Application of MtMscL nanopore system in detecting small molecule drugs

Abstract

The present invention belongs to the field of nanopore detection, and particularly relates to the application of an MtMscL nanopore system in detecting small molecule drugs. The present invention provides an application of a nanopore system in detecting small molecule drugs, 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 applying a driving force between the first medium and the second medium, the small molecule drug located in the first medium interacts with the nanopore; the nanopore comprises Mycobacterium tuberculosis large conductance mechanosensitive channel protein (MtMscL).

Description

[0001] This application claims the priority of the Chinese invention patent application [CN2021110062502], titled "Bio-nanopore system for small molecule drug detection based on MtMscL", filed on August 30, 2021, and the Chinese invention patent application [CN2021116089782], titled "A bio-nanopore system and its application", filed on December 27, 2021. The two priority invention patent applications are incorporated herein by reference in their entireties. Technical Field

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

[0003] Nanopore sensing is a single-molecule sensing technology with a detection principle similar to that of a Kurt counter. This technology has the characteristics of real-time and direct monitoring at the single-molecule level, and generally does not require labeling or modification of the analyte. These advantages make nanopores an emerging technology for biosensing and biodiagnostics. Most biological nanopores have diameters between approximately 1 nm and approximately 4 nm (such as MspA, α-HL20, and phi 29 DNA packaging motors), and are suitable for sensing single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). However, for sensing smaller molecules, labeling or aptamers are usually required, such as site-directed mutagenesis or modification of specific adapters. Taking α-HL as an example, its limited pore diameter is about 1.4 nm, so its application range is limited to the analysis of ssDNA, RNA, or small molecules. By using cyclodextrin modification, it can be used for the direct detection of deoxynucleoside monophosphates dNMPs without fluorescence labeling. However, changing the pore diameter of biological nanopores by modification means requires a large amount of bioengineering technology assistance. Although there are now studies proposing methods for detecting drug molecules 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 a nanopore system in detecting small molecule drugs, 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 small molecule drug located in the first medium interacts with the nanopore; the nanopore includes 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 NO: 1-3.

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

[0007] In one embodiment, the molar mass of the small molecule drug is less than 1000 g / mol.

[0008] In one embodiment, the small molecule drug is an aminoglycoside antibiotic.

[0009] In one embodiment, the detection limit of the small molecule drug is 100 nM.

[0010] In one embodiment, the sequence of the C-terminal truncation of MtMscL is as shown in SEQ ID NO: 2.

[0011] In one embodiment, the first medium is 300 mM sodium chloride solution, 10 mM HEPES and pH 7.0, and the second medium is 30 mM sodium chloride solution, 10 mM HEPES and pH 7.0.

[0012] In one embodiment, the aminoglycoside antibiotic comprises one or more of gentamicin, tobramycin, neomycin, kanamycin or a pharmaceutically acceptable salt thereof.

[0013] In one embodiment, the application includes detecting the small molecule drug in a whole blood sample.

[0014] In one embodiment, the application includes detecting the presence and / or concentration of the small molecule drug.

[0015] Beneficial effects

[0016] In the present invention, the mechanosensitive channel MtMscL is used as a nanopore for biosensing, and it is demonstrated that MtMscL has the ability to precisely sense different small molecule drugs. Specifically, MtMscL can be embedded in an insulating membrane, and a stable channel current can be presented without additional pressure stimulation. After directly adding a small molecule drug to the formed MtMscL nanopore system and applying a driving force, the small molecule drug (such as an aminoglycoside antibiotic) can block the MtMscL nanopore and cause a specific blocking current signal. By analyzing different blocking times and blocking current signals, different small molecule drugs can be detected and distinguished.

