A nanochannel detection device for simultaneous rapid analysis and real-time quantitative monitoring of angiotensin series peptides

Abstract

This invention provides a nanopore detection device, as well as a method and application for detecting biological samples using this device. The nanopore detection device includes an insulator with openings, a phospholipid membrane or polymer membrane located within the openings, nanopores formed by mutant aeromonas lysin passing through the phospholipid membrane or polymer membrane, chambers located on opposite sides of the insulator, an electrolyte contained within the chambers, and a detector disposed within the chambers. The nanopore technology developed in this application enables efficient qualitative or rapid quantitative detection of one or more samples—such as one or more angiotensin peptides—and achieves significant improvements in resolution, sensitivity, capture efficiency, and matrix compatibility. The nanopore technology developed in this application also enables real-time quantitative monitoring of the dynamic changes of one or more samples—such as one or more angiotensin peptides.

Description

Technical Field

[0001] This invention relates generally to the detection of electrical signals in biological samples. Specifically, this invention provides a technique for the simultaneous quantitative detection and real-time monitoring of peptides, components of the renin-angiotensin system, in biological samples using a nanopore detection device. Background Technology

[0002] Real-time and precise analysis of biological materials, including proteins, peptides, and nucleotides, is of great significance in fields such as medicine, genetics, and biology. For example, signaling molecules secreted by multicellular organisms, depending on their classification, may include various proteins, peptides, hormones, etc. These molecules are transported to distant tissues via complex biological processes and exert their regulatory effects on physiological processes and behaviors by binding to specific receptor proteins on target cells, leading to changes in cellular function, such as metabolism, growth and development, stress induction, reproduction, and emotional control. If the types or amounts of signaling molecules in various biological samples can be detected in real time and with precision, the processes of signaling molecule generation, transport, binding, degradation, and transformation can be effectively tracked. Current techniques for detecting these biological materials include fluorescence, chemiluminescence, and mass spectrometry. However, these techniques all have significant drawbacks, such as requiring high concentrations of the sample, being unable to effectively distinguish between multiple analytes within the sample, being susceptible to interference from impurities (e.g., quenching of luminescence signals, masking of peaks), having overly complex and time-consuming detection procedures, and incurring high costs for labor and consumables. Therefore, researchers in related fields urgently hope to develop novel detection technologies to address these problems.

[0003] Cardiovascular disease is currently a leading cause of death and morbidity among non-communicable diseases worldwide. The renin-angiotensin system (RAS) plays a central role in the homeostatic control of the cardiovascular system by regulating blood pressure and electrolyte balance. Hypertension caused by RAS dysregulation under pathophysiological conditions is one of the major risk factors for the development of cardiovascular disease. Angiotensin (Ang), produced by a series of enzymatic reactions in the RAS, is considered its main molecular effector. Pathological increases in Ang II cause vasoconstriction, leading to elevated blood pressure and promoting cardiac and vascular damage. Drug inhibition of the Ang II axis in the RAS is a major therapeutic strategy for hypertension. Although researchers have studied the RAS for over a century, the complexity of its internal and inter-system interactions remains to be fully elucidated. More importantly, recent discoveries of endogenous antagonistic regulatory mechanisms within the RAS system remind us that even processes currently considered well-understood may be far more complex than assumed, requiring further work to systematically and comprehensively analyze the interactions of complex systems and thus fully understand their physiological functions and regulatory mechanisms. However, before conducting comprehensive analysis of the complex components in RAS and other systems, it is necessary to overcome the challenge of real-time, systematic qualitative and quantitative detection of short-half-life peptides (such as one or more angiotensin peptides) that differ only by a single amino acid in structure. There is an urgent need for a novel detection method that can effectively overcome the above problems. Summary of the Invention

[0004] To address the aforementioned problems, the inventors of this invention have conducted in-depth research and successfully developed a novel nanopore detection device, as well as a method and application for using this device to detect biological samples, such as peptides that are components of the Renin-Angiotensin System (RAS).

[0005] The first aspect of this application provides a method for detecting biological samples using a nanopore detection device, the nanopore detection device comprising an insulator with openings, a phospholipid membrane or polymer membrane located in the openings, nanopores formed by mutant aeromonas lysin passing through the phospholipid membrane or polymer membrane, two or more chambers including electrodes located on both sides of the insulator, and a detector connected to the electrodes.

[0006] The method includes the following steps:

[0007] Add electrolyte to the two or more chambers;

[0008] The biological sample is added into at least one of the chambers;

[0009] This allows the biological sample to at least partially enter the nanopores, where displacement occurs and an electrical signal is generated; and

[0010] The electrical signal is detected using a detector, and the biological sample is analyzed based on the measured electrical signal. Preferably, the analysis includes qualitative or quantitative analysis of one or more molecules contained in the biological sample.

[0011] According to one specific embodiment of the first aspect of this application, the biological sample is selected from mammalian body fluids, tissue extracts, intracellular fluids, natural polypeptides, synthetic polypeptides, recombinant polypeptides, and combinations thereof. According to one specific embodiment of the first aspect of this application, the biological sample comprises proteins, polypeptides, nucleotides, amino acids, polysaccharides, their derivatives, and combinations thereof.

[0012] According to another specific embodiment, the biological sample contains a renin-angiotensin system component polypeptide, which includes one or more of the following: Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III, AGT, Ang 1-25, Ang 1-12, Ang 2-10, Ang 3-10, Ang 4-10, Ang 5-10, Ang 6-10, Ang IV, Ang 4-8, Ang 5-8, Ang 1-5, Ang 1-4, Ang 1-3, Ang 1-2, Ang 3-4, Ang 2-7, Ang 3-7, Ang 5-7, Ang A, and Alamandine.

[0013] According to another embodiment of the first aspect of this application, the mutant aeromonas lysin is obtained by replacing one or more original amino acids at one or more sites in wild-type aeromonas lysin with one or more novel amino acids selected from the following: alanine (A), serine (S), threonine (T), glycine (G), cysteine ​​(C), leucine (L), lysine (K), tryptophan (W), arginine (R), histidine (H), glutamine (Q), proline (P), glutamic acid (E), methionine (M), isoleucine (I), valine (V), phenylalanine (F), tyrosine (Y), asparagine (N), and aspartic acid (D); and the type of original amino acid is different from that of the novel amino acid.

[0014] According to another embodiment of the first aspect of this application, the mutant aeromonas lysin is obtained by replacing one or more original amino acids at one or more sites of wild-type aeromonas lysin at positions 224, 226, 228, 230, 232, 234, 236, 260, 262, 264, 266, 268, 270, and 272 with one or more new amino acids selected from the following: lysine (K), arginine (R), and histidine (H).

[0015] According to another embodiment of the first aspect of this application, the mutant aeromonas lysin is selected from one or more of the following mutants: A224K, A224R, A224H, N226K, N226R, N226H, S228K, S228R, S228H, T230K, T230R, T230H, T232K, T232R, T232H, G234K, G234R, G 234H, S236K, S236R, S236H, A260K, A260R, A260H, N262K, N262R, N262H, S264K, S264R, S26 4H, A266K, A266R, A266H, Q268K, Q268R, Q268H, G270K, G270R, G270H, S272K, S272R, S272H.

[0016] According to another embodiment of the first aspect of this application, the method performs qualitative or quantitative detection on the biological sample based on the measured electrical signal.

[0017] According to another embodiment of the first aspect of this application, the qualitative detection is performed based on one or more of the following detection information: electrical signal blocking current, electrical signal blocking degree, blocking time, blocking current fluctuation standard deviation (STD), or a combination thereof.

[0018] According to another embodiment of the first aspect of this application, the quantitative analysis is performed based on a linear relationship between concentration and capture rate. The capture rate is the reciprocal of the interval between specific analyte electrical signal events, or the number of specific analyte electrical signals per effective unit time. According to another embodiment of the first aspect of this application, the detection is performed in real time.

