also provides an amino acid recognition system.

[0018] The system comprises the above-mentioned probe composition and a sample pool, or the above-mentioned probe A main molecule complex and a sample pool; the sample pool comprises two compartments separated by an insulating membrane; the insulating membrane has a through hole with a diameter of 100-150 μm, the through hole is filled with a phospholipid bilayer, and a nanopore exists in the phospholipid bilayer.

[0019] Furthermore, the insulating film is a polytetrafluoroethylene film, and the thickness of the polytetrafluoroethylene film can be 20 μm.

[0020] Furthermore, the nanopore can be selected from biological nanopores with a diameter of less than 2 nm and solid-state nanopores with a diameter of less than 3 nm. Specifically, the nanopore is selected from at least one of α-hemolysin (αHL) and its mutants, MspA and its mutants, Aerolysin and its mutants, Phi29, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopores, glass nanopores, SiNx nanopores, Al2O3 nanopores, graphene nanopores, hybrid nanopores, carbon nanotubes, and DNAOrigami nanopores.

[0021] In a sixth aspect, the present invention also provides the above-mentioned probe or probe Application of host molecule complexes or amino acid recognition systems.

[0022] The application is a probe or a probe The application of the main molecule complex or amino acid recognition system in at least one of the following aspects: 1) protein sequencing; 2) detection of post-translational modification of proteins; 3) proteomics research.

[0023] In a seventh aspect, the present invention also provides an amino acid recognition method based on nanopore technology.

[0024] The present invention uses the probe formed by the designed probe and the main molecule The main molecular complex is analyzed by nanopore single molecule technology to generate characteristic signals to identify 20 amino acids on the polypeptide chain or post-translationally modified amino acids. At this time, the recognition sites in the probe are placed with various amino acids to be tested.

[0025] The amino acid recognition method based on nanopore technology provided by the present invention comprises the following steps: adding the probe and the host molecule into the sample pool of the present invention, forming a probe under host-guest interaction. The main molecule complex uses nanopores to collect characteristic current signals, analyzes and compares the current blockage ratio, signal frequency and blocking time of the current signal, thereby achieving the distinction of amino acids on the probe recognition site.

[0026] Furthermore, an electrolyte solution is pre-added in the compartment of the sample pool, and the electrolyte solution is a KCl electrolyte solution or a NaCl electrolyte solution. + Or Na + The concentration is 0.8-4.0 M, preferably 3.6 M; the pH of the electrolyte solution is 4-6. In a specific embodiment, the electrolyte solution is prepared with 10 mM citric acid buffer.

[0027] Furthermore, the sampling frequency of the characteristic current signal is 100 kHz, and the Bessel low-pass filter cut-off frequency is 10 kHz.

[0028] The present invention provides a more detailed amino acid recognition method based on nanopore technology, comprising the following steps:

[0029] (a) Diphytylphosphatidylcholine (DPhPC) was used to form a synthetic lipid bilayer with a diameter of 100-150 μm on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA).

[0030] (b) embedding the nanopore into a phospholipid bilayer, the phospholipid bilayer dividing the sample pool into two chambers, cis and trans, both chambers containing 1.0 mL of buffer solution; adding the probe into the cis chamber, and adding the main molecule into the cis chamber at the same time;

[0031] The above experiments were performed in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and 23.0 ± 0.5 °C;

[0032] The amount of the main molecule substance is more than 10 times the amount of the probe substance, specifically 10-100:1;

[0033] (c) A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system. Experimental recording is performed at a given voltage. The probe and the host molecule form a probe under host-guest interaction. The main molecule complex enters and passes through the nanopore driven by the electrophoretic force, generating a characteristic current signal; the current blocking ratio, blocking time and signal frequency of the current signal are analyzed and compared, thereby realizing the recognition of the amino acid at the probe recognition site.

[0034] More detailed data processing and analysis steps are as follows:

[0035] c1: Use the Single-Channel Search function in the Clampfit software to scan the recorded data and extract the information of each characteristic current block amplitude (Amplitude) and block duration (Dwell Time);

[0036] d2: Calculate the current blockade ratio of the corresponding signal based on the baseline current;

[0037] d3: Import the obtained data into Origin software, draw a signal block time length-current block ratio diagram, and perform frequency analysis on the signal current block ratio to realize the recognition of amino acids at the probe recognition site.