[0017] The present invention discovers that both the MtMscL nanopores (wild-type MtMscL and MtMscL C-truncated body (△C)) have the ability to detect and distinguish different small molecule drugs. When detecting small molecule drugs, the MtMscL nanopores provided by the present invention do not require aptamer binding or antibody modification, and can directly detect and characterize small molecule drugs with high sensitivity (including detecting the presence of small molecule drugs and / or the concentration of small molecule drugs). In addition, the MtMscL nanopores also exhibit strong anti-interference ability and can directly detect body fluid samples (such as whole blood samples). Therefore, the MtMscL nanopores are suitable for various application scenarios such as detecting blood drug concentration. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0019] Figure 1 It is the SDS-PAGE gel diagram of MtMscL (WT) and the protein structure, expression and purification molecular sieve (A) and the pore-insertion trajectory diagram (B), the SDS-PAGE gel diagram and pore-insertion trajectory diagram (C) of the constructed truncated body, the SDS-PAGE gel diagram of MtMscL (WT) and MtMscL (△C) (D) and the protein structure and expression purification molecular sieve (E) of MtMscL (△C);

[0020] Figure 2 It is the I-V curve diagram of MtMscL (WT) and MtMscL (△C) in the voltage range from -50 mV to +50 mV;

[0021] Figure 3 It is the experimental diagram of MtMscL (WT) nanopore detecting and distinguishing antibiotics at a voltage of -50 mV;

[0022] Figure 4 It is the experimental diagram of MtMscL (△C) nanopore detecting and distinguishing antibiotics at a voltage of -50 mV;

[0023] Figure 5 It is the pore-insertion trajectory diagram (A) and the experimental diagram of detecting tobramycin (B) of MtMscL (△C) mutant A nanopore at a voltage of 50 mV;

[0024] Figure 6 It is the plasmid maps of MtMscL (WT) (A) and MtMscL (△C) (B);

[0025] Figure 7 Experimental diagram for detecting and differentiating Complexin-I polypeptides of different lengths by the MtMscL(WT) nanopore 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 8 Experimental diagram for detecting and differentiating Complexin-I polypeptides of different lengths by the MtMscL(ΔC) nanopore 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);

[0027] Figure 9 Schematic diagram of the electrophysiological experimental device of the embodiment of the present invention;

[0028] Figure 10 Graph of the change in the characteristic signal frequency of the MtMscL(WT) nanopore for detecting different gradient concentrations of tobramycin (A) and Complexin-I (B), and the graph of the change in the characteristic signal frequency of the MtMscL(ΔC) nanopore for detecting different gradient concentrations of Complexin-I (C);

[0029] Figure 11 Experimental diagram for detecting and differentiating Complexin-I polypeptides of different lengths by the MtMscL(WT) nanopore 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);

[0030] Figure 12 Experimental diagram of the MtMscL(WT) nanopore for detecting SNAP-25;

[0031] Figure 13 Experimental diagram of the MtMscL(WT) nanopore for detecting whole blood samples containing tobramycin;

[0032] Figure 14 Experimental diagram of the MtMscL(WT) nanopore for detecting neomycin at a voltage of -50 mV. Detailed implementation manner

[0033] To make the objectives, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part rather than all of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0034] It should be noted that, in this document, the term "comprising", "including" or any other variation thereof is intended to cover a non-exclusive inclusion, such that a process, method, article or device comprising a series of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article or device. Without further limitation, an element defined by the phrase "comprising a..." does not exclude the presence of additional identical elements in the process, method, article or device comprising such element.

[0035] 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% of the stated value, and even more typically + / - 0.5% of the stated value.

[0036] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that such a description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of the range should be considered to have specifically disclosed all possible sub-ranges and the individual numerical values within that range. For example, a description of a range should be considered to have specifically disclosed sub-ranges 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., and the individual numbers within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0037] nanopore

[0038] The nanopores used in the present invention are Mechanosensitive channels of large conductance (MscL), preferably MtMscL (Mycobacterium tuberculosis large conductance mechanosensitive channel) or variants thereof. The variants (which can also be understood as "mutants") can be naturally occurring variants expressed by organisms (such as Mycobacterium tuberculosis). Variants also include non-naturally occurring variants produced by recombinant techniques. In the present invention, "MtMscL variant", "mutant MtMscL", "MtMscL mutant", "MtMscL mutant" have the same meaning unless otherwise specified.

[0039] In one embodiment of the present invention, the nanopore can be wild-type MtMscL.

