Application of an MtMscL nanopore system in detecting polypeptides

Abstract

The present invention belongs to the field of nanopore detection, and particularly relates to the application of an MtMscL nanopore system in detecting polypeptides. The present invention provides an application of a nanopore system in detecting polypeptides, characterized in that the nanopore system includes a nanopore, an insulating membrane, a first medium and a second medium; the nanopore is embedded in the insulating membrane, the insulating membrane separates the first medium from the second medium, the nanopore provides a channel for connecting the first medium and the second medium, and after applying a driving force between the first medium and the second medium, the polypeptide located in the first medium interacts with the nanopore; the nanopore includes Mycobacterium tuberculosis large conductance mechanosensitive channel protein (MtMscL).

Description

[0001] This application claims priority to the Chinese invention patent application [CN2021110062502] filed on August 30, 2021, entitled “Bio-nanopore system for small molecule drug detection based on MtMscL”, and the Chinese invention patent application [CN2021116089782] filed on December 27, 2021, entitled “A biological nanopore system and its application”. The two priority invention patent applications are incorporated by reference in their entirety. Technical Field

[0002] The present invention belongs to the field of nanopore detection, and in particular relates to the application of an MtMscL nanopore system in detecting polypeptides. Background Art

[0003] Nanopore sensing is a single-molecule sensing technology with a detection principle similar to that of a Coulter counter. It offers real-time and direct monitoring at the single-molecule level, generally without the need for labeling or modification of the analyte. These advantages make nanopores an emerging technology for biosensing and bioassays. Most biological nanopores have diameters ranging from approximately 1 nm to approximately 4 nm (e.g., MspA, α-HL20, and the phi 29 DNA packaging motor), making them suitable for sensing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). However, sensing smaller molecules often requires labeling or adaptors, such as site-directed mutagenesis or modification of specific adaptors. For example, α-HL, with its limited pore size of approximately 1.4 nm, has limited its application to the analysis of ssDNA, RNA, or small molecules. Modification with cyclodextrins has enabled the direct detection of deoxyribonucleoside monophosphates (dNMPs) without the need for fluorescent labeling. However, modifying the pore size of biological nanopores through modification requires extensive bioengineering techniques. Although some studies have proposed drug molecule detection methods based on nanopore or drug channel interactions, these methods still require the combination of aptamers. Summary of the Invention

[0004] In view of this, the present invention provides an application of an MtMscL nanopore system in detecting polypeptides, characterized in that the nanopore system comprises a nanopore, an insulating membrane, a first medium, and a second medium; the nanopore is embedded in the insulating membrane, the insulating membrane separates the first medium from the second medium, the nanopore provides a channel connecting the first medium and the second medium, and after a driving force is applied between the first medium and the second medium, the polypeptide located in the first medium interacts with the nanopore; the nanopore comprises the Mycobacterium tuberculosis large conductance mechanosensitive channel protein (MtMscL).

[0005] In one embodiment, the nanopore comprises the amino acid sequence of any one of SEQ ID NOs: 1-3.

[0006] In one embodiment, the nanopore is a MtMscL C-terminal truncation, which is constructed by C-terminally truncating amino acid residues between positions 111 and 151 of SEQ ID NO: 1.

[0007] In one embodiment, the polypeptide has a molecular weight greater than 4 kD.

[0008] In one embodiment, the limit of detection of the polypeptide is 2 μM.

[0009] In one embodiment, the sequence of the MtMscL C-terminal truncation is shown in SEQ ID NO:2.

[0010] In one embodiment, the first medium is a 200 mM potassium chloride solution, 10 mM HEPES, and pH 7.5, and the second medium is a 2 M potassium chloride solution, 10 mM HEPES, and pH 7.5.

[0011] In one embodiment, the polypeptide comprises Complexin-1 or SNAP-25.

[0012] In one embodiment, the use comprises detecting the polypeptide in a whole blood sample.

[0013] In one embodiment, the use comprises detecting the presence and / or concentration of the polypeptide.

[0014] Beneficial effects

[0015] This study utilizes the mechanosensitive channel MtMscL as a nanopore for biosensing, demonstrating its ability to precisely sense peptides of varying lengths and types. Specifically, MtMscL can be embedded in an insulating membrane, exhibiting stable channel currents without the need for additional pressure stimulation. Directly adding a peptide to the resulting MtMscL nanopore system and applying a driving force triggers the peptide to interact with the MtMscL nanopore, generating a specific current signal. By analyzing the distinct current signals, different peptides can be detected and characterized (including their presence and / or concentration).

