Nanopore detection probe generated by connecting target molecule to be detected and load molecule as well as preparation method and application of nanopore detection probe

Abstract

The invention discloses a nanopore detection probe generated by connecting a target molecule to be detected and a load molecule as well as a preparation method and application of the nanopore detection probe. The nanopore detection probe is obtained by coupling a to-be-detected target molecule and a load molecule through a difunctional cross-linking reagent, wherein the load molecule consists of a guest action molecule, a functional linking group and a traction sequence; the guest action molecule provides a specific signal which is generated by nanometer in a combined detection system with the subject action molecule in the solution; the functional linking group provides an action site of the bifunctional crosslinking reagent, so that the bifunctional crosslinking reagent is combined with a target molecule to be detected; the traction sequence provides power for the whole probe molecule to enter a nanopore detection area under the driving of electrophoretic force. By using the nanopore detection probe, a target molecule to be detected is detected by using a nanopore single molecule technology, so that a characteristic signal is generated to perform qualitative and / or quantitative analysis on the target molecule. By taking amino acid-supported molecules as an example, twenty different amino acids can be distinguished by the method.

Description

Technical Field

[0001] The present invention belongs to the technical field of life analysis chemistry and protein sequencing, and specifically relates to a nanopore detection probe generated by connecting a target molecule to be detected and a load molecule, 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 activities. However, gene sequences cannot directly encode information about protein abundance, post-translational modification or shearing. Even slight changes in the main sequence of proteins may have relevant effects. Therefore, proteomics research is in great need of an efficient and economical protein sequencing and post-translational modification simple strategy. In recent years, only two de novo sequencing strategies are feasible: Edman degradation and mass spectrometry. After decades of development, multi-channel parallel detection and automated detection have improved their detection efficiency, but both have obvious limitations: the dynamic range of detection is not large enough, the sequencing length is not long enough, and the detection accuracy is not high enough, which seriously limits the application of these two detection technologies.

[0003] In the past few years, a variety of new protein sequencing methods have been developed, such as fluorescence sequencing, single-molecule peptide fingerprinting, identification of single amino acids using tunneling current, and nanopore protein sequencing. Among these new methods, nanopore technology has maintained a huge potential due to its successful application in DNA sequencing. Compared with DNA sequencing, protein sequencing is more challenging. Proteins are composed of 20 different amino acids, while DNA only contains four nucleotides. It is undoubtedly a huge challenge to read out 20 different signals. In addition, unlike DNA molecules with unique negative charges, proteins have heterogeneous charges, so it is difficult to control the directional passage of polypeptide chains through nanopores.

[0004] Therefore, it is imperative to develop a new probe that can recognize 20 different amino acids and overcome the heterogeneous charge properties of amino acids to enable the probe to move directionally through the nanopore. Summary of the invention

[0005] In view of the defects of the prior art, one object of the present invention is to provide a nanopore detection probe generated by connecting a target molecule to be detected with a load molecule and a preparation method thereof. The present invention uses a bifunctional cross-linking reagent to connect the target molecule to the load molecule using a simple and efficient click chemistry reaction to generate a detection probe, and uses the detection sensitivity of the nanopore single molecule technology to perform qualitative and / or quantitative analysis on the probe, specifically involving the analysis and detection of various biological small molecules (such as amino acids, nucleotides, neurotransmitters, etc.) and inorganic molecules, as well as DNA sequencing, protein sequencing, etc.

[0006] The nanopore detection probe provided by the present invention is generated by connecting a target molecule to be detected and a load molecule, and is obtained by coupling the target molecule to be detected and the load molecule through a bifunctional cross-linking reagent.

[0007] The load molecule is composed of a guest molecule, a functional linking group and a traction sequence. The guest molecule combines with the host molecule and interacts with the nanopore in the detection system to generate a specific signal; the functional linking group provides the action site of the bifunctional cross-linking reagent to enable it to combine with the target molecule to be detected; the traction sequence provides the power for the entire probe molecule to be driven by the electrophoretic force into the nanopore detection area.

