A nanopore probe using host-guest interaction and a preparation method and application thereof

Abstract

The application discloses a nanopore probe utilizing host-guest interaction and a preparation method and application thereof. The nanopore probe is composed of a guest molecule, a recognition site and a pulling sequence; wherein the guest molecule is used for combining with a host molecule, and simultaneously enhances the interaction between the probe and the nanopore and slows down the displacement time of the probe in the nanopore; the pulling sequence provides the power for driving the whole probe molecule into the nanopore detection area under the electrophoretic force; and the recognition site can be used for placing various small molecule analytes (such as amino acids, side chain modified amino acids, nucleotides, neurotransmitters and the like). The probe has the advantages of simple structure, small sample amount requirement, high detection sensitivity, simple operation method, stable and uniform signal output, and strong distinguishing ability for 20 kinds of amino acids and post-translational modifications. The above-mentioned probe host molecule complex is taken as an example, and twenty different amino acids can be distinguished by the method.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of amino acid recognition, protein sequencing, proteomics research and disease diagnosis, and particularly relates to a nanopore probe using host-guest interaction and a preparation method and application thereof. BACKGROUND

[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 body fluid balance in organisms, and providing stable structure for cells and organisms. They are composed of long amino acid chains, the sequence of amino acids is determined by the nucleotide sequence of genes, and the amino acid chains fold to form specific three-dimensional structures to determine their physiological activity. However, the gene sequence cannot directly encode information about protein abundance, post-translational modification or cleavage, and even a small change in the primary sequence of a protein can have an impact. Therefore, proteomics research is in great need of an efficient and economical protein sequencing and post-translational modification detection strategy. Nanopore technology has great potential in the field of protein sequencing due to its successful application in DNA sequencing. In the field of protein sequencing, there are mainly three challenges: protein unfolding, controllable perforation of polypeptide chains and recognition of single amino acids. At present, the community usually uses reverse electrophoretic force and electroosmotic flow to control the displacement speed of the analyte in the nanopore, and at the same time, the interaction force between the nanopore and the analyte is enhanced by point mutation or modification of the nanopore to slow down the perforation time of the analyte, so as to obtain a longer duration and more characteristic recognition signal.

[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 which the host-guest molecules can maintain a relatively moderate interaction strength, which can be applied to the nanopore detection process to produce a characteristic and long-lasting recognition signal, and the interaction can be released under the action of external force, which makes it possible to detect multiple probes simultaneously in the nanopore detection process. Therefore, the application of host-guest interaction to the design of nanopore probes has great detection potential. SUMMARY

[0004] The purpose of the present application is to provide a nanopore probe using host-guest interaction, which can enhance the interaction between the probe and the nanopore while slowing down the displacement time of the probe in the nanopore, thereby increasing the detection capacity of the nanopore and further qualitatively and / or quantitatively analyzing the target molecules.

[0005] In a first aspect, the present application provides a nanopore probe using host-guest interaction, which is composed of a guest molecule, a recognition site and a pulling sequence; wherein the guest molecule is used to bind a host molecule, which enhances the interaction between the probe and the nanopore and slows down the displacement time of the probe in the nanopore; the pulling sequence provides the driving force for the whole probe molecule to enter the nanopore detection area driven by the electrophoretic force; and 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] Further, 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] Further, the pulling sequence on the probe is selected from at least one of polynucleotides (such as PolyT, PolyA), nucleotide random sequences (which can be of any length, such as 10mer, 20mer, 30mer), charged polypeptides (such as PolyR, PolyD, PolyK, PolyE, PolyH, which can be of any number of amino acids, such as 7, 8, 9), and polymeric macromolecules (such as (PEG) n ).

[0008] Further, the host molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other cyclodextrin derivatives, cucurbit[5]uril, cucurbit[6]uril, cucurbit[7]uril, cucurbit[8]uril, cucurbit

[10] uril, and other cucurbituril derivatives.

[0009] According to an embodiment of the present application, the polypeptide probe for amino acid recognition can be FGXD n , wherein X represents any one of the 20 natural amino acids to be detected, and n represents the degree of polymerization of polyaspartic acid (PolyD). The guest molecule in the probe is phenylalanine (F), and the pulling sequence is polyaspartic acid.

[0010] In a second aspect, the present application provides a preparation method of the above-mentioned probe.

[0011] The preparation method of the probe provided by the present application comprises the following steps: coupling the recognition site on the probe to the pulling sequence through an amide reaction or other coupling reaction; and coupling the guest molecule to the recognition site-pulling sequence through an amide reaction, click chemistry reaction or other coupling reaction.

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

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

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

[0015] In a fourth aspect, the present application further provides a probe host molecule complex.