[0016] The present invention discovered that both MtMscL nanopores (wild-type MtMscL and the MtMscL C truncation (ΔC)) possess the ability to detect and characterize peptides. When detecting peptides, the MtMscL nanopores provided by the present invention do not require aptamer binding or antibody modification, and can directly detect and characterize peptides (including detecting the presence and / or concentration of the peptide) with high sensitivity. Furthermore, the MtMscL nanopores exhibit strong anti-interference capabilities and are capable of direct detection in bodily fluid samples (such as whole blood). Therefore, the MtMscL nanopores are suitable for a variety of applications, including detecting biomarkers associated with neurotransmitter release disorders, detecting the presence of synthetic peptides during production, and studying protein-peptide interactions. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be derived from these drawings without inventive effort.

[0018] Figure 1 The SDS-PAGE gel image of MtMscL (WT) and the protein structure and expression and purification molecular sieve ( Figure 1 A) and embedded hole trajectory diagram ( Figure 1 B), SDS-PAGE gel image and embedded hole trajectory map of the constructed truncated body ( Figure 1 C), SDS-PAGE gel images of MtMscL (WT) and MtMscL (△C) ( Figure 1 D) and protein structure and expression and purification of MtMscL (△C) molecular sieve ( Figure 1 E);

[0019] Figure 2 MtMscL (WT, Figure 2 A. Figure 2 B) and MtMscL (△C, Figure 2 A) IV curves in the voltage range of -50 mV to +50 mV.

[0020] Figure 3 Experimental diagram showing the detection and discrimination of antibiotics by MtMscL (WT) nanopore at a voltage of -50 mV;

[0021] Figure 4 This is an experimental diagram of the MtMscL (△C) nanopore detecting and distinguishing antibiotics at a voltage of -50 mV;

[0022] Figure 5This is the embedded pore trajectory diagram of the MtMscL (△C) mutant A nanopore at a voltage of 50 mV ( Figure 5 A) and experimental diagram of tobramycin detection ( Figure 5 B);

[0023] Figure 6 is MtMscL (WT) ( Figure 6 A) and MtMscL (△C) ( Figure 6 B) Plasmid map;

[0024] Figure 7 Figure 3. Experimental diagram of the MtMscL (WT) nanopore detecting and distinguishing Complexin-I peptides of different lengths at a voltage of -50 mV (electrolyte conditions: -trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5).

[0025] Figure 8 Figure 3 shows the experimental diagram of the MtMscL (△C) nanopore detecting and distinguishing Complexin-I peptides of different lengths at a voltage of -50 mV (electrolyte conditions: -trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5);

[0026] Figure 9 Schematic diagram of an electrophysiological experimental device according to an embodiment of the present invention;

[0027] Figure 10 The MtMscL (WT) nanopore detects different gradient concentrations of tobramycin ( Figure 10 A) and Complexin-I ( Figure 10 B) Characteristic signal frequency change diagram, and MtMscL (△C) nanopore detection of different gradient concentrations of Complexin-I ( Figure 10 C) Characteristic signal frequency change diagram;

[0028] Figure 11 Figure 3. Experimental diagram of the MtMscL (WT) nanopore detecting and distinguishing Complexin-I peptides of different lengths at a voltage of -50 mV (electrolyte conditions: -trans chamber: 30 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 300 mM KCl, 10 mM HEPES, pH 7.5).

[0029] Figure 12 Diagram of the experiment for detecting SNAP-25 by MtMscL (WT) nanopore;

[0030] Figure 13 This is an experimental diagram of the MtMscL (WT) nanopore detecting a whole blood sample containing tobramycin;

[0031] Figure 14 Figure 2 shows the experimental detection of neomycin by MtMscL (WT) nanopore at a voltage of -50 mV. DETAILED DESCRIPTION

[0032] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0033] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0034] As used in this specification, the term "about" typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1%, and even more typically + / - 0.5% of the stated value.

[0035] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "being within a range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and independent numerical values ​​within this range. For example, the description of a range of 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., as well as independent numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0036] Nanopore

[0037] The nanopore used in the present invention is a mechanosensitive channel of large conductance (MScL), preferably MtMscL (Mycobacterium tuberculosis large conductance mechanosensitive channel) or a variant thereof. Such variants (also understood as "mutants") can be naturally occurring variants expressed by an organism (e.g., Mycobacterium tuberculosis). Variants also include non-naturally occurring variants produced by recombinant techniques. In the present invention, "MtMscL variant," "mutant MtMscL," "mutant MtMscL," and "MtMscL mutant" have the same meaning unless otherwise indicated.