[0019] A second aspect of this application provides a method for testing enzyme activity, the method comprising:

[0020] This allows one or more substrates to undergo an enzyme-catalyzed reaction under enzyme-catalyzed conditions, generating one or more products;

[0021] Prior to the enzyme-catalyzed reaction, the substrate is qualitatively or quantitatively detected using the method described in the first aspect of this application; and / or during and / or after the enzyme-catalyzed reaction, the substrate and / or the product are qualitatively or quantitatively detected using the method described in the first aspect of this application.

[0022] Enzyme activity is determined based on qualitative or quantitative detection results of the substrate and / or product. According to another embodiment of the second aspect of this application, the method for testing enzyme activity is performed in real time.

[0023] A third aspect of this application provides a nanopore detection device, comprising:

[0024] (a) Insulator with openings

[0025] (b) A phospholipid membrane or polymer membrane located in the opening;

[0026] (c) Nanopores formed by mutant aeromonas lysin through the phospholipid membrane or polymer membrane, said mutant aeromonas lysin being obtained by replacing one or more original amino acids at one or more sites from 1 to 470 in wild-type aeromonas lysin with one or more novel amino acids selected from the following: alanine (A), serine (S), threonine (T), glycine (G), cysteine ​​(C), leucine (L), lysine (K), tryptophan (W), arginine (R), histidine (H), glutamine (Q), proline (P), glutamic acid (E), methionine (M), isoleucine (I), valine (V), phenylalanine (F), tyrosine (Y), asparagine (N), and aspartic acid (D);

[0027] Furthermore, the types of the original amino acids are different from those of the new amino acids;

[0028] (d) Two or more chambers located on both sides of the insulator, each chamber containing an electrode and an electrolyte; and

[0029] (e) Detector.

[0030] According to one embodiment of the third aspect of this application, the mutant aeromonas lysin is as described in the first aspect above.

[0031] A fourth aspect of this application provides the use of the detection device described above to detect biological samples selected from mammalian body fluids, tissue extracts, intracellular fluids, natural polypeptides, synthetic polypeptides, recombinant polypeptides, and combinations thereof, for example, containing one or more of the renin-angiotensin system components polypeptides described above. Attached Figure Description

[0032] The following paragraphs discuss various embodiments of the invention in conjunction with the accompanying drawings. However, it should be noted that the embodiments shown in the drawings and described in detail below are merely some preferred embodiments of the invention, and the scope of protection of the invention is defined by the claims, and not limited to these preferred embodiments.

[0033] Figure 1 A schematic diagram of a nanopore detection device according to one embodiment of this application is shown;

[0034] Figure 2 This diagram illustrates the process by which mutant Aeromonas hydrolysin self-assembles to form nanopores according to one embodiment of this application.

[0035] Figure 3 A schematic diagram of a mutant Aeromonas hydrolysin nanopore is shown according to one embodiment of this application;

[0036] Figure 4 The following is a summary of schematic diagrams of the nanopores of the mutant Aeromonas hydrolysin in the embodiments of the invention;

[0037] Figure 5 A schematic diagram of the process for obtaining mutant Aeromonas hydrolysin according to one embodiment of this application is shown.

[0038] Figure 6 The original current signal diagrams measured in the embodiments and comparative examples of the invention are summarized in the figure;

[0039] Figure 7 The document summarizes the blocking time and capture rate statistics of the current signals measured in the embodiments and comparative examples of the invention;

[0040] Figure 8 This shows a scatter plot of characteristic current blocking I / I0-blocking time measured sequentially for a series of Ang peptides according to an embodiment of the present invention;

[0041] Figure 9 The diagram shows a scatter plot of characteristic current blocking I / I0-blocking time measured for a mixture of a series of Ang peptides according to an embodiment of the present invention.

[0042] Figure 10 The diagram illustrates the current blocking of the original signal measured for a mixture of a series of Ang peptides according to an embodiment of the present invention.

[0043] Figure 11A The diagram shows a scatter plot of STD versus I / I0 for current blocking measured for a mixture of a series of Ang peptides, according to an embodiment of the present invention.

[0044] Figure 11BShowing according to Figure 11A The illustrated example shows 3D plots of I / I0, STD, and blocking time measured for a mixture of a series of Ang peptides;

[0045] Figure 12 The diagram shows a scatter plot of characteristic current blocking I / I0-blocking time measured for a mixture of a series of Ang peptides according to an embodiment of the present invention.

[0046] Figure 13 The diagram shows a scatter plot of characteristic current blocking I / I0-blocking time measured for a mixture of a series of Ang peptides according to an embodiment of the present invention.

[0047] Figure 14A The diagram shows the change over time of the original current trajectory of Ang I sheared by ACE according to one embodiment of this application;

[0048] Figures 14B to 14F The results are shown using scatter plots and Gaussian fitted curves (obtained from I / I0 histograms) respectively. Figure 14A The illustrated embodiment shows the changes in the characteristic current corresponding to Ang I at different times and the newly generated characteristic current;

[0049] Figure 14G Showing according to Figure 14A The illustrated embodiment shows the change of current blocking I / I0 over time.

[0050] Figure 14H Showing according to Figure 14A The example shown illustrates the changes in the concentrations of Ang I and the product Ang polypeptide over time during ACE digestion.

[0051] Figure 15A Showing according to Figure 15A The illustrated embodiment shows the change of current blocking I / I0 over time.

[0052] Figure 15B Showing according to Figure 15A The illustrated example shows the changes in the concentrations of Ang I and the product Ang polypeptide over time during simultaneous enzymatic digestion by ACE and ACE2 enzymes. Detailed Implementation

[0053] The “range” disclosed in this document takes the form of a lower limit and an upper limit. It can be one or more lower limits and one or more upper limits, respectively. A given range is defined by selecting a lower limit and an upper limit. The selected lower and upper limits define the boundaries of the particular range. All ranges that can be defined in this way are inclusive and composable; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60-120 and 80-110 are listed for a specific parameter, it is also expected that ranges of 60-110 and 80-120 are also included. Furthermore, if the minimum range values ​​are listed as 1 and 2, and if the maximum range values ​​are listed as 3, 4, and 5, then the following ranges are all expected: 1-3, 1-4, 1-5, 2-3, 2-4, and 2-5.

[0054] In this invention, unless otherwise stated, the numerical range "ab" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0-5" means that all real numbers between "0-5" have been listed herein, and "0-5" is simply a shortened representation of these numerical combinations.

[0055] In this invention, unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0056] In this invention, unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0057] In this invention, unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0058] In this invention, unless otherwise specified, the term "comprising" as used herein can be open-ended or closed-ended. For example, "comprising" may mean that it may also include other components not listed, or it may only include the listed components.

[0059] In this application, the term "polypeptide" refers to a compound obtained by the condensation of two, three, four, five, six or more amino acid molecules, wherein a polypeptide compound formed by the condensation of two amino acid molecules is called a dipeptide, a polypeptide compound formed by the condensation of three amino acid molecules is called a tripeptide, a polypeptide compound formed by the condensation of four amino acid molecules is called a tetrapeptide, a polypeptide compound formed by the condensation of five amino acid molecules is called a pentapeptide, a polypeptide compound formed by the condensation of six amino acid molecules is called a hexapeptide, and so on.

[0060] The nanopore detection device of this application includes: an insulator with openings, a phospholipid membrane or polymer membrane located in the openings, nanopores formed by mutant aeromonas lysin passing through the phospholipid membrane or polymer membrane, two or more chambers including electrodes located on both sides of the insulator, a detector connected to the electrodes, and an electrolyte added to the two or more chambers.

[0061] In this application, the insulator refers to a component of arbitrary shape made of an electrically insulating material, as long as it can separate the chambers located on both sides of the insulator, allowing only the electrolyte within the chambers to flow through the nanopores. For example, the insulator can be a perforated partition, a perforated membrane, a hollow tube, etc.