[0038] According to one embodiment of the present invention, the object to be detected is an amino acid on the recognition site of the probe, in which case the corresponding main molecule is cucurbituril [7], the guest molecule on the probe is phenylalanine (F), the traction sequence is polyaspartic acid, the recognition site on the probe is located at the third, fourth or fifth position of the probe starting from the guest molecule, and the nanopores used are αHL and mutant M113F.

[0039] Compared with the prior art, the present invention has the following beneficial effects:

[0040] The present invention provides an amino acid recognition probe based on nanopore technology, which has a simple probe structure, a small sample volume requirement (the final probe concentration is only 400nM), a high detection sensitivity, a simple operation method, and a stable and uniform output signal. It has a strong ability to distinguish 20 amino acids and post-translational modifications. Taking the main molecular complex as an example, twenty different amino acids can be distinguished by this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is an experimental record of the probe host-guest complex passing through the nanopore; wherein a is a schematic diagram of the experimental method, and b is a diagram of the actual current signal generated by the probe FGGDDDDDDDD.

[0042] Figure 2 This is the enhancement effect of the host-guest interaction on the recognition of the target molecule in Example 1.

[0043] Figure 3 The interaction between different host and guest molecules in Example 2 improves the recognition function of the nanopore.

[0044] Figure 4 In Example 3, the probe Figure 3. Effects of the main molecule complex, wild-type αHL and mutants on the discrimination of 20 amino acids.

[0045] Figure 5 This is a diagram showing the differentiation effect of the probes corresponding to individual amino acids in the mixed samples in Example 4; wherein a is the first group of mixed samples, including eight amino acids E, H, N, Y, S, W, M and L, and b is the second group of mixed samples, including eight amino acids R, K, Q, T, P, F, V and I. DETAILED DESCRIPTION

[0046] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0047] The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial channels, and the room temperature described in the following examples refers to 23.0±0.5°C.

[0048] In the following examples, the probe uses phenylalanine (F) as the guest molecule, the amino acid recognition site can be located at the third, fourth, or fifth position, the host molecule is cucurbituril [7] (CB [7]), the nanopores are wild-type α-hemolysin (αHL) and αHL mutant M113F, and the traction sequence is polyaspartic acid.

[0049] Wild-type α-hemolysin (αHL) and αHL mutant M113F can be expressed in E. coli, assembled into homogenous heptamers on rabbit erythrocyte membranes, and then purified by 8% SDS-PAGE. Please refer to the following literature: Li, T., Liu, L., Li, Y., Xie, J. and Wu, H.-C. (2015), A Universal Strategy for Aptamer-Based Nanopore Sensing through Host–Guest Interactions inside α-Hemolysin.

[0050] Angew.Chem.Int.Ed.,54:7568-7571.

[0051] Example 1: Using host-guest interaction to extend the capture time of the molecule to be detected by the nanopore

[0052] Experimental sample: Probe containing the molecule to be detected: FGGD8, main molecule: Cucurbituril 7 (CB[7])

[0053] The probe FGGD8 is a polypeptide with an amino acid sequence of FGGDDDDDDDD from N-terminus to C-terminus, as shown in SEQ ID No: 1; it can be purchased from major polypeptide synthesis companies (such as Qiangyao Biotechnology Co., Ltd.), or it can be synthesized by solid phase synthesis technology.

[0054] The experimental steps are as follows:

[0055] 1) Dissolve FGGD8 in water to 400 μM and dissolve cucurbituril [7] in water to a final concentration of 100 mM.

[0056] 2) A synthetic lipid bilayer with a diameter of 100 μm was formed on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA) using diphytylphosphatidylcholine (DPhPC).

[0057] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample pool into two chambers, cis and trans, both of which contained 1.0 mL of buffer solution (composition: 3.0 M KCl, 10 mM citric acid, pH 5.0).