[0038] In one embodiment of the present invention, the nanopore may be a wild-type MtMscL.

[0039] In another embodiment of the present invention, the nanopore may be a MtMscL variant. Specifically, the nanopore may be a MtMscL truncated form. Furthermore, those skilled in the art may modify the aforementioned MtMscL (e.g., by mutation, truncation, fusion, chemical modification, etc.) to obtain corresponding MtMscL variants, depending on practical circumstances. Modification methods are well known in the art.

[0040] Object under test

[0041] The analyte is a charged substance. If the analyte has a net charge, it is charged. The analyte can be negatively or positively charged. If the analyte has a net negative charge, it is negatively charged. If the analyte has a net positive charge, it is positively charged. Suitable analytes are preferably small molecule drugs and peptides.

[0042] In one embodiment of the present invention, the analyte may be a small molecule drug. A small molecule drug may be a chemical compound. More specifically, a "small molecule drug" may be a drug having a molecular weight of 1000 g / mol or less (e.g., less than 800, 700, 600, 500, 400, 300, or 200 g / mol). Preferably, the small molecule drug may be an aminoglycoside antibiotic.

[0043] In another embodiment of the present invention, the analyte may be a polypeptide. "Polypeptide" refers to a peptide or protein comprising two or more amino acids linked by peptide bonds. Polypeptides may contain natural, modified, or synthetic amino acids. Polypeptides may also be modified naturally (e.g., through post-translational processing) or chemically (e.g., by amidation, acylation, cross-linking, etc.). Preferably, the polypeptide is greater than 4 kDa.

[0044] Nanopore system

[0045] A "nanopore system" comprises a nanoscale pore (abbreviated as "nanopore"), an insulating membrane, a first medium, and a second medium. In one embodiment of the present invention, the nanoscale pore is the Mycobacterium tuberculosis large conductance mechanosensitive channel (MtMscL) nanopore. Wild-type MtMscL is a homopentamer composed of two domains. Each monomer consists of a cytoplasmic N-terminal amphipathic helix, two transmembrane helices (TM1 and TM2), and a cytoplasmic C-terminal helix. TM1 is located on the inner side of the pore, forming a funnel-shaped structure; TM2 is located on the outer side of the pore, providing support. MtMscL has a single pore with a pore diameter (channel size) ranging from approximately 0.3 to 3.5 nm. MtMscL (ΔC) is structurally identical to wild-type MtMscL, except for the lack of the cytoplasmic C-terminal helix. The nanoscale pore allows the analyte to translocate from one side of the insulating membrane to the other.

[0046] In one embodiment of the present invention, the nanoscale pores are embedded in the insulating membrane. The insulating membrane (or, alternatively, the composite of the nanoscale pores and the insulating membrane) separates the first medium from the second medium, with the nanoscale pores providing a channel connecting the first and second media. When a driving force is applied between the first and second media, the analyte in the first medium interacts with the MscL nanopore, generating an electric current (i.e., an electrical signal). In this invention, the "first medium" refers to the medium in which the analyte is located when introduced into the nanopore system; the "second medium" refers to the other side of the "first medium," separated by the insulating membrane. In this invention, the driving force refers to the force that drives the interaction between the analyte and the nanopore, such as through electric potential, electroosmotic flow, or concentration gradient.

[0047] The first and second media may be the same or different, and may include a conductive liquid. The conductive liquid is an aqueous solution of an alkali metal halide, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), or sodium bromide (NaBr). In one embodiment of the present invention, the first and second media contain different concentrations of the conductive liquid. In other words, there is a difference in the concentration of the conductive liquid in the first and second media, which results in a difference in the osmotic pressure across the insulating membrane. The first and / or second media may also include a buffer, such as HEPES. The concentration of the first and / or second media may range from 30 mM to 3 M.

[0048] An insulating membrane is a membrane that has the ability to carry nanopores and block ionic currents that flow through non-nanopores. The insulating membrane can include a phospholipid membrane and / or a polymer membrane. Exemplary phospholipid membranes include DPHPC, DOPC, and E. coli lipids, and exemplary polymer membranes include triblock copolymer polymer membranes.

[0049] In one embodiment of the present invention, the nanopore system includes two electrolyte chambers separated by an insulating membrane to form a trans (-trans) compartment and a cis (-cis) compartment. The pores of the MtMscL channels are embedded in the insulating membrane, and only the MtMscL channels on the insulating membrane connect the two electrolyte chambers. When an electric potential is applied to the two electrolyte chambers, electrolyte ions in the solutions in the electrolyte chambers migrate through the MtMscL channels by electrophoresis.