[0062] According to some embodiments of this application, the insulator can be formed from any suitable insulating material, as long as it has the required insulating properties and does not negatively affect the electrolyte added to the device or the biological sample being tested. For example, the materials used to manufacture the insulator may include one or more of the following: acetal resin, phenolic resin, urea-formaldehyde resin, aniline-formaldehyde resin, melamine-formaldehyde resin, glycerol resin, polyvinyl chloride, polyethylene, polytetrafluoroethylene, chloroprene rubber, polyvinyl alcohol, silicone resin, polyester, epoxy resin, glass fiber, mica products, polyimide, polyamide-imide, polyimide, polymaleimide, polydiphenyl ether, modified epoxy resin, unsaturated polyester, polyaramid fiber paper, and impregnated fiber materials, composite film materials, and laminated materials made from the above insulating materials.

[0063] The insulator in the detection device of this application is described below using a partition with openings as an example, but the structure of the insulator of this invention is not limited thereto, and the scope of protection of this invention can include any desired shape of insulator.

[0064] In this application, the two sides of the partition are referred to as the trans side and the cis side, respectively. The cis side refers to the side where mutant aeromonas lysin is added to the chamber during the formation of the nanopores. The mutant aeromonas lysin inserts into the lipid bilayer membrane from this side and self-assembles to form the nanopores. The trans side refers to the opposite side. According to another embodiment of this application, the analyte enters the nanopores at least partially from the cis side, undergoes relative displacement within the nanopores, and generates specific electrical signals related to the molecular structure of the analyte. Based on these specific electrical signals, qualitative or quantitative testing of the analyte molecules can be performed.

[0065] Although Figure 1 In the illustrated embodiment, a chamber is provided on both the CIS side and the trans side of the partition. However, the detection device of the present invention can be configured in various different structures as needed. For example, according to the exemplary embodiment, one or more chambers can be designed on both the CIS side and the trans side of the partition as needed. This allows for the addition of one or more test samples or auxiliary reagents to these chambers as required. When multiple chambers are provided on the CIS side and / or the trans side, chambers on the same side are directly fluidly connected (i.e., fluidly connected without nanopores); chambers on different sides require nanopores for fluid communication. Depending on specific needs, each chamber can have a specially designed shape and size, and the shapes and sizes of the chambers can be the same or different from each other.

[0066] According to one embodiment of this application, the chamber and the partition are independent units; while according to another embodiment, the chamber and the partition may be integrated.

[0067] In addition, although Figure 1 In the illustrated embodiment, the two chambers on either side of the partition are arranged side-by-side with the partition between them. However, the arrangement of these chambers can be changed as needed. For example, according to another exemplary embodiment, the chambers can be arranged horizontally or vertically, such that one chamber is sandwiched between several chambers, or one chamber is nested within another chamber, provided that the requirement described above is met: "chambers on the same side are directly fluidly connected on the same side; chambers on different sides need to be fluidly connected through nanopores."

[0068] The following text will be based on Figure 1 The embodiments shown are examples of the detection device of this application, but the scope of protection of this application is not limited thereto.

[0069] Figure 1A nanopore detection device according to one embodiment of the present invention is shown, the device comprising a trans chamber (detection cell) and a cis chamber (detection cell) separated by a partition. Figure 1 The tans chamber, cis chamber, and partition shown are integrated into a single structure, but it is also possible to consider using three separate structures. Figure 1 The diagram shows a fill port in fluid communication with the CIS chamber, but other structures can be used as needed. For example, it is possible to consider not using a fill port, or using multiple fill ports in fluid communication with the CIS chamber, or designing one or more fill ports in fluid communication with the trans chamber.

[0070] One or more pores are provided in the separator, preferably one pore. First, a lipid or polymer is applied into the pore to form a lipid or polymer membrane, such as a lipid bilayer. Then, one or more mutant aeromonas lysins are applied to the lipid or polymer membrane. The interaction between the lipid or polymer membrane and the mutant aeromonas lysins causes the mutant aeromonas lysins to self-assemble and form nanopores that penetrate the membrane within the pore. According to one embodiment of this application, the size of the pores in the separator can be designed as needed. For example, the diameter of the pores can be 0.01 μm to 5 mm, such as 0.1 μm to 2 mm, or 0.5 μm to 1 mm, or 1 μm to 800 μm, or 10 μm to 500 μm, or 20 μm to 400 μm, or 40 μm to 200 μm, or 50 μm to 100 μm, or within a range obtained by combining any two of the above values. Figure 1 The right side shows a schematic diagram of the nanopores. According to one embodiment of this application, lipids are added to this chamber, causing the lipids to form a lipid membrane within the pores. Then, mutant aeromonas lysin is added to this chamber, causing the mutant aeromonas lysin to self-assemble into nanopores that penetrate the lipid membrane. This can include, for example, the following steps: first, water (e.g., distilled water, double-distilled water, deionized water, ultrapure water, etc.) is added to the trans and cis chambers; then, a hydrophobic lipid is added to at least one of the trans and cis chambers, causing the lipids to form a lipid membrane within the pores of the separator, preferably a lipid bilayer membrane, which fully extends within the pores of the separator to completely separate the trans and cis chambers. It is not intended to be limited to any specific theory, such as... Figure 1As shown on the right, this lipid bilayer membrane is a bilayer membrane in which lipid molecules in each layer have their hydrophilic portions facing outwards, while the hydrophobic portions of the molecules are located inside the membrane. According to one embodiment of this application, the lipids may include oils (triglycerides), phospholipids, glycolipids, sterols, etc. Examples of glycolipids include monogalactosyldiglyceride and digalactosyldiglyceride. Preferably, the lipids include phospholipids, such as glycerophospholipids, lecithin, cephalin, sphingomyelin, etc.; more specifically, the phospholipids may include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, diphosphatidylglycerol, phosphatidylinositol, and any combination thereof. According to a specific embodiment, the phospholipid used in this invention is 1,2-diphydanoylphospholipid (DMPC). According to one embodiment of this application, the lipids can be directly added into the pores of the separator to form a lipid membrane, or the lipids can be first dissolved in an organic solvent to form a solution, and then the solution is used to form a lipid membrane. Solvents used to dissolve lipids may include conventional organic solvents, such as decane, and the concentration of lipids in the solvent may be 0.1-100 mg / mL, for example 0.5-50 mg / mL, or 1-20 mg / mL, or 5-10 mg / mL. According to another embodiment of this application, polymeric materials having properties similar to lipids may also be used as needed, and these polymeric materials can be used in combination with suitable solvents, such as the solvents for lipids described above.

[0071] Subsequently, activated mutant aeromonas lysin was added to the CIS chamber. The hydrophobic portion of the molecule spontaneously flipped and coiled, embedding itself into and penetrating the lipid membrane, thereby forming nanopores.

[0072] Figure 2 A schematic diagram illustrating the self-assembly of mutant aeromonas lysin to form nanopores is shown, illustrating four states (i) to (iv). State (i) shows the original state of the mutant aeromonas lysin molecule, at which point the mutant aeromonas lysin has not yet undergone any subsequent flipping or coiling. For simplicity, the molecule is shown as a planar representation in the figure. Those skilled in the art will understand that the molecule can exhibit any configuration in its conventional state and that amino acid residues are attached at various points on the molecule (in... Figure 2(A portion of this is highlighted with a dashed circle). These amino acid residues are used to interact with the analyte in subsequent detection. These amino acid residues can be modified through site-directed mutagenesis to adjust and improve the analyte capture capacity, resolution, and other properties of the nanochannel. During nanochannel formation, after the mutant aerolysin is added to the cis chamber, its hydrophobic portion spontaneously flips (ii) and coils (iii), binding together through the interaction of hydrophobic amino acid side chains to form head-to-head concentric β-barrel structures. This is activated by protease cleavage of the C-terminal peptide (CTP) of proaerolysin, spontaneously assembling to form a heptamer transmembrane protein channel. The amino acid residues inside the barrels interact with the analyte during subsequent detection, generating corresponding electrical signals. The hydrophobic barrel-shaped portion of the mutant aerolysin (called the β-barrel) self-assembles and penetrates the lipid membrane, forming... Figure 2 The nanopores shown.