[0008] Furthermore, the guest interaction molecule on the load molecule 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.

[0009] Furthermore, the functional linking group on the load molecule is selected from at least one of allyl, thiol, amino, succinimide ester and maleimide ester.

[0010] Furthermore, the pulling sequence on the load molecule is selected from: polynucleotides (such as PolyT, PolyA), random nucleotide sequences, charged polypeptides (such as PolyR, PolyD, PolyK, PolyE), polymers (such as (PEG) n ) at least one of.

[0011] According to one embodiment of the present invention, the load molecule may be FGCD8, FGGCD8, FGGGCD8, FGGCGGD8, FGK(AZA)D8, an organic transition metal compound-amino acid-polynucleotide complex structure with the sequence of ferrocene-GC-TTTTTTTTTTT, etc.

[0012] Specifically, the FGCD8 is a polypeptide with an amino acid sequence of FGCDDDDDDDD from N-terminus to C-terminus; C in the load molecule provides a functional group for connection, and G and GG, GGG, etc. in the following load molecules can change the position of the functional group, thereby meeting the probe requirements in different situations.

[0013] The FGGCD8 is a polypeptide having an amino acid sequence of FGGCDDDDDDDD from N-terminus to C-terminus, as shown in SEQ ID No: 1;

[0014] The FGGGCD8 is a polypeptide having an amino acid sequence of FGGGCDDDDDDDD from N-terminus to C-terminus, as shown in SEQ ID No: 2;

[0015] The FGGCGGD8 is a polypeptide having an amino acid sequence of FGGCGGDDDDDDDD from N-terminus to C-terminus, as shown in SEQ ID No: 3;

[0016] The FGK(AZA)D8 is a polypeptide with an amino acid sequence of FGKDDDDDDDD from N-terminus to C-terminus; wherein the third K is an azide-modified lysine, as shown in SEQ ID No: 4.

[0017] The load molecule in the present invention can be synthesized using solid phase synthesis technology, or purchased from major peptide synthesis companies, such as Qiangyao Biotechnology Co., Ltd.

[0018] The charged polypeptide in the traction sequence can be synthesized by a peptide synthesizer, the polynucleotide can be synthesized by a DNA synthesizer, and the polymer can be purchased directly. The functional linking group on the load molecule can be connected to the traction sequence through a coupling reaction such as an amide reaction. The guest molecule can be connected to the load molecule-traction sequence through a coupling reaction such as an amide reaction and a click chemistry reaction.

[0019] Further, the bifunctional cross-linking reagent is selected from at least one of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate (SMCC), 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide) ester (DSP), maleimidoacetic acid succinimide ester (AMAS), iodoacetic acid-N succinimide (SIA), bromoacetic acid N-hydroxysuccinimide ester (SBA), and disuccinimidyl carbonate (DSC).

[0020] Furthermore, the main active molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other types of cyclodextrin and their derivatives, cucurbituril [5], cucurbituril [6], cucurbituril [7], cucurbituril [8], cucurbituril

[10] , and other types of cucurbituril derivatives.

[0021] Furthermore, the target molecules to be detected can be various types of biological small molecules (such as amino acids, nucleotides, neurotransmitters, etc.) as well as inorganic molecules, DNA, proteins, etc.

[0022] The present invention also provides a method for preparing a nanopore detection probe generated by connecting a target molecule to be detected and a load molecule.

[0023] The method comprises the following steps:

[0024] 1) reacting the target molecule to be detected, the bifunctional cross-linking reagent and the organic base to form a target molecule to be detected-bifunctional cross-linking reagent complex;

[0025] 2) reacting the target molecule-bifunctional cross-linking reagent complex with a load molecule to obtain the nanopore detection probe.

[0026] In step 1) of the above method, the organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, 2,6-Lutidine (2,6-dimethylpyridine), Imidazole and NMI.