[0050] The interaction between the nanopore and the analyte

[0051] The object to be detected may be in contact with the nanopore on either side of the insulating film. The object to be detected may be in contact with either side of the insulating film so that the object to be detected passes through the channel of the nanopore to reach the other side of the insulating film. In this case, the object to be detected interacts with the nanopore when it passes through the insulating film via the channel of the hole. Alternatively, the object to be detected may be in contact with the side of the insulating film, and the side of the insulating film may allow the object to be detected to interact with the nanopore so that it is separated from the nanopore and stays on the same side of the insulating film. The object to be detected may interact with the nanopore in any way and at any site. The object to be detected may also collide with the nanopore and interact with the nanopore so that it is separated from the nanopore and stays on the same side of the insulating film.

[0052] During the interaction between the analyte and the nanopore, the analyte affects the current flowing through the nanopore in a manner specific to the analyte, i.e., the current flowing through the nanopore is characteristic for the specific analyte. Control experiments can be performed to determine the effect of a specific analyte on the current flowing through the nanopore, and then to identify the specific analyte in the sample or to determine whether the specific analyte is present in the sample. More specifically, the current pattern obtained by detecting the analyte can be compared with a known current pattern obtained under the same conditions using a known analyte to identify the presence or concentration of the analyte.

[0053] The nanopore system of the present invention may further comprise one or more measuring devices for measuring the current flowing through the nanopore, such as a patch clamp amplifier or a data acquisition device.

[0054] Example 1: Materials and Methods

[0055] Chemicals: Sodium chloride (NaCl, >99.0%, CAS#7647-14-5), yeast extract (CAS#8013-01-2), trypsin (CAS#73049-73-7), ampicillin sodium salt (≥98.5%, CAS#69-52-3), Tris (≥99.9%, CAS#77-86-1), imidazole (≥99%, CAS#288-32-4), n-dodecyl-β-D-maltose Dimethicone (DDM) (≥99%, CAS#69227-93-6), isopropyl-β-D-thiogalactopyranoside (IPTG) (≥99%, CAS#367-93-1), phenylmethylsulfonyl fluoride (PMSF) (≥99.%, CAS#329-98-6), and 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES, >99.5%, CAS#7365-45-9) were purchased from Sigma-Aldrich. Escherichia coli polar lipid extract (100600P) was purchased from Avanti.

[0056] MtMscL expression and purification: The corresponding plasmid of the Mycobacterium tuberculosis large conductance mechanosensitive channel (MscL) protein (i.e., MtMscL) was constructed using the pET28b-6His vector. Coli BL21 (DE3) cells containing pET28b-MscL-6His were cultured in Luria-Bertani (LB) medium with a kanamycin concentration of 50 μg / mL and a culture temperature of 37°C. When the OD 600When the p-value reached 0.8-1.0, protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 20°C for 12 h. Bacteria were harvested by centrifugation at 3,800 rpm for 15 minutes and resuspended in lysis buffer A (20 mM Tris, 100 mM NaCl, 5 mM β-mercaptoethanol, pH 7.4). Cells were disrupted and lysed by high pressure with 1 mM phenylmethylsulfonyl fluoride (PMSF). Extraction was performed with 1% (wt / vol) n-dodecyl-β-D-maltopyranoside (DDM) for 2 h. After centrifugation at 18,000 rpm for 1 h, the supernatant was incubated with Ni-NTA resin (Qiagen) for 2 h. The mixture was washed with buffer B (50 mM Tris, 100 mM NaCl, 20 mM imidazole, 0.025% DDM, pH 7.4), and the target protein was eluted with buffer C (50 mM Tris, 100 mM NaCl, 500 mM imidazole, 0.025% DDM, pH 7.4). The protein was further purified by size exclusion chromatography (Superdex 200 increase 10 / 300 GL, GE Healthcare). The column and purified protein were pre-equilibrated with buffer D (200 mM NH4Ac, 0.025% DDM, 10% glycerol, pH 7.4). Peak fractions were identified by SDS-PAGE analysis. This method was applicable to both wild-type and mutant proteins.

[0057] The proteins involved in the embodiments of the present invention include MtMscL (WT) and MtMscL (△C). First, an MtMscL vector or a MtMscL C-terminally truncated vector is constructed, a plasmid map is constructed using SnapGene, and then a plasmid is synthesized. The plasmid is then used to express and purify the MtMscL (WT) or MtMscL (△C) protein. The plasmid maps of MtMscL (WT) and MtMscL (△C) involved in the present invention are as follows: Figure 6 A and Figure 6 As shown in B.