[0058] 4) Pipette 2 μL of FGGD8 solution and add it into the cis chamber of the nanopore single-channel test system. The conditions of the nanopore single-channel test system are: 3.0 M KCl, 10 mM citric acid, pH 5.0, bias voltage +200 mV, and record data for more than 1 hour. Figure 2 a.

[0059] 5) Pipette 10 μL CB[7] solution into the cis chamber of the nanopore single-channel test system. The conditions of the nanopore single-channel test system are: 3.0 M KCl, 10 mM CA, pH 5.0, bias voltage +200 mV, and record data for more than 1 hour. Figure 2 b.

[0060] 6) Pipette 2 μL FGGD8 and 10 μL CB[7] solution and add them into the cis chamber of the nanopore single-channel test system. The conditions of the nanopore single-channel test system are: 3.0 M KCl, 10 mM CA, pH 5.0, bias voltage +200 mV, and record data for more than 1 hour. Figure 2 c. It can be seen that with the assistance of host-guest interaction, FGGD8 produces obvious characteristic signals in the patch clamp test system.

[0061] Example 2: Enhancement of Nanopore Recognition Function by Different Types of Host-Guest Interactions

[0062] Experimental samples: probes containing the molecules to be tested: YGK-DNA complex, FGK-DNA complex, ferrocene-DNA complex, adamantane-DNA complex, amFGK-DNA complex (amF is 4-aminomethyl-L-phenylalanine);

[0063] Main molecules: Cucurbituril 6 (CB[6]), Cucurbituril 7 (CB[7]).

[0064] The experimental steps are as follows:

[0065] 1) Construction of the complex: The short peptide structures in the above complexes were purchased from Qiangyao Biotechnology Co., Ltd., in which the side chain of lysine K was modified with a maleimide group. This structure can also be synthesized by using a peptide solid phase synthesizer. The DNA random sequence modified with a thiol group at the end (5'-CATATTACACTCTCACGACTC-3') and the DNA random sequence modified with ferrocene and adamantane (5'-CATATTACACTCTCACGACTC-3') were all obtained from Shenggong Biotechnology Co., Ltd. Among them, the short peptide modified with maleimide needs to be added with the DNA modified with a thiol group at the end under pH 9 (100mM CBS, pH 9.0) at a molar ratio of 1:1 and reacted for 20 minutes to obtain the complex used for the experiment. The solution was diluted to 400μM with water. The main molecules cucurbituril [6] and cucurbituril [7] were dissolved in water to 100mM.

[0066] 2) A synthetic lipid bilayer with a diameter of 100 μm was formed on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA) using diphytylphosphatidylcholine (DPhPC).

[0067] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample pool into two chambers, cis and trans, both of which contained 1.0 mL of buffer solution (composition: 3.0 M KCl, 10 mM citric acid, pH 5.0).

[0068] 4) Pipette 2 μL of the above different guest complex solutions (concentration 400 μM) and 10 μL of the host molecule solution (concentration 50 mM) and add them into the cis chamber of the nanopore single-channel test system at the same time. The conditions of the nanopore single-channel test system are: 3.0 M KCl, 10 mM CA, pH 5.0, bias voltage +200 mV, and record data for more than 1 hour. Figure 3 It can be seen that with the assistance of the interaction between different host and guest molecules, the recognition function of the nanopore is significantly improved.

[0069] Example 3: Nanopore detection of amino acids at the recognition site in the probe

[0070] The specific method is as follows:

[0071] 1) Prepare an aqueous solution (100 mM) of the host molecule CB[7] and an aqueous solution (concentration 750 μM) of the amino acid recognition probe FGXD8, where X in FGXD8 represents any one of the 20 natural amino acids to be detected.

[0072] 2) A synthetic lipid bilayer with a diameter of 100 μm was formed on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA) using diphytylphosphatidylcholine (DPhPC).

[0073] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample pool into two chambers, cis and trans. Both chambers contained 1.0 mL of buffer solution. 2 μL of probe FGXD8 solution was added to the cis chamber, and 10 μL of cucurbituril was added to the cis chamber at the same time [7]. All experiments were performed in a 3.6 M KCl, 10 mM CA buffer at a pH of 5.0 and a temperature of 23.0 ± 0.5 °C.