[0027] In step 1) of the above method, the molar ratio of the target molecule to be detected, the bifunctional cross-linking reagent and the organic base is (1-100):1:(2-10), and the reaction time is 1-4h; specifically, the molar ratio of the target molecule to be detected, the bifunctional cross-linking reagent and the organic base is 1:1:2.

[0028] The target molecule to be detected is added in the form of an aqueous solution (the concentration can be 1 mM), the bifunctional cross-linking reagent is added in the form of a DMF solution (the concentration can be 4 mM), and the organic base is also added in the form of a DMF solution (the concentration can be 8 mM).

[0029] In step 2) of the above method, the molar ratio of the target molecule to be detected-bifunctional cross-linking reagent complex to the load molecule is (1-100):1, and the reaction is performed at room temperature for 1-8 hours; specifically, the molar ratio of the target molecule to be detected-bifunctional cross-linking reagent complex to the load molecule is 1:1, and the reaction is performed at room temperature for 1 hour.

[0030] The load molecule is added in the form of an aqueous solution (the concentration may be 1 mM).

[0031] The present invention also provides a method for qualitatively and / or quantitatively detecting a target molecule using the nanopore detection probe prepared above. The method uses a bifunctional cross-linking reagent to connect the target molecule to a load molecule using a simple and efficient click chemistry reaction to generate a detection probe, and uses nanopore single molecule technology to analyze it, thereby generating a characteristic signal to perform qualitative and / or quantitative analysis on it.

[0032] The method for qualitative and / or quantitative detection of the target molecule to be detected provided by the present invention is a detection performed using a nanopore, comprising the following steps: firstly combining the guest acting molecule with the host acting molecule in the nanopore detection probe generated by connecting the target molecule to be detected and the load molecule, and then performing nanopore single channel recording.

[0033] Furthermore, the nanopore single-channel recording refers to the collection of characteristic electrical signals using a nanopore.

[0034] Furthermore, the characteristic electrical signal is collected at room temperature of 25.0±3.0°C. The signal sampling frequency is 100kHz, and the Bessel low-pass filter cut-off frequency is 10kHz. The characteristic current signal is analyzed by single-channel data analysis using Clampfit software to collect characteristic signals, and the data is statistically analyzed and analyzed using origin software.

[0035] Furthermore, the nanopore can be selected from biological nanopores with a diameter of less than 2 nm and solid nanopores with a diameter of less than 3 nm. Specifically, the nanopore is selected from α-HL and its mutants, MspA and its mutants, Phi29, Aerolysin, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopores, glass nanopores, SiN nanopores, x At least one of nanopore, Al2O3 nanopore, graphene nanopore, hybrid nanopore, carbon nanotube, DNAOrigami nanopore.

[0036] The present invention provides a more detailed method for qualitative and / or quantitative detection of a target molecule, comprising the following steps:

[0037] (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).

[0038] (b) embedding a 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 nanopore detection probe generated by connecting the target molecule to be detected and the load molecule to the cis chamber, and adding the main action molecule to the cis chamber at the same time;

[0039] The above experiments were performed in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and 25.0 ± 3.0 °C;

[0040] The molar ratio of the nanopore detection probe generated by connecting the target molecule to be detected and the load molecule to the main molecule can be greater than 1:10.

[0041] (c) A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system and recorded. The load molecules and the host molecules move toward the nanopore driven by the host-guest interaction and the electrophoretic force, generating a characteristic current signal.

[0042] (d) The current blockade ratio, signal frequency and blocking time of the current signal are analyzed and compared to obtain the characteristic information and concentration information of the target molecule to be measured.

[0043] According to an embodiment of the present invention, the molecules to be tested can be 20 kinds of natural amino acid molecules, in which case the corresponding bifunctional cross-linking reagent is SPDP, the guest molecule of the load molecule is phenylalanine (F), the functional linking group is thiol (-SH), the traction sequence can be DDDDDDDD, the main molecule is cucurbituril [7], and the nanopore is α-HL (WT).