[0040] In another embodiment of the present invention, the nanopore can be a MtMscL variant. Specifically, the nanopore can be a MtMscL truncation. Additionally, those skilled in the art can also modify the above-mentioned MtMscL according to the actual situation (for example, any mutation, truncation, fusion, chemical modification, etc.) to obtain the corresponding MtMscL variant, and the modification means are well-known in the art.

[0041] Analyte

[0042] The analyte is a charged substance. It is charged if it has a net charge. The analyte can be negatively charged or positively charged. It is negatively charged if it has a net negative charge. It is positively charged if it has a net positive charge. Suitable analytes are preferably small molecule drugs and polypeptides.

[0043] In one embodiment of the present invention, the analyte can be a small molecule drug. A small molecule drug can be a compound. More specifically, a "small molecule drug" can be a drug with a molecular weight of 1000 g / mol or lower (for example, less than 800, 700, 600, 500, 400, 300 or 200 g / mol). Preferably, the small molecule drug can be an aminoglycoside antibiotic.

[0044] In another embodiment of the present invention, the analyte can be a polypeptide. A "polypeptide" refers to a peptide or protein containing two or more amino acids linked by peptide bonds. Polypeptides can contain natural, modified or synthetic amino acids. Polypeptides can also be naturally modified (such as by post-translational processing) or chemically modified (such as amidation, acylation, crosslinking, etc.). Preferably, the polypeptide can be a polypeptide with a molecular weight greater than 4 kD.

[0045] Nanopore system

[0046] The "nanopore system" includes pores with nanoscale dimensions (abbreviated as "nanopores"), an insulating membrane, a first medium, and a second medium. In one embodiment of the present invention, the pores with nanoscale dimensions are Mycobacterium tuberculosis large conductance mechanosensitive channel (MtMscL) nanopores. Wild-type MtMscL is a homotetramer composed of two domains, and 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 inside the pore channel and forms a funnel-shaped structure; TM2 is located outside the pore channel and serves as a support. MtMscL has a pore channel, and its pore diameter (channel size) ranges from approximately 0.3 - 3.5 nm. MtMscL(△C) has the same structure as wild-type MtMscL except for lacking the cytoplasmic C-terminal helix. The pores with nanoscale dimensions allow the analyte to translocate from one side of the insulating membrane to the other side.

[0047] In one embodiment of the present invention, the pores with nanoscale dimensions are embedded in the insulating membrane, and the insulating membrane (which can also be understood as the complex of the pores with nanoscale dimensions and the insulating membrane) separates the first medium from the second medium, and the pore channel of the pores with nanoscale dimensions provides a channel connecting the first medium and the second medium; after applying a driving force between the first medium and the second medium, the analyte in the first medium interacts with the MscL nanopore to form a current (i.e., an electrical signal). In the present invention, the "first medium" refers to the medium in which the analyte is located when it is added to the nanopore system; the "second medium" refers to the other side of the "first medium" among the two parts of the medium separated by the insulating membrane. In the present invention, the driving force refers to the force that drives the interaction between the analyte and the nanopore through methods such as electric potential, electroosmotic flow, and concentration gradient.

[0048] The first medium and the second medium can be the same or different, and the first medium and the second medium can include a conducting liquid. The conducting liquid is an aqueous solution of alkali metal halide, specifically sodium chloride (NaCl), lithium chloride (LiCl), cesium chloride (CsCl), potassium chloride (KCl), sodium bromide (NaBr). In one embodiment of the present invention, the concentrations of the conducting liquids contained in the first medium and the second medium are different. In other words, there is a difference in the concentrations of the conducting liquids in the first medium and the second medium, thereby resulting in a difference in the osmotic pressure on both sides of the insulating membrane. The first medium and / or the second medium can also include a buffer solution, such as HEPES. The concentration range of the first medium and / or the second medium can be 30 mM - 3 M.

[0049] An insulating membrane refers to a membrane that has the ability to carry nanopores and block the ionic current passing through non-nanopores. The insulating membrane may include a phospholipid membrane and / or a polymer membrane. Exemplary phospholipid membranes include DPHPC, DOPC, E. coli lipid, and exemplary polymer membranes include triblock copolymer polymer membranes.