[0074] 4) Experimental recording was performed at a voltage of +200 mV. The probe FGXD8 and the host molecule formed a host-guest complex under host-guest interaction. X is any one of 20 different amino acids, which enters and passes through the nanopore driven by electrophoretic force to generate a characteristic current signal. The current blockade ratio and blocking time of the current signal are analyzed and compared, and the αHL mutant M113F is used to further distinguish the undistinguished polypeptides, and finally all the polypeptides are distinguished.

[0075] In this embodiment, the probe The effect of the main molecule complex and wild-type αHL and mutants on the discrimination of 20 amino acids is shown in the figure Figure 4 As shown. Figure 4 It can be seen that the probe can completely distinguish 20 natural amino acids.

[0076] Example 4: Utilization The host-guest complex and wild-type αHL distinguish the amino acid at the third position on the polypeptide chain in the mixed sample

[0077] 1) Prepare an aqueous solution (100 mM) of the main molecule CB[7], and divide the mixed sample into two groups, each containing 8 peptide probes. The amino acids to be tested in the first group are H, N, Y, S, W, M and L, and the amino acids to be tested in the second group are R, K, Q, T, P, F, V and I.

[0078] 2) A synthetic lipid bilayer with a diameter of 100 μm was formed on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA) using diphytylphosphatidylcholine (DPhPC).

[0079] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample pool into two chambers, the cis and trans chambers, both containing 1.0 mL of buffer solution. Each peptide probe was added to the cis chamber, with a final concentration of 150 nM for FGRD8, FGKD8, and FGHD8, a final concentration of 90 nM for FGED8, and a final concentration of 100 nM for the remaining peptides. At the same time, 10 μL of cucurbituril was added to the cis chamber [7]. All experiments were performed in a 3.6 M KCl, 10 mM citrate buffer at a pH of 5.0 and a temperature of 23.0 ± 0.5 °C.

[0080] 4) Experimental recording was performed at a voltage of +200mV. The peptide probe and the host molecule formed a host-guest complex under the host-guest interaction. Driven by the electrophoretic force, the amino acids enter and pass through the nanopores, generating characteristic current signals. The current blocking ratio and blocking time of the current signals are analyzed and compared to obtain the type information of the amino acids to be tested.

[0081] In mixed samples, the differentiation effect of probes corresponding to single amino acids is shown in the figure below: Figure 5 As shown. Figure 5 It can be seen that the difference in I / I0 of each polypeptide signal in each group of current signal graphs is very obvious. Each polypeptide signal can be classified very clearly. The I / I0 of the corresponding polypeptide is consistent with that when detected alone. This result further confirms the excellent performance of this probe.

[0082] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

[0083] References

[0084] 1.Asandei A,Di Muccio G,Schiopu I,et al.Nanopore-based proteins sequencing using biopores:Current achievements and open challenges[J].SmallMethods:2020,4:1-13.

[0085] 2. Domon, B. & Aebersold, R. Options and considerations when selecting aquantitative proteomics strategy. Nat. Biotechnol. 28, 710-721 (2010).

[0086] 3.Xiao,Y.,Vecchi,MM&Wen,D.Distinguishing between leucine andisoleucine by integrated LC-MS analysis using an orbitrap fusion massspectrometer.Anal.Chem.88,10757-10766(2016).

[0087] 4. Allen, G. Sequencing of proteins and peptides. Vol. 9 161-234 (Elsevier, 2011).

[0088] 5.Hu ZL,Huo MZ,Ying YL,et al.Biological nanopore approach for single-molecule protein sequencing[J].Angew Chem Int Ed:2021,60:14738-14749.

[0089] 6.Ouldali H,Sarthak K,Ensslen T,et al.Electrical recognition of thetwenty proteinogenic amino acids using an aerolysin nanopore[J].NatBiotechnol:2020,38:176-181.