[0044] Compared with the prior art, the present invention has the following beneficial effects:

[0045] The method provided by the present invention for connecting the detection target molecule and the load molecule to generate a nanopore detection probe can detect small molecules qualitatively and quantitatively, has a simple reaction, a short reaction time, a low detection limit, a stable and uniform output signal, and has a strong ability to distinguish molecules with similar structures. Taking the above-mentioned amino acid-load molecule as an example, twenty different amino acids can be distinguished by this method. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 The basic process of using bifunctional cross-linking reagents to connect target molecules to load molecules through simple and efficient click chemistry reactions to generate detection probes;

[0047] Figure 2 HPLC-MS charts of several target molecules prepared in Example 1;

[0048] Figure 3 The nanopore experiment records the synthetic amino acid-loaded molecules; wherein, Figure 3 a is a schematic diagram of the experimental method. Figure 3 b is the actual current signal diagram generated by the probe;

[0049] Figure 4 This is a concentration test curve for the probe formed by the amino acid molecule (glycine) and the load molecule in Example 1; the test sample concentrations are 40pM, 400pM, 4nM, 40nM, and 400nM, respectively, and the detection limit is 4nM (measured as a signal greater than 1 per minute). DETAILED DESCRIPTION

[0050] 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.

[0051] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out 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 25.0±3.0°C.

[0052] Embodiment 1:

[0053] Experimental samples: In the following example, the target is a natural amino acid molecule, the bifunctional cross-linking reagent is SPDP, the load molecule is FGGCD8, the guest molecule is phenylalanine (F), the functional linking group is thiol (-SH), the traction sequence is DDDDDDDD, the host molecule is cucurbituril [7], and the nanopore is α-HL (WT).

[0054] The load molecule FGGCD8 is a polypeptide FGGCDDDDDDDD (N-terminus → C-terminus), which is customized by Qiangyao Biotechnology Co., Ltd. It can be purchased from major polypeptide synthesis companies or synthesized by itself using solid-phase synthesis technology.

[0055] Cross-linking of amino acid AV IL with load molecules:

[0056] according to Figure 1 The steps in the above are to couple the amino acid molecule to be tested with the load molecule, and the detailed method includes the following steps:

[0057] 1) Prepare solutions of the amino acid molecule to be tested (solvent water, concentration 1 mM), the load molecule (solvent water, concentration 1 mM), the bifunctional cross-linking reagent SPDP (solvent DMF, concentration 4 mM), and the organic base NMM (solvent DMF, concentration 8 mM).

[0058] 2) reacting the amino acid molecule to be tested, the bifunctional cross-linking reagent and the organic base at a molar ratio of 1:1:2 for 2 hours to form an amino acid-bifunctional reagent complex.

[0059] 3) reacting the amino acid-bifunctional reagent complex with the load molecule at a molar ratio of 1:1 for 1 hour to obtain the amino acid molecule-load molecule composite probe to be detected.

[0060] The probe prepared according to the above connection method was characterized by HPLC-MS, and the following Figure 2 The molecular purity information shown in the figure shows that the purity of the molecular probes obtained by this method is higher than 95%. The four target amino acid molecules can be efficiently connected to the load molecules to form the probes to be detected.

[0061] Example 2: Nanopore single molecule measurement of the probe obtained in Example 1

[0062] The specific method is as follows:

[0063] 1) DPhPc was used to form a synthetic lipid bilayer with a diameter of 100 μm on a 25 μm thick polytetrafluoroethylene membrane (Goodfellow, Malvern, PA).

[0064] 2) The α-HL nanopore was embedded in a phospholipid bilayer, which divided the sample pool into two chambers, the cis and trans chambers, both of which contained 1.0 ml of buffer solution. 2 μL of amino acid molecule-load molecule composite probe (concentration of 200 μM) was added to the cis chamber, and 10 uL of cucurbituril [7] (concentration of 5 mM) was added to the cis chamber. The experiments were carried out in a 3.6 M KCl, 10 mM citric acid buffer (i.e., the buffer in the two chambers) at a pH of 5.0 and a temperature of 25.0 ± 3.0 °C.