[0073] Figure 3 A schematic diagram of nanopores formed by the self-assembly of mutant Aeromonas hydrolysin according to one embodiment of this application is shown. For emphasis and simplification, the lipid layer is not shown in the figure. Figure 3 As can be seen, the mutant aeromonas lysin molecule is rivet-shaped overall, with the barrel-shaped portion (β-barrel) embedded in the lipid layer, while the remaining hydrophilic portion (called the cap region) protrudes outside the membrane on the cis side. Due to factors such as the molecular structure, molecular size, internal mutations, and process conditions of the mutant aeromonas lysin molecule, the inner diameter of the self-assembled nanopore is approximately 1.1-1.7 nm, which effectively allows only a single analyte molecule to pass through, thus making the pore a monomolecular nanopore. According to one embodiment of this application, one or more nanopores are formed throughout the lipid membrane, for example, 1-20, 1-15, 1-10, 1-5, or 1-2 nanopores, preferably only one nanopore.

[0074] According to one embodiment of this application, during the process of forming single-molecule nanopores, water is present in the cis and trans chambers to promote the spontaneous formation of the lipid membrane within the pores of the septum and the self-assembly of the mutant aeromonas lysin molecules into the lipid membrane to form nanopores. Optionally, one or more reagents, such as pH adjusters, electrolytes, etc., may be included in the water as needed. According to a preferred embodiment, an electrolyte, such as an aqueous KCl solution, is added to the cis and trans chambers on both sides of the septum. According to one embodiment of this application, the mutant aeromonas lysin molecules used in this invention are obtained by mutating at least one amino acid at one or more sites of unmutated (i.e., wild) aeromonas lysin. According to one embodiment of this application, the mutant aeromonas lysin is obtained by replacing one or more original amino acids at one or more sites in wild-type aeromonas lysin with one or more novel amino acids selected from the following: alanine (A), serine (S), threonine (T), glycine (G), cysteine ​​(C), leucine (L), lysine (K), tryptophan (W), arginine (R), histidine (H), glutamine (Q), proline (P), glutamic acid (E), methionine (M), isoleucine (I), valine (V), phenylalanine (F), tyrosine (Y), asparagine (N), and aspartic acid (D), wherein the original amino acids are different from the novel amino acids. In this application, "original amino acid" refers to the amino acid originally present in wild-type aeromonas lysin, while "novel amino acid" refers to an amino acid molecule newly introduced in mutant aeromonas lysin to replace the original amino acid. According to an exemplary embodiment, one or more original amino acids at the following sites of wild-type Aeromonas hydrolysin can be replaced with one or more of the aforementioned novel amino acids: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120121、122、123、124、125、126、127、128、129、130、131、132、133、134、135、136、137、138、139、140、141、142、143、144、154、146、147、148、149、150、151、152、153、154、155、156、157、158、159、160、161、162、163、164、165、166、167、168、169、170、171、172、173、174、175、176、177、178、179、180、181、182、183、184、185、186、187、188、189、190、191、192、193、194、195、196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、3287、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, ​​383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 421, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470.

[0075] According to one embodiment of this application, the mutant aeromonas lysin is obtained by replacing one or more original amino acids at one or more sites of the wild-type aeromonas lysin at positions 224, 226, 228, 230, 232, 234, 236, 260, 262, 264, 266, 268, 270, and 272 with one or more new amino acids selected from the following: lysine (K), arginine (R), and histidine (H).

[0076] According to another embodiment of this application, the mutant aeromonas lysin is selected from one or more of the following mutants: A224K, A224R, A224H, N226K, N226R, N226H, S228K, S228R, S228H, T230K, T230R, T230H, T232K, T232R, T232H, G234K, G234R, G234 H, S236K, S236R, S236H, A260K, A260R, A260H, N262K, N262R, N262H, S264K, S264R, S264H , A266K, A266R, A266H, Q268K, Q268R, Q268H, G270K, G270R, G270H, S272K, S272R, S272H. In the above-described display of mutants, numbers represent the sites of the mutated amino acids in the molecule, such as site 224, site 266, etc.; the letters preceding the numbers indicate the types of amino acids before the mutation, such as A for alanine, N for asparagine, S for serine, T for threonine, G for glycine, and Q for glutamine; the letters following the numbers indicate the types of amino acids after the mutation, such as K for lysine, R for arginine, H for histidine, etc. These letter representations of amino acids are known in the art. For example, a mutation site according to an exemplary embodiment of this application is as follows: Figure 4 As shown.

[0077] The Aeromonas lysin precursor used for mutation is a toxin protein secreted by Aeromonas, with a molecular weight of 52 kDa, existing as a dimer in solution. In this invention, specific amino acid groups on the monomolecular interface of the pores are site-specifically modified to regulate the chemical environment of key regions within the pores, enhancing the sensing characteristics of the nanopores to better meet specific measurement requirements. The main preparation process of the mutant Aeromonas lysin precursor is as follows: Figure 5 As shown: Based on actual needs and combined with molecular dynamics simulations, the positions and types of amino acids requiring modification were first selected. Then, site-directed mutagenesis was performed using genetic engineering to obtain mutant aerolysin. The mutagenesis process is described as follows: Based on the characteristics of aerolysin nanopores, the Pet-22b-Proaerolysin expression vector was designed and constructed and transformed into *E. coli* cells for expression. Subsequently, the *E. coli* cells were sonicated to break them up. The obtained product was purified to obtain high-purity mutant aerolysin precursor Proaerolysin. The C-terminal peptide (CTP) of Proaerolysin was cleaved using trypsin to obtain activated mutant aerolysin monomers. Then, as described above, these activated mutant aerolysin protein monomers self-assembled on a planar phospholipid membrane to form nanopores.

[0078] When operating the apparatus of the present invention, an electrolyte solution, preferably an aqueous electrolyte solution, is added to the CIS and tanS chambers. The aqueous solvent used can be distilled water, double-distilled water, deionized water, ultrapure water, etc., preferably ultrapure water. Conventional electrolytes, such as KCl and NaCl, can be used. The concentration of the electrolyte solution can be adjusted as needed, for example, it can be 0.01-5 mol / L, 0.1-2 mol / L, 0.5-1.5 mol / L, 0.8-1.2 mol / L, or 0.9-1 mol / L, or within the numerical range formed by any combination of the above two extreme values.

[0079] Suitable electrodes can be selected in the CIS chamber and the trans chamber as needed, such as platinum electrodes, gold electrodes, graphite electrodes, Ag / AgCl electrodes, etc. According to a specific embodiment of this application, an identical Ag / AgCl electrode is provided in both the CIS chamber and the trans chamber. The electrode in each chamber is connected to an external detection device. Figure 1 The diagram shows a preamplifier, A / D converter, amplifier, etc., which are directly or indirectly connected to the electrodes. However, other devices can also be used as needed to connect them directly or indirectly to the electrodes. These other devices may include power supplies, resistors, ammeters, voltmeters, multimeters, protective resistors, etc.

[0080] According to one embodiment of this application, when the detection device of this application is in operation, a voltage is applied to the electrodes in the CIS chamber and the trans chamber. The electrolyte in these two chambers moves under the action of the voltage, and the nanopores become the only channels for conducting ions. When one or more molecules in the sample to be tested enter the channel at least partially and undergo relative displacement therein, the ion concentration inside the channel changes due to the space occupied by the molecules, causing blockage of the ion flow inside the channel. In addition, since different positions in the nanopores have different amino acid designs (thus generating different interactions), the analyte may also generate different specific electrical signals when it displaces to different positions in the nanopores. Furthermore, if the molecule can completely pass through the channel, the current will return to the open state after the molecule has completely passed through. Figure 1The small image in the upper right corner illustrates a schematic diagram of the current being blocked as molecules pass through nanopores. However, it should be noted that this is merely a schematic representation of the principle, not the actual detection signal of the technical solution in this application. The parameters of this blocking current signal, such as the degree of blocking, blocking time, blocking current, standard deviation of blocking current fluctuation, interval time distribution, and number of blocking electrical signals, are closely related to factors such as the size, structure, conformational changes, interactions, passage speed, and concentration of molecules passing through the nanopores. This invention can achieve the detection of various parameter details of individual molecules passing through nanopores, such as qualitative or quantitative detection, by utilizing the aforementioned parameter characteristics of the blocking current signal. A single protein nanopore is a precisely tunable single-molecule sensing interface. Electrochemically controllably confines a single analyte molecule within an independent nanopore, thus allowing the reading of the "intrinsic information" of a single molecule closest to itself without labeling. On the other hand, based on the continuous recording of electrochemical ion currents, nanopore technology can read the behavior of each molecule in the system in high throughput, achieving both high resolution and high throughput detection. According to one embodiment of this application, the method of the present invention performs qualitative detection of the molecules of the analyte, for example, based on one or more of the following detection information: electrical signal blocking current, electrical signal blocking degree, blocking time, standard deviation of blocking current fluctuation (STD), or a combination of two or more of the above information. According to another embodiment of this application, the method of the present invention performs quantitative detection of the molecules of the analyte, based on the linear relationship between the number of electrical signals detected at intervals or effective unit times and their concentration.