[0050] In a specific embodiment of the present invention, the nanopore system includes two electrolyte chambers separated by an insulating membrane to form a trans compartment and a cis compartment. The pore of the MtMscL channel is embedded in the insulating membrane, and only the MtMscL channel on the insulating membrane connects the two electrolyte chambers. When an electric potential is applied to the two electrolyte chambers, the electrolyte ions in the solution in the electrolyte chambers move electrophoretically and pass through the MtMscL channel.

[0051] The interaction between the nanopore and the analyte

[0052] The analyte can be in contact with either side of the insulating membrane on both sides of the nanopore. The analyte can be in contact with either side of the insulating membrane, such that the analyte passes through the channel of the nanopore to reach the other side of the insulating membrane. In this case, the analyte interacts with the nanopore when it passes through the insulating membrane via the channel of the pore. Alternatively, the analyte can be in contact with the side surface of the insulating membrane, and the side surface of the insulating membrane can enable the analyte to interact with the nanopore, causing it to separate from the nanopore and remain on the same side of the insulating membrane. The analyte can interact with the nanopore in any manner and at any site. The analyte can also strike the nanopore, interact with the nanopore, causing it to separate from the nanopore and remain on the same side of the insulating membrane.

[0053] During the interaction between the analyte and the nanopore, the analyte will affect the current flowing through the nanopore in a manner specific to the analyte, i.e., the current flowing through the nanopore is characteristic of a specific analyte. Control experiments can be conducted to determine the effect of a specific analyte on the current flowing through the nanopore, and then to identify a specific analyte in a sample or to determine the presence of a specific analyte in a sample. More specifically, the presence, concentration, etc. of the analyte can be identified by comparing the current pattern obtained by detecting the analyte with the known current pattern obtained using a known analyte under the same conditions.

[0054] The nanopore system of the present invention may further include one or more measuring devices for measuring the current flowing through the nanopore, such as a patch clamp amplifier or a data acquisition device.

[0055] Example 1: Materials and Methods

[0056] 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-maltoside (DDM) (≥99%, CAS#69227-93-6), isopropyl-β-D-thiogalactoside (IPTG) (≥99%, CAS#367-93-1), phenylmethylsulfonyl fluoride (PMSF) (≥99.%, CAS#329-98-6), 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.

[0057] Expression and purification of MtMscL: Using the pET28b-6His vector, the corresponding plasmid of the large conductance mechanosensitive channel (MscL) protein of Mycobacterium tuberculosis (i.e., MtMscL) was constructed. The coli BL21(DE3) cells containing pET28b-MscL-6His were cultured in Luria-Bertani (LB) medium with a kanamycin concentration of 50 μg / mL at a culture temperature of 37 °C. When OD 600When it reached 0.8 - 1.0, protein expression was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 20 °C for 12 h. Bacteria were collected by centrifugation at 3,800 rpm for 15 min and resuspended in lysis buffer A (20 mM Tris, 100 mM NaCl, 5 mM β-mercaptoethanol, pH 7.4). The cells were disrupted and lysed under high pressure after adding 1 mM phenylmethylsulfonyl fluoride (PMSF). 1% (wt / vol) dodecyl-β-D-maltopyranoside (DDM) was added for extraction for 2 h. Centrifugation was carried out at 18,000 rpm for 1 h, and then 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), and the column and the purified protein were pre-equilibrated with buffer D (200 mM NH4Ac, 0.025% DDM, 10% glycerol, pH 7.4). The peak fractions were determined by SDS-PAGE analysis. The above method is applicable to both wild-type and mutant proteins.

[0058] The proteins involved in the examples of the present invention include MtMscL (WT) and MtMscL (ΔC). First, construct the MtMscL vector or the vector with the C-terminal truncated MtMscL, use SnapGene to construct the plasmid map, then synthesize the plasmid, and then use the plasmid to express and purify the MtMscL (WT) or MtMscL (ΔC) protein. The plasmid maps of MtMscL (WT) and MtMscL (ΔC) involved in the present invention are as Figure 6 A and Figure 6 shown in B.