[0058] The sequence information of MtMscL (WT) is: MLKGFKEFLARGNIVDLAVAVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQVVLLTEIRDLLAQTNGDSPGRHGGRGTPSPTDGPRASTESQ (SEQ ID NO: 1)

[0059] The sequence information of MtMscL (△C) is: MLKGFKEFLARGNIVDLAVAVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQ (SEQ ID NO: 2)

[0060] The MtMscL (△C) protein is a truncated mutant with its C-terminus (cytoplasmic region) shortened. The C-terminus is structurally more prominent, and after truncation, the majority of the MtMscL (△C) protein is the transmembrane region. Furthermore, this truncation results in higher protein expression and enhanced pore-forming capacity, making it suitable for detection and analysis.

[0061] Membrane-bound and single-channel recordings: Experiments were performed in a vertical sampling cell provided by Warner Instruments. All current traces were recorded using a HEKAepc10 USB patch clamp amplifier at a sampling frequency of 9900 Hz. A 150 μm cuvette was pre-coated with 1 μL of 25 mg / mL phospholipids from an Escherichia coli extract. Then, 1 mL of electrolyte solution (30 mM NaCl, 10 mM HEPES, pH 7.0 in the -trans chamber and 300 mM NaCl, 10 mM HEPES, pH 7.0 in the -cis chamber) was added to each side of the cell. A 1 mL pipette was then used to aspirate approximately two-thirds of the electrolyte solution from the -cis chamber. This electrolyte solution was then pumped into the -cis chamber of the cell. When the average current approached 0 pA, a planar phospholipid bilayer was formed (i.e., the electrolyte solution in the cis chamber was pulled up by the pipette to form the phospholipid bilayer). After the planar phospholipid bilayer was formed, a solution of MtMscL protein (wild-type and / or mutant) was added to the -cis chamber. When MtMscL was embedded in a planar phospholipid bilayer membrane, the current changed significantly, leading to subsequent experiments.

[0062] Antibiotic testing: Antibiotics were detected using MtMscL nanopores (wild-type and mutant forms). Once a stable MtMscL nanopore was formed on a planar phospholipid bilayer membrane, a background signal was recorded and observed at -50 mV for 20 minutes. The antibiotic to be tested was then added to the -cis chamber of the sample reservoir, and a voltage was applied, and the current signal was recorded.

[0063] Protein peptide detection: MtMscL nanopores are used to detect proteins and peptides. Once a stable MtMscL nanopore is formed on a planar phospholipid bilayer membrane, the background signal is recorded and observed at -50 mV for 20 minutes. The protein or peptide to be detected is then added to the cis chamber of the sample reservoir. A voltage is then applied and the current signal is recorded.

[0064] Electrophysiological data analysis: In this experiment, Clampfit software was used to process the electrophysiological data, and Origin software was used to draw the graphs.

[0065] Example 2: Structure of MtMscL channel and its electrophysiological testing

[0066] Structure of the MtMscL channel:

[0067] like Figure 1 As shown in Figure A, the MtMscL (WT) channel is a homopentamer composed of two domains. Each monomer consists of a cytoplasmic N-terminal amphipathic helix, two transmembrane helices (TM1 and TM2), and a cytoplasmic C-terminal helix. TM1 is located on the inner side of the pore, forming a funnel-shaped structure; TM2 is located on the outer side of the pore, providing support. MtMscL has a single pore with a pore diameter (channel size) ranging from approximately 0.3 to 3.5 nm.

[0068] Electrophysiological testing of MtMscL channels:

[0069] Single-channel electrophysiological studies of MtMscL channels were performed in planar phospholipid bilayer membranes. The schematic diagram of the electrophysiological apparatus is shown in Figure 2. Figure 9 As shown. Figure 1 As shown in Figure B, in an electrolyte solution (-trans chamber: 30 mM NaCl, 10 mM HEPES, pH 7.5; -cis chamber: 300 mM NaCl, 10 mM HEPES, pH 7.5), the MtMscL (WT) channel generates stable channel currents of 38 pA and 175 pA at voltages of +50 mV and +200 mV, respectively (this recording is a representative event from a single pore). These experiments demonstrate that the MtMscL channel can be used for electrophysiological sensing of planar phospholipid membranes.