[0081] During testing, one or more additional reagents, such as pH adjusters, buffers, chelating agents, surfactants, etc., can be added to at least one of the CIS and TANS chambers as needed.

[0082] According to one embodiment of this application, the method of this application can be used to perform qualitative or quantitative analysis on one or more suitable biological sample molecules to be tested. For example, according to one embodiment, a pure biological molecule to be tested can be qualitatively or quantitatively analyzed. According to another embodiment, a mixture of two, three, four, five, six, seven, eight, nine, ten or more biological molecules to be tested can be qualitatively or quantitatively analyzed simultaneously.

[0083] According to one embodiment of this application, biological samples that can be tested using the methods of this application may include mammalian body fluids, synthetic peptides, recombinant peptides and combinations thereof, and may also include any other suitable biological samples.

[0084] According to one embodiment of this application, the biological sample detected by the method of the present invention can be a sample obtained or extracted from any organism or microorganism, which is typically an archaea, prokaryotic, or eukaryotic microorganism and generally belongs to one of the following five kingdoms: plant, animal, fungi, prokaryotes, and protists. The invention can be performed in vitro on samples obtained or extracted from any virus. The sample is preferably a fluid sample. The sample typically contains bodily fluids from a patient. The sample can be urine, lymph, saliva, mucus, or amniotic fluid, but is preferably blood, plasma, or serum. Typically, the sample is of human origin, but alternatively it can be from another mammal, such as commercially raised animals like horses, cattle, sheep, fish, chickens, or pigs, or alternatively, it can be a pet, such as a cat or dog. Simultaneously, the sample can be of plant origin, such as samples obtained from economic crops, like cereals, legumes, fruits, or plants, such as wheat, barley, oats, canola, corn, soybeans, rice, rhubarb, bananas, apples, tomatoes, potatoes, grapes, tobacco, kidney beans, lentils, sugarcane, cocoa, and cotton. The sample can also be abiotic. Abiotic samples are preferably fluid samples. Examples of abiotic samples include surgical fluids, water (such as drinking water, seawater, or river water), and reagents for laboratory testing. According to one embodiment of this application, the biological sample comprises proteins, polypeptides, nucleotides, amino acids, polysaccharides, their derivatives, and combinations thereof. In this application, the derivatives include protein derivatives, polypeptide derivatives, nucleotide derivatives, amino acid derivatives, and polysaccharide derivatives. The derivative refers to a derivative compound formed by removing, adding, or forming one or more substituents in the molecule of the above-mentioned compound. Examples of substituents may include any one or more of the following: amino acid residues, carboxyl, hydroxyl, mercapto, alkyl, alkenyl, alkoxy, alkenyloxy, ester, ether, amino, nitro, nitroso, nitrate, phosphate, phosphonic acid, phosphate ester, phosphonate, phosphite, phosphonite, and halogens (fluorine, chlorine, bromine, iodine, etc.). For example, the amino acid derivative may include adrenaline, thyroid hormone, plant growth regulators, etc.

[0085] According to a specific embodiment of this application, the method of the present invention can be used to detect analyte molecules in biological samples, including peptides that are components of the renin-angiotensin system, which may include one or more of the substances shown in Table 1 below.

[0086] Table 1: Specific examples of polypeptides that make up the Renin-Angiotensin System

[0087]

[0088]

[0089] *The table shows the net charge of the polypeptide at pH 8.0.

[0090] Through long-term in-depth research, researchers have discovered that electrostatic interactions, van der Waals interactions (including dispersion forces between transient induced dipoles, Debye forces between permanent and induced dipoles, and Keesom forces between permanent molecular dipoles), hydrogen bonding, and volume exclusion within the pores all affect the blocking of ion current in the nanopores to varying degrees. In other words, numerous factors significantly influence the measured electrical signal. The technology of this invention can even identify differences in individual amino acids on analyte molecules (e.g., proteins, peptides), thereby enabling qualitative and quantitative analysis of mixtures of the same series of analyte molecules with very similar compositions and structures. For example, according to one embodiment of this application, multiple peptide molecules belonging to the Ang series can be distinguished in real time and accurately, achieving high sensitivity, high capture efficiency, and high compatibility. It should be particularly noted that the achievement of the technical objectives of this invention overcame significant difficulties. First, as shown in Table 1, many analyte molecules in the samples targeted by this application, such as Ang series peptides, can be electrically neutral molecules, and the detection of such electrically neutral molecules has always been a challenge. For example, it is known in the art that typical polynucleotides contain a large number of nucleotide units, each with a negative charge on its phosphate backbone. Therefore, the negative charge of conventional polynucleotides or oligonucleotides is typically at least -3 or more, while the electronegativity of long nucleotide chains like DNA is much higher. Existing detection techniques are often designed for these highly charged analytes, and these techniques are often ineffective for the lower-charged or electroneutrally neutral analytes shown in Table 1 above. The method of this invention is suitable for conventional analytes with a large number of positive or negative charges, and also pioneers a detection technique particularly suitable for the lower-charged or electroneutrally neutral analytes shown in Table 1 above. Secondly, this application achieves high capture efficiency and real-time, accurate, and efficient qualitative or quantitative detection, enabling precise and rapid qualitative and quantitative testing of one or more molecules, which is of great significance for research in related fields.

[0091] In addition, this application also develops a method for testing enzyme activity, the method comprising:

[0092] This allows one or more substrates to undergo an enzyme-catalyzed reaction under enzyme-catalyzed conditions, generating one or more products;

[0093] Prior to the enzyme-catalyzed reaction, the substrate is qualitatively or quantitatively detected using the detection method described in this invention, preferably quantitatively, to determine the substrate content in the system before the catalytic reaction; and during and / or after the enzyme-catalyzed reaction, the substrate is qualitatively or quantitatively detected using the detection method described in this invention, preferably quantitatively, to track the substrate content in the system during and / or after the reaction; and

[0094] The rate of enzyme-catalyzed reaction is tracked based on the qualitative or quantitative detection results of the substrate, preferably the quantitative detection results, thereby determining the enzyme activity.

[0095] The enzymes tested in the above methods may include any number of targeted substrates or products that can be qualitatively or quantitatively tested using the methods of this application, such as various proteases, peptidases, nucleotidases, aminoacidases, and polysaccharideases. They may also include enzymes with various functions, such as invertases, polymerases, dissociative enzymes, transcriptases, reverse transcriptases, hydrolases, lysins, and synthases. According to a particularly preferred embodiment, the enzyme may be angiotensin-converting enzyme.

[0096] Example

[0097] To better understand the present invention, the following description, in conjunction with embodiments and accompanying drawings, further illustrates the invention. The following embodiments are only for further illustrative purposes and should not be construed as limiting the scope of the invention. Any non-essential improvements and adjustments made to the inventive concept and technical solutions based on the present invention will be covered within the protection scope of the present invention.

[0098] Unless otherwise specified, all reagents used in the following examples are of analytical grade.

[0099] In the following embodiments, if it is stated that "the same steps as in a certain embodiment above are followed, except that...", it means that the process conditions and steps described below are used, while other process conditions and steps not mentioned are as described in the previous embodiments cited.