[0059] The sequence information of MtMscL (WT) is: MLKGFKEFLARGNIVDLAVAVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQVVLLTEIRDLLAQTNGDSPGRHGGRGTPSPTDGPRASTESQ (SEQ ID NO:1)

[0060] The sequence information of MtMscL(△C) is: MLKGFKEFLARGNIVDLAVAVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQ (SEQ ID NO:2)

[0061] The MtMscL(△C) protein is a truncated mutant with the C-terminus (cytoplasmic region) truncated. The C-terminus is structurally prominent. After truncating the C-terminus, most of the MtMscL(△C) protein is transmembrane region. In addition, after truncation, the protein expression level is higher and the pore-forming ability is stronger, which is suitable for detection and analysis.

[0062] Membrane binding and single-channel recording: The experiment was carried out in a vertical sampling chamber provided by Warner Instruments. All current traces were recorded using a HEKA epc10 USB patch clamp amplifier with a sampling frequency of 9900 hz. 1 μL of 25 mg / mL Escherichia coli extract phospholipids was pre-coated on the mouth of a 150 μm cup, and then 1 mL of electrolyte solution was added to both sides of the sample chamber (-trans chamber: 30 mM NaCl, 10 mM HEPES, pH 7.0; -cis chamber: 300 mM NaCl, 10 mM HEPES, pH 7.0). Then, about 2 / 3 of the electrolyte solution was aspirated from the -cis chamber using a 1 mL pipette, and the electrolyte solution was then driven into the -cis chamber of the sample chamber. When the average current approached 0 pA, a planar phospholipid bilayer membrane was formed (i.e., by lifting the electrolyte solution in the cis chamber with a pipette to form a phospholipid bilayer). After the planar phospholipid bilayer membrane was formed, the MtMscL protein (wild-type and / or mutant) solution was added to the -cis chamber. When MtMscL was embedded in the planar phospholipid bilayer membrane, a significant change in current occurred, and subsequent experiments were carried out.

[0063] Antibiotic detection: Use MtMscL nanopores (wild-type and mutant) to detect antibiotics. When a stable MtMscL nanopore is formed on the planar phospholipid bilayer membrane, the background signal is recorded and observed for 20 min at -50 mV, and then the antibiotic to be detected is added to the cis chamber (-cis chamber) of the sample cell, and then a voltage is applied and the current signal is recorded.

[0064] Protein and polypeptide detection: Use MtMscL nanopores to detect protein polypeptides. When a stable MtMscL nanopore is formed on the planar phospholipid bilayer membrane, the background signal is recorded and observed for 20 min at -50 mV, and then the protein polypeptide to be detected is added to the cis chamber of the sample cell, and then a voltage is applied and the current signal is recorded.

[0065] Electrophysiological data analysis: In this experiment, Clampfit software was used to process electrophysiological data, and Origin software was used for plotting.

[0066] Example 2: Structure of MtMscL Channel and Its Electrophysiological Testing

[0067] Structure of the MtMscL channel:

[0068] As Figure 1 shown in A, the MtMscL (WT, wild type) channel is a homotetramer 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 inside the pore, forming a funnel-shaped structure; TM2 is located outside the pore, playing a supporting role. MtMscL has a pore with a pore diameter (channel size) ranging from approximately 0.3 - 3.5 nm.

[0069] Electrophysiological testing of the MtMscL channel:

[0070] The single-channel electrophysiological study of the MtMscL channel was carried out in a planar phospholipid bilayer membrane. A schematic diagram of the electrophysiological apparatus is as Figure 9 shown. As Figure 1 shown in B, in the 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 can form stable channel currents at voltages of +50 mV and +200 mV, which are 38 pA and 175 pA respectively (this record is a representative event of a single-channel insertion). The above experiments indicate that the MtMscL channel can perform planar phospholipid membrane electrophysiological sensing.

[0071] The present invention also constructed various truncations based on MtMscL (WT), and used SDS-PAGE gel images and single-channel insertion traces to verify whether the constructed truncations can be expressed and inserted into the membrane. The experimental results are as Figure 1 shown in C, Figure 1 D. Among them, the truncation with the N-terminal truncated cannot be expressed, and the truncation with the C-terminal truncated (removing amino acids 97 - 151) cannot be inserted into the membrane. Only the truncation with the C-terminal truncated (removing amino acids 111 - 151, designated as MtMscL (△C)) can be both expressed and inserted into the membrane. As Figure 1 shown in E, MtMscL (△C) lacks the cytoplasmic C-terminal helix.