[0070] The present invention also constructed a variety of truncations based on MtMscL (WT), and verified whether the constructed truncations could be expressed and embedded by SDS-PAGE gel images and embedded hole trajectory diagrams. The experimental results are as follows Figure 1 C. Figure 1 As shown in D. Among them, the truncated form with truncated N-terminus cannot be expressed, the truncated form with truncated C-terminus (cut off amino acids 97-151) cannot embed into the hole, and only the truncated form with truncated C-terminus (cut off amino acids 111-151, expressed as MtMscL (△C)) can be expressed and embed into the hole. Figure 1 E shows that MtMscL (△C) lacks the cytoplasmic C-terminal helix.

[0071] Figure 2Shown are the IV curves of MtMscL (WT) and MtMscL (ΔC) in the voltage range of −50 mV to +50 mV in electrolyte solution (−trans chamber: 30 mM NaCl, 10 mM HEPES, pH 7.5; −cis chamber: 300 mM NaCl, 10 mM HEPES, pH 7.5). Figure 2 The IV curves of A-2B show that MtMscL (WT) and MtMscL (△C) remain stable within this voltage range without high voltage gating (each value is represented by the mean ± SD, MtMscL (WT) is from 5 repeated experiments; MtMscL (△C) is from 7 repeated experiments). Figure 2 The slope of the IV curve shown in A-2B can be used to obtain the conductance of MtMscL (WT) and MtMscL (△C) (the slope is the conductance value), which are 0.616±0.007 nS and 0.683±0.007 nS, respectively.

[0072] Both MtMscL (WT) and MtMscL (△C) proteins are pentameric. MtMscL (WT) is the full-length protein, while MtMscL (△C) lacks the C-terminal domain. As shown above, MtMscL (WT) and MtMscL (△C) exhibit similar electrophysiological properties, except for a slightly larger conductance in MtMscL (△C) compared to MtMscL (WT).

[0073] Example 3: Small molecule drug detection based on MtMscL channel

[0074] In this example, MtMscL channels (MtMscL (WT) and MtMscL (ΔC)) were used to detect small molecule drugs (molar mass less than 1000 g / mol), namely gentamicin sulfate (MW: 561.65), tobramycin sulfate (MW: 565.595), and kanamycin sulfate (MW: 582.577).

[0075] When a stable MtMscL nanopore is formed on a planar phospholipid bilayer (a type of insulating membrane), a single antibiotic to be tested is added to the -cis chamber of the sample reservoir. A voltage of -50 mV is then applied and the current signal is recorded. Detection experiments were performed in 300 mM NaCl (-cis chamber) and 30 mM NaCl (-trans chamber) in the electrolyte solution of 10 mM HEPES, pH 7.0.

[0076] When the analyte passes through the MtMscL nanopore, a specific current signal is generated ( Figure 3 and Figure 4). Among them, the typical peak current blockade rate of gentamicin signal of MtMscL (WT) is 40%, and the peak retention time is 2 ms; the typical current blockade rate of tobramycin signal is 32%, and the peak retention time is 5 ms; the typical current blockade rate of kanamycin signal is 30%, and the peak retention time is 5 ms ( Figure 3 The typical peak current blockade rate of gentamicin signal in MtMscL (△C) is 40%, and the peak retention time is 2 ms; the typical current blockade rate of tobramycin signal is 35%, and the peak retention time is 2 ms; the typical current blockade rate of kanamycin signal is 31%, and the peak retention time is 1 ms ( Figure 4 In addition, the current blockade rate of the typical neomycin (MW: 614.644) signal of MtMscL (WT) was 35%, and the peak retention time was 1.5 ms ( Figure 14 ).

[0077] When a bias voltage is applied, the nanopore captures the analyte antibiotic, and due to differences in the size, structure, and charge of the antibiotics, a unique current blockade event is observed. Gentamycin sulfate, tobramycin sulfate, and kanamycin sulfate are all hydrophilic and positively charged, with similar molecular weights, but the MtMscL nanopore can directly distinguish these aminoglycoside antibiotics, demonstrating the ability to robustly and sensitively detect and distinguish different small molecule drugs. In other words, the MtMscL nanopore provided by the present invention can be used not only to detect and distinguish aminoglycoside antibiotics (such as gentamicin, tobramycin, and kanamycin), but also other small molecule drugs with a molar mass of less than 1000 g / mol (such as epinephrine hydrochloride), and has a wide range of applications.

[0078] like Figure 10 As shown in Figure A, the MtMscL (WT) nanopore detects different concentration gradients of tobramycin sulfate, generating distinct signal frequencies. This linear relationship exists between the tobramycin sulfate concentration and the corresponding signal frequency. Therefore, the resulting signal frequency can also be used to measure the concentration of small molecule drugs (such as tobramycin sulfate). Furthermore, the detection limit of the MtMscL nanopore for small molecule drugs is 100 nM.