[0100] Materials and reagents

[0101] The following reagents were used in the examples:

[0102] Decane (purity ≥99.98%), potassium chloride (purity ≥99.99%), and Trypsin-EDTA were purchased from Sigma-Aldrich; tris(hydroxymethyl)aminomethane (TRIS, purity ≥99.8%) and ethylenediaminetetraacetic acid (EDTA, purity ≥99%) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 1,2-diphydanylphospholipid was purchased from Avanti (USA). All buffer solutions used in the experiments were prepared using a Milli-Q water purifier (Millipore, USA) with a conductivity of 18 MΩcm. -1 ProAerolysin was prepared with ultrapure water at 25°C. ProAerolysin was prepared with Tris-HCl buffer. 1,2-Diphycoylphospholipid was prepared into a solution with a concentration of 10 mg / mL using decane, aliquoted under a nitrogen atmosphere, and stored in a sealed container at 4°C.

[0103] The peptides used as test subjects in the experiment were all provided by Jier Biochemical (Shanghai) Co., Ltd., and had already been purified by HPLC. The inventors used them directly without further purification after purchasing them.

[0104] Experimental instruments

[0105] The nanopore detection cell was custom-designed by Warner Instruments and features... Figure 1 The structure shown is made of acetal resin and includes a cis-end detection cell (chamber) and a trans-end detection cell (chamber). The two detection cells are integrally manufactured with a partition and connected by a 50 μm diameter hole to support the phospholipid membrane. The Axopatch 200B amplifier was purchased from Molecular Devices, Inc., USA; the Digidata 1440A low-noise data acquisition system was purchased from Molecular Devices, Inc., USA; the CV203BU preamplifier probe was purchased from Molecular Devices, Inc., USA; the Faraday shielding box was independently developed by the inventor's laboratory; the analytical balance was purchased from METTLER TOLEDO, Inc., USA; the silver wire (1.0 mm in diameter) was purchased from Alfa Aesar, Inc., USA; the MOSAIC 1.3 was purchased from the NIH, Inc., USA; the Origin 9.0 analysis software was purchased from OriginLab, Inc., USA; and the angiotensin-converting enzyme (ACE) and angiotensin-converting enzyme 2 (ACE2) were purchased from Sigma-Aldrich.

[0106] Example 1: Synthesis of a detection device including T232K mutant Aeromonas hydrolysin nanopores

[0107] In Example 1, a mutant aeromonas hydrolysin T232K for fabricating nanopores was synthesized and used to form nanopores. Specifically, the codons of the Proaerolysin reference sequence (PDB number 1PRE) in the Protein Data Bank were optimized, and the wild-type Proaerolysin sequence was synthesized in its entirety by Shanghai Paisenuo Co., Ltd. The front primer sequence was designed as 5'-TATCGGAATTAATTCGGATCCGGATCCGGCAGAACC-3'; the back primer sequence was designed as 5'-TCGAGTGCGGCCGCAAGCTTAAGCTTTTGATTTGC-3', and the primers were provided by Shanghai Sangon Biotech Co., Ltd. The Proaerolysin sequence was amplified by PCR using Pfu DNA polymerase under the following conditions: 94°C pre-denaturation for 3 minutes, followed by the following cycles: 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 70°C extension for 1.5 minutes, and 35 cycles followed by a final extension at 72°C for 10 minutes. The empty vector pET-22b(+) was double-digested with restriction endonucleases BamHI and HindIII, and the digested vector was recovered using a gel extraction kit. The Proaerolysin sequence and the digested vector were ligated using a one-step cloning kit to obtain the pET-22b-Proaerolysin recombinant expression vector. This expression vector was then transformed into DH5α competent E. coli cells, and sequencing was performed by Shanghai Sangon Biotech Co., Ltd. Plasmids were extracted using a plasmid miniprep kit for later use. The mutant Proaerolysin expression vector T232K was constructed using KOD-Plus PCR polymerase via point mutation inverse PCR. The first primer sequence was 5'-AAACCAATAAATACGGTCTGAGCGAGAAAG-3', and the second primer sequence was 5'-AGACCGTATTTATTGGTTTTGCTCCAG-3'. The primers were synthesized by Shanghai Sangon Biotech Co., Ltd. In a 50 μL PCR system, the following were added: 5 μL KOD Plus polymerase buffer, 1 μL KOD Plus polymerase (1 u / μL), 5 μL dNTPs (0.2 mM), 2 μL MgSO4 (1.0 mM), 1.5 μL each of the front primer (10 μM) and the back primer (10 μM), 0.5 μL wild-type Proaerolysin plasmid (200 ng / μL), and 33.5 μL sterile water. The reaction conditions were: pre-denaturation at 94 °C for 3 minutes, followed by the following cycles: denaturation at 94 °C for 15 seconds, extension at 68 °C for 8 minutes, and a final extension at 72 °C for 10 minutes after 30 cycles. The PCR product was digested with DPnl enzyme to remove the template plasmid. In a 20 μL digestion system, the following were added: 2 μL DPnl enzyme buffer, 1 μL DPnl enzyme, and 17 μL PCR product; incubation was carried out at 37 °C for 2 hours.The digestion product was used for T4 DNA ligase circularization to form a plasmid. In a 20 μL circularization system, 2 μL of T4 DNA ligase buffer, 1 μL of T4 PNK (10 u / mL), 1 μL of T4 DNA ligase (400 u / mL), 2 μL of digestion product, and 14 μL of sterile water were added, and the mixture was incubated at 16°C for 3 hours. The circularized product was transformed into DH5α *E. coli* competent cells, and the plasmid was extracted using a plasmid miniprep kit. These *E. coli* cells were incubated at 37°C for approximately 2 hours. Subsequently, 1.0 mM IPTG was added to the culture medium to induce *E. coli* expression of the target protein for 5 hours. After centrifugation at 4°C and 8000 rpm for 30 minutes, the cells were collected, and then sonicated on ice for 30 minutes to lyse the cells and extract the protein. The supernatant was collected and the target mutant *Aeromonas hydrolysin* precursor, *Proaerolysin*, was extracted using a Ni-NTA affinity resin column and stored at -80°C for later use. 1.0 mg of the mutant aeromonas lysin precursor Proaerolysin was mixed with 0.03 units of trypsin agarose and incubated at 20 °C for 6 hours. Then, the trypsin agarose was removed by centrifugation at 12000 g for 10 minutes. The resulting active mutant aeromonas lysin T232K was stored at -20 °C for later use.

[0108] A few drops of a 10 mg / mL solution of 1,2-diphydanyl phospholipid in decane were added to the nanopore detection cell, causing a lipid bilayer membrane to form on the 50 μm micropores of the phospholipid acetal resin detection cell. Activated aeromonas lysin monomer protein was then added to the cis chamber, causing it to spontaneously self-assemble on the constructed phospholipid membrane to form transmembrane individual nanopores. The device thus fabricated is referred to hereinafter as "Inventive Device 1".

[0109] Comparative Example 1: Synthesis of a detection device including K238Q mutant Aeromonas hydrolysin nanopores

[0110] In this Comparative Example 1, the steps of Example 1 were repeated, except that the mutant Proaerolysin expression vector K238Q was constructed. The device thus created is referred to hereinafter as "Comparative Device 1".

[0111] Comparative Example 2: Synthesis of a detection device including N226Q mutant Aeromonas hydrolysin nanopores

[0112] In Comparative Example 2, the steps of Example 1 were repeated, with the only difference being the construction of the mutant Proaerolysin expression vector N226Q. The device thus created is referred to hereinafter as "Comparative Device 2".

[0113] Comparative Example 3: Synthesis of a detection device including wild-type Aeromonas hydrolysin nanopores

[0114] In Comparative Example 3, 1.0 mg of unmutated wild-type Aeromonas lysin precursor was mixed with 0.03 units of trypsin agarose, incubated at 20°C for 6 hours, and then centrifuged at 12000g for 10 minutes to remove trypsin agarose. The resulting active wild-type Aeromonas lysin was stored at -20°C for later use.