[0072] Figure 2Shows the I-V curves of MtMscL(WT) and MtMscL(△C) 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) in the voltage range from -50 mV to +50 mV. Figure 2 The I-V curves of A-2B show that MtMscL(WT) and MtMscL(△C) remain stable in this voltage range without high-voltage gating (each value is represented as mean ± SD, MtMscL(WT) from 5 repeated experiments; MtMscL(△C) from 7 repeated experiments). And through Figure 2 The conductance magnitudes of MtMscL(WT) and MtMscL(△C) can be obtained from the slopes of the I-V curves shown in A-2B (the slope is the conductance value), which are 0.616 ± 0.007 nS and 0.683 ± 0.007 nS, respectively.

[0073] Both MtMscL(WT) and MtMscL(△C) proteins are pentameric structures, where MtMscL(WT) is the full-length protein and MtMscL(△C) lacks the C-terminal domain. As can be seen above, except that the conductance of MtMscL(△C) is slightly larger than that of MtMscL(WT), the other electrophysiological properties of MtMscL(WT) and MtMscL(△C) are similar.

[0074] Example 3: Detection of small molecule drugs based on the MtMscL channel

[0075] In this example, the MtMscL channels (MtMscL(WT) and MtMscL(△C)) were used to detect small molecule drugs (molar mass less than 1000 g / mol), namely gentamicin sulfate (molecular weight MW: 561.65), tobramycin sulfate (molecular weight MW: 565.595), and kanamycin sulfate (MW: 582.577).

[0076] When a stable MtMscL nanopore is formed on a planar phospholipid bilayer membrane (one of the insulating membranes), the single antibiotic to be detected is added to the -cis chamber of the sample cell, then a voltage of -50 mV is applied and the current signal is recorded. The detection experiment was carried out under the electrolyte conditions of 300 mM NaCl (-cis chamber) and 30 mM NaCl (-trans chamber), 10 mM HEPES, pH 7.0.

[0077] Specific current signals were generated respectively when the analytes passed through the MtMscL nanopore ( Figure 3 and Figure 4) Among them, the peak current blockage rate of the typical gentamicin signal of MtMscL(WT) is 40%, and the peak residence time is 2 ms; the current blockage rate of the typical tobramycin signal is 32%, and the peak residence time is 5 ms; the current blockage rate of the typical kanamycin signal is 30%, and the peak residence time is 5 ms( Figure 3 ) The peak current blockage rate of the typical gentamicin signal of MtMscL(△C) is 40%, and the peak residence time is 2 ms; the current blockage rate of the typical tobramycin signal is 35%, and the peak residence time is 2 ms; the current blockage rate of the typical kanamycin signal is 31%, and the peak residence time is 1 ms( Figure 4 ) In addition, the current blockage rate of the typical neomycin (molecular weight MW: 614.644) signal of MtMscL(WT) is 35%, and the peak residence time is 1.5 ms( Figure 14 )

[0078] When a bias voltage is applied, the nanopore captures the analyte antibiotic. Due to differences in the size, structure, and charge of the antibiotic, unique current blocking events can be observed. Gentamicin sulfate, tobramycin sulfate, and kanamycin sulfate all have hydrophilicity, positive charge, and similar molecular weights, but the MtMscL nanopore can directly distinguish the above 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 not only be used to detect and distinguish aminoglycoside antibiotics (such as gentamicin, tobramycin, and kanamycin), but also to detect and distinguish other small molecule drugs with a molar mass less than 1000 g / mol (such as adrenaline hydrochloride), with broad application scenarios.

[0079] As Figure 10 shown in A, when the MtMscL(WT) nanopore detects tobramycin sulfate at different concentration gradients, different signal frequencies will be generated, and the concentration of tobramycin sulfate has a linear relationship with the corresponding signal frequency. Therefore, the concentration of small molecule drugs (such as tobramycin sulfate) can also be detected by the obtained signal frequency. In addition, it can also be seen that the detection limit of the MtMscL nanopore for detecting small molecule drugs is 100 nM.