[0079] In addition, a mutation was performed on MtMscL (△C) by mutating the 20th amino acid alanine (A) of MtMscL (△C) to cysteine ​​(C) (denoted as mutant A20C). The mutation site is located within the pore of MtMscL (△C). The sequence information of this mutant is: MLKGFKEFLARGNIVDLAVCVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQ (SEQ ID NO: 3)

[0080] The mosaic diagram of mutant A20C at 50 mV and the result diagram of tobramycin detection are shown as follows: Figure 5 A and Figure 5 As shown in B. The above experimental results show that even if MtMscL (△C) is mutated (such as amino acid substitution), the resulting mutant still has properties similar to MtMscL (△C) and the mutant still has the ability to detect small molecule drugs.

[0081] Example 4: Protein and peptide detection based on MtMscL channel

[0082] In this example, MtMscL channels (MtMscL (WT) and MtMscL (ΔC)) were used to detect and characterize Complexin-I polypeptides of different lengths.

[0083] The sequence information and molecular weight of Complexin (1-134) are: MEFVMKQALGGATKDMGKMLGGDEEKDPDAAKKEEERQEALRQAEEERKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQAAMEANSEGSLTRPKKAIPPGCGDEPEEEDESILDTVIKYLPGPLQDMFKK (15.122 kDa) (SEQ ID NO: 4)

[0084] The sequence information and molecular weight of Complexin (26-134) are: KDPDAAKKEEERQEALRQAEEERKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQAAMEANSEGSLTRPKKAIPPGCGDEPEEEDESILDTVIKYLPGPLQDMFKK (12.466 kDa) (SEQ ID NO: 5)

[0085] The sequence information and molecular weight of Complexin (48-83) are: RKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQ (4.355 kDa) (SEQ ID NO: 6)

[0086] When a stable MtMscL nanopore is formed on a planar phospholipid bilayer membrane, Complexin-I polypeptides of different lengths to be detected are added to the -cis chamber of the sample reservoir (the polypeptide is charged in the nanopore system), and then a voltage of -50 mV is applied and the current signal is recorded.

[0087] This example tests the detection of Complexin-I of different lengths using the MtMscL (WT) nanopore at a voltage of -50 mV and two electrolyte conditions. The detection results in the -trans chamber: 30 mM KCl, 10 mM HEPES, pH 7.5; the -cis chamber: 300 mM KCl, 10 mM HEPES, pH 7.5 electrolyte conditions are shown in Figure 2. Figure 11 As shown, the peak current blockade rate of the typical Complexin (1-134) signal of MtMscL (WT) is 35%, and the peak retention time is 5 ms; the current blockade rate of the typical Complexin (26-134) signal is 25%, and the peak retention time is 1 ms; the current blockade rate of the typical Complexin (48-83) signal is 17%, and the peak retention time is 0.5 ms.

[0088] The test results were as follows: the electrolyte conditions of -trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5 Figure 7 The peak current blockade rate for the typical Complexin (1-134) signal of MtMscL (WT) was 9.91%, with a peak retention time of 4.94 ms; the current blockade rate for the typical Complexin (26-134) signal was 31.32%, with a peak retention time of 7.76 ms; and the current blockade rate for the typical Complexin (48-83) signal was 6.65%, with a peak retention time of 4.75 ms. The experimental results demonstrated that MtMscL (WT) was more effective in distinguishing different peptides under the electrolyte conditions of "-trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5," with significant improvements in the peak retention time of some peptide signals.

[0089] Based on this, under the electrolyte conditions of "-trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5", MtMscL (△C) was used to detect Complexin-I of different lengths. The peak current blockade rate of the typical Complexin (1-134) signal was 20.08%, and the peak retention time was 4.94 ms; the current blockade rate of the typical Complexin (26-134) signal was 30.03%, and the peak retention time was 5.01 ms; the current blockade rate of the typical Complexin (48-83) signal was 15.18%, and the peak retention time was 4.85 ms ( Figure 8 The above experiments showed that both MtMscL (WT) and MtMscL (△C) have the ability to detect and distinguish different peptides.