[0065] 3) Experimental recording was performed at a voltage of +200 mV. The load molecules and the main molecules moved toward the nanopore driven by the host-guest interaction and the electrophoretic force, generating characteristic current signals, such as Figure 3 As shown in b (the current signal generated by the probe obtained by coupling glycine with the load molecule FGGCD8). The current blocking ratio, signal frequency and blocking time of the current signal are analyzed and compared to obtain the characteristic information and concentration information of the target to be measured.

[0066] Embodiment 3:

[0067] The concentration curve of the probe obtained in Example 1 (the probe obtained by coupling glycine with the load molecule FGGCD8) was measured, and its signal frequency was counted to obtain the number of characteristic signals generated per minute. The results are shown in Figure 4 The gradient concentrations of the samples to be detected were 40pM, 400pM, 4nM, 40nM, and 400nM, respectively, and the detection limit was 4nM (measured as a signal greater than 1 per minute).

[0068] The detection limit was determined as follows: 10 μL of test sample (FGGC(G)D8) with concentrations of 4 nM, 40 nM, 400 nM, 4 μM, and 40 μM and 10 μL of the main molecule cucurbituril [7] (concentration: 100 mM) were added to the nanopore single-channel detection system, and a voltage of +200 mV was applied for single-channel recording for more than 1 h. The recording results were subjected to signal frequency analysis, and the signal frequency (min) in the recording was calculated. -1 ), with a signal frequency of 1.52 at 4 nM as the sample detection limit, when the final sample concentration is lower than 4 nM, the signal frequency will be less than 1 min -1 , which cannot satisfy signal statistics and processing.

[0069] 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.

Claims

1. A nanopore detection probe generated by connecting a target molecule to be detected and a load molecule, which is obtained by coupling the target molecule to be detected and the load molecule through a bifunctional cross-linking reagent; in, The load molecule is composed of a guest interaction molecule, a functional linking group and a pulling sequence; The guest action molecule combines with the host action molecule and interacts with the nanopore in the detection system to generate a specific signal; the functional linking group provides the action site of the bifunctional cross-linking reagent, allowing it to combine with the target molecule to be detected; the traction sequence provides the power for the entire probe molecule to be driven by the electrophoretic force into the nanopore detection area.

2. The nanopore detection probe according to claim 1, characterized in that: The guest interaction molecule on the load molecule 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 functional linking group on the load molecule is selected from at least one of: allyl, thiol, amino, succinimide ester, and maleimide ester; Alternatively, the traction sequence on the load molecule is selected from at least one of: a polynucleotide, a random nucleotide sequence, a charged polypeptide, and a polymer.

3. The nanopore detection probe according to claim 1 or 2, characterized in that: The bifunctional cross-linking reagent is selected from at least one of 3-(2-pyridyldithio) propionic acid N-hydroxysuccinimide ester (SPDP), trans-2,5-dioxapyrrolidin-1-yl 4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexanecarboxylate (SMCC), 3,3'-dithiodipropionic acid di(N-hydroxysuccinimide) ester (DSP), maleimidoacetic acid succinimide ester (AMAS), iodoacetic acid-N succinimide (SIA), bromoacetic acid N-hydroxysuccinimide ester (SBA), and disuccinimidyl carbonate (DSC); Or, the main active molecule is selected from at least one of α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, other types of cyclodextrin and their derivatives, cucurbituril[5], cucurbituril[6], cucurbituril[7], cucurbituril[8], cucurbituril[10], and other types of cucurbituril derivatives; Alternatively, the target molecule to be detected is selected from any one of the following: small biological molecules, inorganic molecules, DNA, and proteins.