[0016] The probe provided by the present application host molecule complex is formed by the above-mentioned probe of the present application and the host molecule through host-guest interaction, so that the guest molecule in the probe is combined with the host molecule to form a probe host molecule complex. The probe host molecule complex temporarily suspends the recognition site of the probe in the sensing area of the nanopore, so as to improve the recognition ability of the nanopore to the test molecule.

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

[0018] The system comprises the above-mentioned probe composition and a sample cell, or the above-mentioned probe host molecule complex and a sample cell; the sample cell comprises two compartments separated by an insulating film; the insulating film 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] Further, the insulating film is a polytetrafluoroethylene film. The thickness of the polytetrafluoroethylene film can be 20 μm.

[0020] Further, 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, and specifically the nanopore is selected from at least one of alpha-hemolysin (alphaHL) and its mutants, MspA and its mutants, Aerolysin and 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 application further provides the use of the above-mentioned probe or probe host molecule complex or amino acid recognition system.

[0022] The use is the use of the probe or probe Applications of host molecular complex or amino acid recognition systems in at least one of the following: 1) protein sequencing; 2) detection of post-translational modifications 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] This invention utilizes a probe formed by a designed probe and the host molecule. The host molecular complex is analyzed using nanopore single-molecule technology to generate characteristic signals, enabling the identification of 20 amino acids or post-translational modified amino acids on the polypeptide chain. The recognition sites in the probe contain the various amino acids to be tested.

[0025] The amino acid recognition method based on nanopore technology provided by this invention includes the following steps: adding the probe and the host molecule into the sample cell of this invention, forming a probe under host-guest interaction. The host molecular complex utilizes nanopores to collect characteristic current signals, and analyzes and compares the current blocking ratio, signal frequency, and retardation time of the current signals to distinguish amino acids at the probe recognition sites.

[0026] Furthermore, the sample cell compartment is pre-filled with an electrolyte solution, which is either a KCl electrolyte solution or a NaCl electrolyte solution. + Or Na + The concentration of the electrolyte 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 using 10 mM citrate buffer.

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

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

[0029] (a) Using diaphytylphosphatidylcholine (DPhPC) to form a synthetic lipid bilayer with a diameter of 100-150 micrometers on a 25-micrometer thick polytetrafluoroethylene film (Goodfellow, Malvern, PA).

[0030] (b) The nanopores are embedded in a phospholipid bilayer, which divides the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution; the probe is added to the cis chamber, and the host molecules are added to the cis chamber at the same time;

[0031] The above experiments were conducted in 0.8–4.0 M KCl and 10 mM citrate buffer at a pH of 5.0–8.0 and a temperature of 23.0 ± 0.5 °C.

[0032] The amount of the main molecule is more than 10 times greater than the amount of the probe, for example, 10-100:1.

[0033] (c) Apply a fixed bias voltage in the range of +80–250 mV to the nanopore single-channel testing system, and record the experimental data at the given voltage. The probe and the host molecule form a probe under the host-guest interaction. The host molecular complex enters and passes through the nanopore under the drive of electrophoretic force, generating a characteristic current signal; the current blocking ratio, hindrance time and signal frequency of the current signal are analyzed and compared, thereby realizing the recognition of amino acids on the probe recognition site.

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

[0035] c1: Use the Single-Channel Search function in Clampfit software to scan the recorded data and extract the Amplitude and Dwell Time information for each characteristic current.

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

[0037] d3: Import the obtained data into Origin software, plot the signal hindrance time-current hindrance ratio graph, and perform frequency analysis on the signal current hindrance ratio to achieve the recognition of amino acids at the probe recognition site.

[0038] According to an embodiment of the present invention, the test object is an amino acid on the probe recognition site, the corresponding host 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 on the probe starting from the guest molecule, and the nanopore used is αHL and mutant M113F.

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

[0040] This invention provides an amino acid recognition probe based on nanopore technology. The probe has a simple structure, requires a small sample volume (final probe concentration is only 400 nM), exhibits high detection sensitivity, is easy to operate, and produces a stable and uniform signal. It demonstrates strong distinguishing ability for 20 amino acids and post-translational modifications. The probe described above... For example, this method can be used to distinguish twenty different amino acids in a host molecular complex. Attached Figure Description

[0041] Figure 1 This is an experimental record of the probe host-guest complex passing through a nanopore; where 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 enhanced effect of host-guest interaction on the recognition of the test molecule in Example 1.

[0043] Figure 3 This is to illustrate how the interaction between different host and guest molecules in Example 2 enhances the recognition function of nanopores.

[0044] Figure 4 In Example 3, a probe was used. A graph showing the effect of the main molecular complex, wild-type αHL, and mutants in distinguishing 20 amino acids.