[0090] like Figure 10 B and Figure 10 As shown in C, the MtMscL nanopore (MtMscL (WT) and MtMscL (△C)) detects Complexin with different concentration gradients, which will produce different signal frequencies, and the concentration of Complexin is linearly related to the corresponding signal frequency. Therefore, the concentration of polypeptides (such as Complexin) can also be detected by the obtained signal frequency. Among them, MtMscL (WT) has higher sensitivity. In other words, when detecting lower concentrations of analytes or analytes of the same concentration, MtMscL (WT) detects more signals (for example, more signal frequencies). In addition, the lowest polypeptide concentration at which a signal can be detected in the experiment of this embodiment is 2 μM. Figure 10 B It can also be seen that the detection limit of the MtMscL nanopore for peptide detection is 2 μM.

[0091] This example also uses MtMscL (WT) to detect SNAP-25. The experimental results are as follows: Figure 12 The sequence information and molecular weight of SNAP-25 are: MAEDADMRNELEEMQRRADQLADESLESTRRMLQLVEESKDAGIRTLVMLDEQGEQLERIEEGMDQINKDMKEAEKNLTDLGKFCGLCVCPCNKLKSSDAYKKAWGNNQDGVVASQPARVVDEREQMAISGGFIRRVTNDARENEMDENLEQVSGIIGNLRHMALDMGNEIDTQNRQIDRIMEKADSNKTRIDEANQRATKMLGSG (23.315 kDa) (SEQ ID NO: 7).

[0092] The potential principles of MtMscL detection of peptides may include: (1) amino acids on the peptide molecule interact with amino acids in or near the MtMscL nanopore (e.g., charge attraction, hydrophilic / hydrophobic effects, etc.) and generate current signals; (2) peptide molecules move in the electrolyte solution, collide with the MtMscL nanopore and generate current signals; (3) linear peptide molecules pass through the MtMscL nanopore and generate current signals.

[0093] In summary, this example demonstrates the ability of MtMscL to detect and characterize various peptides, such as Complexin-I and SNAP-25. Both Complexin-I and SNAP-25 are involved in vesicle trafficking. Therefore, the MtMscL provided by the present invention can be used in a variety of applications, including detecting biomarkers associated with neurotransmitter release disorders, detecting the presence of synthetic peptides during production, and studying protein-peptide interactions.

[0094] Example 5: Whole blood detection based on MtMscL channel

[0095] This example tests the ability of the MtMscL (WT) nanopore to detect small molecule drugs in whole blood samples. The whole blood sample used in this example is a purchased rabbit whole blood sample, to which a certain concentration of small molecule drug (in this example, tobramycin) is added to prepare a stock solution with a concentration of 100 μM of the small molecule drug. 10 μL of the stock solution is added to the detection system (to achieve a final concentration of 1 μM of the small molecule drug in the whole blood sample) for detection. Figure 13 As shown, the above experimental results indicate that MtMscL nanopores can directly detect small molecule drugs (such as tobramycin) in whole blood samples.

[0096] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. Use of a nanopore system for detecting polypeptides for non-diagnostic purposes, Characterized in that, The nanopore system includes a nanopore, an insulating membrane, a first medium and a second medium; the nanopore is embedded in the insulating membrane, the insulating membrane separates the first medium from the second medium, the nanopore provides a channel connecting the first medium and the second medium, and after applying a driving force between the first medium and the second medium, the polypeptide in the first medium interacts with the nanopore; the nanopore includes Mycobacterium tuberculosis large conductance mechanosensitive channel protein (MtMscL); the nanopore includes the amino acid sequence of any one of SEQ ID NO: 1-3.

2. The use according to claim 1, Characterized in that, The nanopore is a C-terminal truncation of MtMscL, and the construction method of the C-terminal truncation of MtMscL is to truncate the amino acid residues between positions 111 and 151 of SEQ ID NO: 1 at the C-terminus.

3. The use according to claim 1, Characterized in that, The molecular weight of the polypeptide is greater than 4 kD.

4. The use according to claim 1, Characterized in that, The detection limit of the polypeptide is 2 μM.

5. The use according to claim 2, Characterized in that, The sequence of the C-terminal truncation of MtMscL is shown in SEQ ID NO:

2.

6. The use according to claim 1, Characterized in that, The first medium is 200 mM potassium chloride solution, 10 mM HEPES and pH 7.5, and the second medium is 2 M potassium chloride solution, 10 mM HEPES and pH 7.

5.

7. The use according to claim 1, Characterized in that, The polypeptide includes Complexin-I or SNAP-25.

8. The use according to claim 1, Characterized in that, The use includes detecting the polypeptide in a whole blood sample.

9. The use according to claim 1, Characterized in that, The use includes detecting the presence and / or concentration of the polypeptide.

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