4. The method for preparing a nanopore detection probe generated by connecting a target molecule to be detected and a load molecule according to any one of claims 1 to 3, comprising the following steps: 1) reacting the target molecule to be detected, the bifunctional cross-linking reagent and the organic base to form a target molecule to be detected-bifunctional cross-linking reagent complex; 2) reacting the target molecule-bifunctional cross-linking reagent complex with a load molecule to obtain the nanopore detection probe.

5. The preparation method according to claim 4, characterized in that: In the step 1), the organic base is selected from at least one of TEA, NMM, DIPEA, Py, DBU, 2,6-lutidine, imidazole and NMI; Or, in the step 1), the molar ratio of the target molecule to be detected, the bifunctional cross-linking reagent and the organic base is (1-100):1:(2-10), and the reaction time is 1-4h; the target molecule to be detected is added in the form of an aqueous solution, the bifunctional cross-linking reagent is added in the form of a DMF solution, and the organic base is added in the form of a DMF solution; Or, in step 2), the molar ratio of the target molecule to be detected-bifunctional cross-linking reagent complex to the load molecule is (1-100):1, the reaction is carried out at room temperature for 1-8 hours; the load molecule is added in the form of an aqueous solution.

6. A method for qualitative and / or quantitative detection of a target molecule to be detected using the nanopore detection probe described in any one of claims 1 to 3, which is detection performed using a nanopore, comprising the following steps: firstly combining the guest acting molecule in the nanopore detection probe generated by connecting the target molecule to be detected and the load molecule with the host acting molecule, and then performing nanopore single channel recording.

7. The method according to claim 6, characterized in that: The nanopore single-channel recording refers to the collection of characteristic electrical signals using a nanopore; Furthermore, the characteristic electrical signal is collected at room temperature of 23±0.5°C; the signal sampling frequency is 100kHz, and the Bessel low-pass filter cut-off frequency is 10kHz; the characteristic current signal is analyzed by single-channel data using Clampfit software to collect the characteristic signal, and the data is statistically analyzed and analyzed using origin software.

8. The method according to claim 6 or 7, characterized in that: The nanopore is selected from biological nanopores with a diameter of less than 2 nm or solid nanopores with a diameter of less than 3 nm; the nanopore is specifically selected from α-HL and its mutants, MspA and its mutants, Phi29, Aerolysin, CsgG, PA63, ClyA, FhuA, SPP1, PET nanopores, glass nanopores, SiN x At least one of nanopore, Al2O3 nanopore, graphene nanopore, hybrid nanopore, carbon nanotube, DNA Origami nanopore.

9. The method according to any one of claims 6 to 8, characterized in that: The method specifically 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) embedding a 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 nanopore detection probe generated by connecting the target molecule to be detected and the load molecule to the cis chamber, and adding the main action molecule to the cis chamber at the same time; The above experiments were performed in 0.8–4.0 M KCl, 10 mM citrate buffer at pH 5.0–8.0 and 25.0 ± 3.0 °C; The molar ratio of the nanopore detection probe generated by connecting the target molecule to be detected and the load molecule to the main molecule is less than 1:10; (c) A fixed bias voltage in the range of +80–250 mV is applied to the nanopore single-channel test system and recorded. The load molecules and the host molecules move toward the nanopore driven by the host-guest interaction and the electrophoretic force, generating a characteristic current signal. (d) The current blockade ratio, signal frequency and blocking time of the current signal are analyzed and compared to obtain the characteristic information and concentration information of the target molecule to be measured.

10. The load molecule of claim 1, wherein the load molecule is composed of a guest interaction molecule, a functional linking group and a pulling sequence; The guest action molecule combines with the host action molecule and interacts with the nanopore in the detection system to generate a specific signal; the functional linking group provides the action site of the bifunctional cross-linking reagent, allowing it to combine with the target molecule to be detected; the traction sequence provides the power for the entire probe molecule to be driven by the electrophoretic force into the nanopore detection area.

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