[0080] In addition, mutations were made on the basis of MtMscL(ΔC). The mutation method was to mutate the 20th amino acid, alanine (A), of MtMscL(ΔC) to cysteine (C) (denoted as mutant A20C). This mutation site is located within the pore of MtMscL(ΔC). The sequence information of this mutant is: MLKGFKEFLARGNIVDLAVCVVIGTAFTALVTKFTDSIITPLINRIGVNAQSDVGILRIGIGGGQTIDLNVLLSAAINFFLIAFAVYFLVVLPYNTLRKKGEVEQPGDTQ (SEQ ID NO:3)

[0081] The pore insertion map and tobramycin detection result map of mutant A20C at 50 mV are shown in Figure 5 A and Figure 5 B, respectively. The above experimental results show that even if MtMscL(ΔC) is mutated (such as amino acid substitution), the resulting mutant is still similar in properties to MtMscL(ΔC), and the mutant still has the ability to detect small molecule drugs.

[0082] Example 4: Detection of protein polypeptides based on the MtMscL channel

[0083] In this example, the MtMscL channels (MtMscL(WT) and MtMscL(ΔC)) were used to detect and characterize Complexin-I polypeptides of different lengths.

[0084] Among them, the sequence information and molecular weight of Complexin(1-134) are: MEFVMKQALGGATKDMGKMLGGDEEKDPDAAKKEEERQEALRQAEEERKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQAAMEANSEGSLTRPKKAIPPGCGDEPEEEDESILDTVIKYLPGPLQDMFKK (15.122 kDa) (SEQ ID NO:4)

[0085] The sequence information and molecular weight of Complexin(26-134) are: KDPDAAKKEEERQEALRQAEEERKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQAAMEANSEGSLTRPKKAIPPGCGDEPEEEDESILDTVIKYLPGPLQDMFKK (12.466 kDa) (SEQ ID NO:5)

[0086] The sequence information and molecular weight of Complexin(48-83) are: RKAKYAKMEAEREVMRQGIRDKYGIKKKEEREAEAQ (4.355 kDa) (SEQ ID NO: 6)

[0087] When stable MtMscL nanopores are formed on a planar phospholipid bilayer membrane, different lengths of Complexin-I polypeptides to be detected are added to the -cis chamber of the sample cell (the polypeptides are charged in the nanopore system), and then a voltage of -50 mV is applied and the current signal is recorded.

[0088] In this example, MtMscL(WT) nanopores were tested for detecting different lengths of Complexin-I at a voltage of -50 mV and under two electrolyte conditions. In the -trans chamber: 30 mM KCl, 10 mM HEPES, pH 7.5; in the -cis chamber: 300 mM KCl, 10 mM HEPES, pH 7.5. The detection results are as follows Figure 11 shown. Among them, the peak current blockage rate of the typical Complexin(1-134) signal of MtMscL(WT) is 35%, and the peak residence time is 5 ms; the current blockage rate of the typical Complexin(26-134) signal is 25%, and the peak residence time is 1 ms; the current blockage rate of the typical Complexin(48-83) signal is 17%, and the peak residence time is 0.5 ms.

[0089] In the -trans chamber: 200 mM KCl, 10 mM HEPES, pH 7.5; in the -cis chamber: 2 M KCl, 10 mM HEPES, pH 7.5. The detection results are as follows Figure 7 shown. Among them, the peak current blockage rate of the typical Complexin(1-134) signal of MtMscL(WT) is 9.91%, and the peak residence time is 4.94 ms; the current blockage rate of the typical Complexin(26-134) signal is 31.32%, and the peak residence time is 7.76 ms; the current blockage rate of the typical Complexin(48-83) signal is 6.65%, and the peak residence time is 4.75 ms. The experimental results show that 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(WT) has a better effect in distinguishing different polypeptides, and the "peak residence time" in some polypeptide signals is significantly improved.