[0045] Figure 5 This is a diagram showing the differentiation effect of probes for individual amino acids in a mixed sample in Example 4; where 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 Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0047] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available. The room temperature mentioned 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 nanopore is wild-type α-hemolysin (αHL) and αHL mutant M113F, and the traction sequence is polyaspartic acid.

[0049] Wild-type α-hemolysin (αHL) and the αHL mutant M113F were expressed in *E. coli*, assembled into homoheptamers on rabbit erythrocyte membranes, and purified by 8% SDS-PAGE. See the following reference: 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: Extending the trapping time of analyte molecules in nanopores using host-guest interactions

[0052] Experimental sample: Probe containing the analyte molecule: FGGD8, host molecule: cucurbituril 7 (CB[7])

[0053] The probe FGGD8 is a polypeptide with an amino acid sequence of FGGDDDDDDDD from the N-terminus to the 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 synthesized by ourselves using solid-phase synthesis technology.

[0054] The experimental procedure is 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 film (Goodfellow, Malvern, PA) using diaphytophosphatidylcholine (DPhPC).

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

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

[0059] 5) Add 10 μL of CB[7] solution to 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) Take 2 μL of FGGD8 and 10 μL of LCB[7] solution and add them simultaneously to 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 is evident that, with the assistance of the interaction between the subject and object, FGGD8 generates a distinct characteristic signal in the patch-clamp testing system.

[0061] Example 2: Enhancement of nanopore recognition function by different types of host-guest interactions

[0062] Experimental samples: Probes containing the analyte molecules: YGK-DNA complex, FGK-DNA complex, ferrocene-DNA complex, adamantane-DNA complex, amFGK-DNA complex (where amF is 4-aminomethyl-L-phenylalanine);

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

[0064] The experimental procedure is as follows:

[0065] 1) Construction of the complex: The short peptide structures in the above complex were all purchased from Qiangyao Biotechnology Co., Ltd. The side chain of lysine K was modified with maleimide group, which can also be synthesized by peptide solid phase synthesis instrument. The random DNA sequence with thiol-modified terminal group (5'-CATATTACACTCTCACGACTC-3') and the random DNA sequence modified with ferrocene and adamantane (5'-CATATTACACTCTCACGACTC-3') were all purchased from Sangon Biotech Co., Ltd. The short peptide modified with maleimide needs to be reacted with the DNA with thiol-modified terminal group at pH 9 (100mM CBS, pH 9.0) at a molar ratio of 1:1 for 20 min to obtain the complex used in the experiment. The volume was adjusted 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 film (Goodfellow, Malvern, PA) using diaphytophosphatidylcholine (DPhPC).

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

[0068] 4) Simultaneously add 2 μL (400 μM) of the above-mentioned different guest complex solutions and 10 μL (50 mM) of the host molecule solution to the cis chamber of the nanopore single-channel assay system. The conditions of the nanopore single-channel assay system are: 3.0 MkCl, 10 mM CA, pH 5.0, bias voltage +200 mV, and record data for more than 1 hour. Figure 3 It is evident that the recognition function of nanopores is significantly enhanced with the assistance of interactions between different host and guest molecules.

[0069] Example 3: Nanopore detection of amino acids at recognition sites in probes

[0070] The specific method is as follows:

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

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

[0073] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample cell into cis and trans chambers, each containing 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 carried out in 3.6 M KCl and 10 mM CA buffer at pH 5.0 and temperature 23.0 ± 0.5 °C.

[0074] 4) Experimental data was recorded at a voltage of +200mV. The probe FGXD8 formed a host-guest complex with the host molecule through host-guest interaction. X is any one of 20 different amino acids. Driven by electrophoretic force, it enters and passes through the nanopore, generating a characteristic current signal. The current blocking ratio and retardation time of the current signal are analyzed and compared. The αHL mutant M113F is used to further distinguish the undifferentiated peptides, and finally all peptides are separated.

[0075] This embodiment utilizes a probe. The effect of the main molecular complex, wild-type αHL, and mutants on the differentiation of 20 amino acids is shown in the figure below. Figure 4 As shown. By Figure 4 It is known that this probe can completely distinguish 20 kinds of natural amino acids.

[0076] Example 4: Utilizing Host-guest complexes and wild-type αHL distinguish amino acids at the third site on the polypeptide chain in mixed samples.

[0077] 1) Prepare an aqueous solution (100 mM) of the main molecule CB[7]. The mixed sample is divided into two groups, each containing 8 polypeptide 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 film (Goodfellow, Malvern, PA) using diaphytophosphatidylcholine (DPhPC).