[0115] A few drops of a 10 mg / mL solution of 1,2-diphydanyl phospholipid in decane were added to the nanopore detection cell, causing a lipid bilayer membrane to form on the 50 μm micropores of the phospholipid acetal resin detection cell. Activated aeromonas lysin monomer protein was then added to the cis chamber, causing it to spontaneously self-assemble on the constructed phospholipid membrane to form transmembrane individual nanopores. The device thus fabricated is referred to hereinafter as "Comparative Device 3".

[0116] Example 2: Single-molecule nanochannel detection of Ang I peptide

[0117] In this second embodiment, the inventive device 1 manufactured in Example 1 was used. A small amount of 1.0M KCl solution with pH 8.0, buffered with 10mM Tris and 1.0mM EDTA, was added to the chamber, such that the KCl liquid level just covered the upper edge of the pore in the partition of the inventive device. Ag / AgCl electrodes were placed in both chambers, and a voltage of +100mV was applied to the electrodes (the cis side chamber was grounded). The operation was carried out at ambient temperature, and a stable current of approximately 50.0pA was obtained as a baseline. After a single stable mutant aeromonas lysin monomer protein nanopore was formed, the current trajectory was recorded for 5-10s at 10mV intervals within a voltage range of -120 to +160mV, to obtain the opening current of the aeromonas lysin bio-nanopore at different voltages. An IV curve was plotted to characterize the properties of the nanopore. Subsequently, the Ang I peptide analyte was added to the cis chamber at a concentration of 4.0μM, and single-channel events were measured using an applied potential. When recording picoampere-level microcurrents, the weak current was first pre-amplified using a CV 203BU amplifier, then further amplified using a patch-clamp amplifier (Axon 200B), and finally converted from analog to digital using a Digidata 1440A. In this experiment, the signal was filtered at 5kHz, and the current was recorded using Clampex 10.4 software at a sampling rate of 100kHz.

[0118] Recorded single-channel current blocking signals were processed and analyzed using MOSAIC 1.3 and Origin 9.0 software. During data processing, current fluctuations with blocking currents less than twice the RMS value were considered baseline noise and not analyzed further. Single-molecule blocking events with blocking currents greater than twice the RMS value were analyzed sequentially using MOSAIC software to obtain the blocking current and blocking time for each event. The blocking time of a single-molecule blocking event was defined as Duration, the blocking current as I, the orifice current as I0, and the blocking current degree as I / I0. The blocking time, blocking current, and interval time of characteristic single-molecule blocking events were statistically analyzed, and the data results were plotted into a two-dimensional blocking time-blocking current scatter plot and a one-dimensional histogram of blocking time, blocking current, and interval time. The overall distribution of blocking time and blocking current for single-molecule blocking events can be obtained from the two-dimensional blocking time-blocking current scatter plot. Exponential or Gaussian fitting was performed on the one-dimensional histogram to obtain the specific statistical average values ​​of blocking time and blocking current. The error is the standard deviation of three repeated experimental results.

[0119] In this Example 2, the analyte Ang I peptide was added to the CIS chamber at a concentration of 4.0 μM. The test was performed according to the steps and conditions described above, and the raw current-time (It) signal graph is shown below. Figure 6 As shown. Additionally, the statistical results of the blocking time and capture rate of the current signal at +140mV are as follows: Figure 7 As shown.

[0120] Comparative Example 4: Single-molecule nanochannel detection of Ang I peptide

[0121] In Comparative Example 4, the steps of Example 2 were repeated to detect the Ang I peptide, the only difference being the use of the comparison apparatus 1-3 prepared in Comparative Examples 1-3. The original signal graph of the measured current-time (It) curve is shown below. Figure 6 As shown. Additionally, the statistical results of the blocking time and capture rate of the current signal at +140mV are as follows: Figure 7 As shown.

[0122] from Figure 6As shown in the result curves of Example 2 and Comparative Example 4, compared with the unmutated wild-type Aeromonas hydrolysin nanopores and the two mutant Aeromonas hydrolysin nanopores (K238Q and N226Q) that do not fall within the scope of protection of this application, the T232K mutant nanopores, which fall within the scope of protection of this invention, can all form and function stably on the phospholipid bilayer, and each exhibits different changes in characteristic signals, achieving varying degrees of improvement compared to the comparative examples. In particular, the T232K mutant nanopores maintain excellent stability and repeatability over a wide voltage range (-120mV to 160mV) with less background signal.

[0123] from Figure 6 As can be seen from the result curves of Example 2 and Comparative Example 4, compared with the unmutated wild aeromonas lysin nanopores and the two mutant aeromonas lysin nanopores (K238Q and N226Q) that do not fall within the scope of protection of this application, the T232K mutant nanopores that fall within the scope of protection of this invention have achieved extremely significant improvements in both the blocking time and capture rate of the current signal.

[0124] The above results show that the design of mutations in nanopore proteins is not something that can be done obviously and once and for all. A mutant nanopore that is suitable for one type of test subject may not be suitable for another. Therefore, it is necessary to carefully and rationally design the single-molecule measurement interface of biological nanopores according to the structural characteristics of the specific target.

[0125] Example 3: Single-molecule nanochannel detection of different Ang peptides

[0126] In this third embodiment, the inventive apparatus 1 manufactured in Example 1 was used to detect a series of Ang peptides (Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III) using the same steps as in Example 2, with each peptide having a concentration of 4.0 μM. Specifically, in this embodiment, the Ang series peptides were first added sequentially to the CIS detection cell at the same concentration as in Example 2, and a scatter plot of the blocking degree I / I0 versus blocking time Duration of the characteristic current blocking signal was obtained as shown below. Figure 8 As shown, multiple distributions gradually appear, with each scatter point corresponding to a specific Ang series polypeptide. This indicates that detection can be performed very effectively by sequentially adding a series of Ang polypeptides.

[0127] Example 4: Single-molecule nanochannel detection of different Ang peptide mixtures

[0128] In this Example 4, the inventive apparatus 1 manufactured in Example 1 was used to perform the detection of a series of Ang peptides (Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III) using the same steps as in Example 2. Specifically, the five Ang peptides were premixed at the same concentration, with each peptide having a concentration of 4.0 μM, and the premixed mixture was added to the CIS detection cell. The scatter plot of the I / I0-Duration of the measured current blocking signal is shown below. Figure 9 As shown in the figure, the mixture of these five Ang peptides exhibited five characteristic current blockages in the T232K mutant nanopores, thus proving that this mutant nanopores can effectively identify five very similar samples in the mixture.

[0129] Figure 10 The original signals showing the distinct current blocking characteristics of different Ang series peptides in the mixture are demonstrated. The significant differences in these characteristic current blocking characteristics prove that the device of this application can be used to identify each different Ang peptide molecule in the mixture with great efficiency.

[0130] The scatter plots of STD and I / I0 for current blocking in the hybrid system, as well as the 3D plots of I / I0, STD, and Duration, are shown in [the figures]. Figure 11A and Figure 11B . Figure 11A The scatter plot shown depicts five distinct clusters, corresponding from left to right to Ang I, Ang 1-9, Ang II, Ang 1-7, and Ang III, respectively. Figure 11B The three-dimensional images shown illustrate the fluctuations in the blocking current of different peptides, further demonstrating that the five Ang series peptides in the mixture can be distinguished from each other very effectively.

[0131] Example 5: Synthesis of a detection device including N226K mutant Aeromonas hydrolysin nanopores

[0132] In this Example 5, the steps of Example 1 were repeated, with the only difference being the construction of the mutant Proaerolysin expression vector N226K. The device thus created is referred to below as "Inventive Device 2".

[0133] Example 6: Single-molecule nanochannel detection of different Ang peptide mixtures

[0134] Example 6 was performed in exactly the same manner as Example 4, except that the N226K mutant Aeromonas hydrolysin nanopores of the inventive device 2 manufactured in Example 5 were used to detect a series of Ang peptides (Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III). The scatter plot of the I / I0-Duration of the measured current blocking signal is shown below. Figure 12 As shown in the figure, the mixture of these five Ang peptides exhibited five characteristic current blockages in the N2262K mutant nanopores, thus proving that this mutant nanopore can effectively identify five very similar samples in a mixture.