Claims

1. A nanopore probe, comprising a guest molecule, a recognition site and a pull sequence; wherein: The guest molecule is used to bind to the host molecule; the traction sequence provides the power for the entire probe molecule to be driven by the electrophoretic force into the nanopore detection area; the recognition site is used to place various small molecule analytes.

2. The nanopore probe according to claim 1, characterized in that: The guest molecule of the nanopore probe is selected from at least one of ferrocene and its derivatives, adamantane and its derivatives, phenylalanine and its derivatives, tyrosine and its derivatives, histidine and its derivatives, and methionine and its derivatives; Or, the pulling sequence of the nanopore probe is selected from at least one of a polynucleotide, a random nucleotide sequence, a charged polypeptide, and a polymer; Alternatively, the small molecule analytes include the following molecules: amino acids, side chain-modified amino acids, nucleotides, and neurotransmitters.

3. The polypeptide probe for amino acid recognition according to claim 1 or 2, characterized in that: The main molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other types of cyclodextrin derivatives, cucurbituril [5], cucurbituril [6], cucurbituril [7], cucurbituril [8], cucurbituril [10], and other types of cucurbituril derivatives. 4 . A probe composition, comprising the nanopore probe according to any one of claims 1 to 3 and the host molecule.

5. A probe The host molecule complex is a probe formed by the nanopore probe according to any one of claims 1 to 3 and the host molecule through host-guest interaction, so that the guest molecule in the probe is combined with the host molecule Host molecular complex.

6. An amino acid recognition system comprising the probe composition and sample pool according to claim 4, or the probe according to claim 5 host molecule complex and sample pool; The sample pool comprises two compartments separated by an insulating membrane; the insulating membrane is provided with a through hole with a diameter of 100-150 μm, the through hole is filled with a phospholipid bilayer, and a nanopore exists in the phospholipid bilayer.

7. The amino acid recognition system according to claim 6, characterized in that: The nanopore is selected from a biological nanopore with a diameter of less than 2 nm or a solid nanopore with a diameter of less than 3 nm; the nanopore is specifically selected from at least one of α-hemolysin and its mutants, MspA and its mutants, Aerolysin and its mutants, Phi29, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopore, glass nanopore, SiNx nanopore, Al2O3 nanopore, graphene nanopore, hybrid nanopore, carbon nanotube, and DNAOrigami nanopore.

8. The polypeptide probe according to any one of claims 1 to 3, the probe composition according to claim 4, or the probe according to claim 5 Application of the main molecule complex or the amino acid recognition system described in claim 6 or 7 in at least one of the following aspects: 1) protein sequencing; 2) detection of post-translational modification of proteins; 3) proteomics research.

9. A method for amino acid recognition based on nanopore technology, comprising the following steps: adding the polypeptide probe for amino acid recognition and the host molecule into the sample pool of the amino acid recognition system according to claim 6, forming a probe under host-guest interaction. The main molecule complex uses nanopores to collect characteristic current signals, analyzes and compares the current blocking ratio, blocking time and signal frequency of the current signal, thereby realizing the distinction of amino acids on the recognition site of the polypeptide probe.

10. The amino acid recognition method based on nanopore technology according to claim 9, characterized in that: The amino acid recognition method based on nanopore technology comprises the following steps: (a) using diphytylphosphatidylcholine to form a synthetic lipid bilayer with a diameter of 100-150 μm on a 25 μm thick polytetrafluoroethylene film; (b) The nanopore is embedded in a phospholipid bilayer, which divides the sample pool into two chambers, cis and trans, both of which contain 1.0 mL of buffer solution; the nanopore probe is added to the cis chamber, and the main molecule is added to the cis chamber at the same time. The above experiments were carried out in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and temperature 23.0 ± 0.5 °C; The amount of the main molecule substance is more than 10 times the amount of the probe substance; (c) A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system. Experimental recording is performed at a given voltage. The nanopore probe and the host molecule form a probe under host-guest interaction. The main molecular complex enters and passes through the nanopore driven by the electrophoretic force, generating a characteristic current signal; The current blocking ratio, blocking time and signal frequency of the current signal are analyzed and compared, so as to realize the recognition of the amino acid at the probe recognition site.

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