[0090] 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 blockage rate of the typical Complexin(1-134) signal was 20.08%, and the peak residence time was 4.94 ms; the current blockage rate of the typical Complexin(26-134) signal was 30.03%, and the peak residence time was 5.01 ms; the current blockage rate of the typical Complexin(48-83) signal was 15.18%, and the peak residence time was 4.85 ms( Figure 8 ). The above experiments showed that both MtMscL(WT) and MtMscL(△C) had the ability to detect and distinguish different polypeptides.

[0091] As Figure 10 shown in Figure 10 B and Figure 10 C, when MtMscL nanopores (MtMscL(WT) and MtMscL(△C)) detected Complexin with different concentration gradients, different signal frequencies would be generated, and there was a linear relationship between the concentration of Complexin and the corresponding signal frequency. Therefore, the concentration of polypeptides (such as Complexin) could also be detected by the obtained signal frequencies. Among them, MtMscL(WT) had higher sensitivity. In other words, when detecting lower concentrations of analytes or the same concentration of analytes, MtMscL(WT) detected more signals (for example, more signal frequencies). In addition, the lowest polypeptide concentration that could be detected in the experiments of this example was 2 μM. Combining

[0092] with Figure 12 B, it could also be seen that the detection limit of MtMscL nanopores for polypeptides was 2 μM.

[0093] The potential principles for detecting polypeptides by MtMscL may include: (1) Amino acids on the polypeptide molecule interact with amino acids inside or near the MtMscL nanopore (such as charge attraction, hydrophilic / hydrophobic effects, etc.) and generate an electric current signal; (2) The polypeptide molecule moves in the electrolyte solution, collides with the MtMscL nanopore and generates an electric current signal; (3) The linear polypeptide molecule passes through the MtMscL nanopore and generates an electric current signal.

[0094] In summary, this example verifies the ability of MtMscL to detect and characterize various polypeptides (such as Complexin-I and SNAP-25). Both Complexin-I and SNAP-25 are related to vesicle trafficking. Therefore, the MtMscL provided by the present invention can be used in various application scenarios such as detecting biomarkers related to neurotransmitter release disorders, detecting the presence of such polypeptides during the production process of synthetic polypeptides, and studying protein-polypeptide interactions.

[0095] Example Five: Whole blood detection based on the MtMscL channel

[0096] 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, and then a certain concentration of small molecule drug (tobramycin is used in this example) is added to it to prepare a mother liquor with a small molecule drug concentration of 100 μM. 10 μL of the mother liquor is added to the detection system (to make the final concentration of the small molecule drug in the whole blood sample 1 μM) for detection. As Figure 13 shown, the above experimental results show that the MtMscL nanopore can directly detect small molecule drugs (such as tobramycin) in whole blood samples.

[0097] The embodiments of the present invention have been described above in conjunction with the accompanying drawings. However, the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. These all fall within the protection scope of the present invention.

Claims

1. Use of a nanopore system in detecting small molecule drugs, characterized in that, The nanopore system includes a nanopore, an insulating film, a first medium, and a second medium; the nanopore is embedded in the insulating film, the insulating film 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 small molecule drug in the first medium interacts with the nanopore; the nanopore includes Mycobacterium tuberculosis large conductance mechanosensitive channel protein (MtMscL); the amino acid sequence of the Mycobacterium tuberculosis large conductance mechanosensitive channel protein is as shown in SEQ ID NO:2 or SEQ ID NO:

3.

2. The application according to claim 1, wherein The molar mass of the small molecule drug is less than 1000 g / mol.

3. The application according to claim 1, wherein The small molecule drug is an aminoglycoside antibiotic.

4. The application according to claim 1, characterized in that The detection limit of the small molecule drug is 100 nM.

5. The application according to claim 1, wherein The first medium is a 300 mM sodium chloride solution, 10 mM HEPES, and pH 7.0, and the second medium is a 30 mM sodium chloride solution, 10 mM HEPES, and pH 7.

0.

6. The application according to claim 3, wherein The aminoglycoside antibiotics include one or more of gentamicin, tobramycin, neomycin, kanamycin, or a pharmaceutically acceptable salt thereof.

7. The application according to claim 1, characterized in that, The application includes detecting the small molecule drug in a whole blood sample.

8. The application according to claim 1, characterized in that, The application includes detecting the presence and / or concentration of the small molecule drug.

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