[0079] 3) A single nanopore (wild-type αHL) was embedded in a phospholipid bilayer, which divided the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution. Each peptide probe was added to the cis chamber, with FGRD8, FGKD8 and FGHD8 added to a final concentration of 150 nM, FGED8 to a final concentration of 90 nM, and the remaining peptides to a final concentration of 100 nM. At the same time, 10 μL of cucurbituril was added to the cis chamber [7]. All experiments were carried out in 3.6 M KCl and 10 mM citrate buffer at pH 5.0 and 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 through host-guest interaction. Driven by electrophoretic force, the amino acid enters and passes through the nanopore, generating a characteristic current signal. By analyzing and comparing the current blocking ratio and retardation time of the current signal, the type of amino acid to be measured can be obtained.

[0081] The image shows the differentiation effect of probes for individual amino acids in a mixed sample. Figure 5 As shown. By Figure 5 It can be seen that the differences in I / I0 of each polypeptide signal in each group of current signal graphs are very obvious, and each polypeptide signal can be clearly classified. The I / I0 of the corresponding polypeptide is consistent with that when detected individually. This result further confirms the excellent performance of this probe.

[0082] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied 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 traction sequence; wherein, Guest molecules are used to bind host molecules; the traction sequence provides the propulsion for the entire probe molecule to enter the nanopore detection region by electrophoretic force; recognition sites are used to place various small molecule analytes. The guest molecules of the nanopore probe are selected from at least one of the following: 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. The traction sequence of the nanopore probe is selected from polymers; The small molecule analytes include the following molecules: amino acids and nucleotides; The main molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other cyclodextrin derivatives, cucurbita[5], cucurbita[6], cucurbita[7], cucurbita[8], cucurbita[10], and other cucurbita derivatives.

2. The nanopore probe according to claim 1, characterized in that: The small molecule analytes also include neurotransmitters.

3. A probe composition comprising the nanopore probe of claim 1 and the host molecule.

4. A probe The host molecule complex is a probe formed by the nanopore probe of claim 1 and the host molecule through host-guest interaction, resulting in the binding of the guest molecule in the probe to the host molecule. The main molecular complex.

5. An amino acid recognition system, comprising the probe composition and sample cell of claim 3, or the probe of claim 4. The main molecular complex and the sample cell; The sample cell includes 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.

6. The amino acid recognition system according to claim 5, characterized in that: The nanopores are selected from biological nanopores with a diameter of less than 2 nm or solid nanopores with a diameter of less than 3 nm; specifically, the nanopores are 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 nanopores, glass nanopores, SiNx nanopores, Al2O3 nanopores, graphene nanopores, hybrid nanopores, carbon nanotubes, and DNA Origami nanopores.

7. The probe of claim 1 or 2, the probe composition of claim 3, or the probe of claim 4. The host molecular complex or the amino acid recognition system of claim 5 or 6 may be used in at least one of the following aspects: 1) protein sequencing; 2) detection of post-translational modifications of proteins; 3) proteomics research.

8. An amino acid recognition method based on nanopore technology, comprising the following steps: adding the probe of claim 1 and the host molecule into the sample cell of the amino acid recognition system of claim 5, thereby forming a probe through host-guest interaction. The main molecular complex utilizes nanopores to collect characteristic current signals, and analyzes and compares the current blocking ratio, hindrance time, and signal frequency of the current signals to distinguish amino acids at the recognition sites of peptide probes.

9. The amino acid recognition method based on nanopore technology according to claim 8, characterized in that: The amino acid recognition method based on nanopore technology includes the following steps: (a) Using diaphytylphosphatidylcholine to form a synthetic lipid bilayer with a diameter of 100-150 micrometers on a 25-micrometer-thick polytetrafluoroethylene film; (b) The nanopore is embedded in a phospholipid bilayer, which divides the sample cell into cis and trans chambers, each containing 1.0 mL of buffer solution; the nanopore probe is added to the cis chamber, and the host molecule is added to the cis chamber at the same time; The above experiments were conducted in a 0.8–4.0 M KCl, 10 mM citrate buffer solution at a pH of 5.0–8.0 and a temperature of 23.0 ± 0.5 ℃. The amount of the main molecular substance is more than 10 times greater than the amount of the probe substance; (c) Apply a fixed bias voltage in the range of +80 – 250 mV to the nanopore single-channel testing system and record the results at the given voltage. The nanopore probe and the host molecule form a probe under the host-guest interaction. The host molecular complex enters and passes through the nanopore under the drive of electrophoretic force, generating a characteristic current signal; The current blocking ratio, hindrance time and signal frequency of the current signal are analyzed and compared to achieve the recognition of amino acids at the probe recognition site.

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