[0135] Example 7: Synthesis of a detection device including A260R mutant Aeromonas hydrolysin nanopores

[0136] In this embodiment 7, the steps of embodiment 1 are repeated, with the only difference being the construction of the mutant Proaerolysin expression vector A260R. The device thus created is referred to hereinafter as "inventive device 3".

[0137] Example 8: Single-molecule nanochannel detection of different Ang peptide mixtures

[0138] Example 8 was performed in exactly the same manner as Example 4, except that the A260R mutant Aeromonas hydrolysin nanopores of the inventive device 3 manufactured in Example 7 were used to detect a series of Ang peptides (Ang I, Ang 1-9, Ang II, Ang 1-7, Ang III). The scatter plot of the I / I0-Duration of the measured current blocking signal is shown below. Figure 13 As shown in the figure, the mixture of these five Ang peptides exhibited five characteristic current blockages in the A260R mutant nanopores, thus proving that this mutant nanopores can effectively identify five very similar samples in a mixture.

[0139] Based on the results of Examples 4, 6, and 8, the T232K mutation was specifically designed for angiotensin-based peptides. First, to ensure the entry of angiotensin peptides into the pore, the T232K mutation enhances the electroosmotic flow of the pores, thereby increasing the capture efficiency of the angiotensin peptides. Therefore, introducing a positively charged amino acid to increase electroosmotic flow through a mutation is beneficial for detection. Second, considering the pore resolution, the T232K mutation reduces the diameter of the pore center, improving the resolution of peptides with individual amino acid differences. Therefore, reducing the pore diameter in and around the pore center is beneficial for detection. Furthermore, the results of Examples 6 and 8 can be extrapolated to other mutant aeromonas lysin pores with positively charged amino acids mutated in the pore center, such as... Figure 4As shown, one or more original amino acids at one or more sites among the wild-type Aeromonas hydrolysin 224, 226, 228, 230, 232, 234, 236, 260, 262, 264, 266, 268, 270, and 272 are mutated to one or more of lysine (K), arginine (R), and histidine (H).

[0140] Example 9

[0141] In this embodiment, the inventive device 1 manufactured in Example 1 above was used. A small amount of 1.0M KCl solution containing 10μM ZnCl2 at pH 8.0, buffered with 10mM HEPES, was added to the chamber, such that the KCl liquid level just covered the upper edge of the pore in the partition of the inventive device 1. ZnCl2 was used to maintain enzyme activity. After completing the above preparation steps, Ang I was added dropwise to the CIS detection cell, followed by ACE, so that the concentration of ACE was 36nM and the concentration of Ang I substrate was 4.0μM. Under these conditions, the enzymatic catalytic effect of ACE on the hydrolysis of Ang I was studied, with the current sampling rate and filtering rate being 100kHz and 5kHz, respectively. Figure 14A The original current trajectories of Ang I sheared by ACE at 0, 20, 40, 90, and 180 minutes are shown. Figures 14B to 14F The changes in characteristic currents corresponding to Ang I and the changes in newly generated characteristic currents at different time points are shown. With the addition of ACE to the nanopore detection system, the gradual disappearance of the characteristic current blockage corresponding to Ang I was observed, accompanied by the gradual appearance of characteristic current signals with smaller blockage degrees (larger I / I0 values) and shorter blockage times. The new characteristic current blockage was generated by Ang II entering the T232K mutant Aerolysin nanopores, proving the hydrolysis of Ang I by ACE. Furthermore, only two characteristic current blocks were detected during the entire ACE hydrolysis of Ang I process (approximately 3 hours), corresponding to Ang I and Ang II respectively, indicating that Ang II is the only product of ACE hydrolysis of Ang I. Based on continuous detection of current changes, we can further infer the complete evolution process of Ang I and Ang II throughout the entire enzymatic reaction by observing the changes in the I / I0 of the blockage signal and the frequency of event occurrence. The concentrations of Ang I and Ang II can be calculated by combining the capture efficiency of Ang I and Ang II in the T232K mutant Aerolysin nanopores. Figure 14G The graph shows the change of current blocking I / I0 over time. Figure 14H The graph shows the changes in the concentrations of Ang I and the product Ang polypeptide over time during ACE digestion, with a quantification time interval of 1 minute.

[0142] This embodiment 9 demonstrates that the inventive device 1 manufactured in embodiment 1 can be used to monitor the entire process of ACE hydrolysis of Ang I in real time very effectively.

[0143] Example 10

[0144] Example 10 was performed in exactly the same manner as Example 9, except that a mixture of ACE and ACE2 enzymes was added dropwise to Ang I in the CIS detection cell, so that the concentration of ACE was 36 nM, the concentration of ACE2 enzyme was 23 nM, and the concentration of Ang I substrate was 4.0 μM. Under these conditions, the enzymatic catalytic pathway of Ang I hydrolysis by the mixture of ACE and ACE2 enzymes was studied. Figure 15A The graph shows the change of current blocking I / I0 over time. Figure 15B The graph shows the changes in the concentrations of Ang I and the product Ang peptide over time during simultaneous ACE and ACE2 digestion, with a quantification time interval of 1 minute.

[0145] Example 10 demonstrates that the inventive device 1 manufactured in Example 1 can be used to very effectively monitor in real time the process of simultaneous hydrolysis of Ang I by a mixture of multiple enzymes.

[0146] In summary, this invention has developed a high-performance detection device based on the nanopores of mutant aeromonas lysin nanoparticles. It exhibits a sensitive and unique characteristic blocking current, enabling effective identification of analyte biomolecules with very similar composition and structure, as well as real-time monitoring of dynamic changes. This provides researchers exploring biological processes with a highly effective and reliable research tool.

Claims

1. A method for detecting biological samples using a nanopore detection device. The nanopore detection device includes an insulator with openings, a phospholipid membrane or polymer membrane located in the openings, nanopores formed by a mutant aeromonas lysin passing through the phospholipid membrane or polymer membrane, two or more chambers including electrodes located on both sides of the insulator, and a detector connected to the electrodes. The mutant aeromonas lysin is selected from one or more of the following mutants: T232K, A260R, N226K; the biological sample contains a renin-angiotensin system component polypeptide, which includes one or more of the following: Ang I, Ang 1-9, Ang II, Ang 1-7, and Ang III. The method includes the following steps: Add electrolyte to the two or more chambers; The biological sample is added into at least one of the chambers; This allows the biological sample to at least partially enter the nanopores, where displacement occurs and an electrical signal is generated. as well as The electrical signal is detected using a detector, and the biological sample is analyzed based on the measured electrical signal.

2. The method as described in claim 1, characterized in that, The biological samples are selected from mammalian body fluids, tissue extracts, intracellular fluids, natural polypeptides, synthetic polypeptides, recombinant polypeptides, and combinations thereof.

3. The method as described in claim 1, characterized in that, The method performs qualitative or quantitative detection on the biological sample based on the measured electrical signal; The qualitative detection is performed based on one or more of the following detection information: electrical signal blocking current, electrical signal blocking degree, blocking time, standard deviation of blocking current fluctuation (STD), or a combination thereof; The quantitative detection is performed based on the linear relationship between concentration and capture rate, where the capture rate is the reciprocal of the time interval between electrical signal events of a specific analyte, or the number of electrical signals of a specific analyte per unit time. The detection is performed in real time.

4. A method for testing enzyme activity, the method comprising: This allows one or more substrates to undergo an enzyme-catalyzed reaction under enzyme-catalyzed conditions, generating one or more products; Prior to the enzyme-catalyzed reaction, the substrate is qualitatively or quantitatively detected using the method of any one of claims 1-3; and / or during and / or after the enzyme-catalyzed reaction, the substrate and / or the product are qualitatively or quantitatively detected using the method of any one of claims 1-3. The enzyme activity is determined based on the qualitative or quantitative detection results of the substrate and / or product; The method for testing enzyme activity is performed in real time.

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