Anthrax virus protein nanopore electrochemical detection and analysis device and its application

Abstract

The present invention belongs to the field of electrochemical detection, and relates to an anthrax virus protein nanopore electrochemical detection and analysis device and its application. The device is formed by embedding a nanopore formed by anthrax virus protein into a membrane, and the membrane divides the resin chamber into two small chambers, cis and trans. Each chamber contains a buffer solution of a specific pH, and electrodes are placed separately to form a current loop. During the detection process, the device forms an electric field force by applying voltage to promote the interaction between amino acids and nanopores, and collects current signals for analysis to distinguish amino acid chiral isomers. The present invention solves the technical problem that amino acid chiral isomers are difficult to distinguish, and provides a highly sensitive detection method with simple operation, fast and accurate detection.

Description

Technical Field

[0001] The invention belongs to the field of electrochemical detection, and relates to an anthrax virus protein nanopore electrochemical detection and analysis device and application thereof. Background Art

[0002] The research history of anthrax virus protein (PA63) can be traced back to the 1980s. Initially, scientists discovered that the toxin produced by the anthrax virus was cleaved into PA63 monomers in the host cells. This discovery prompted people to begin to explore its structure and function in depth. Over time, researchers have gradually revealed the key role of PA63 protein in the pathogenic mechanism of anthrax virus through various biochemical and biophysical methods. Some scientists have also applied anthrax virus protein to nanopore single-molecule detection. The research direction mainly focuses on the interaction between anthrax virus protein and edema factors and pathogenic factors, and to date there is still the problem of instability of the nanopore channel formed. Therefore, it is necessary and urgent to construct a stable anthrax virus antigen protein nanopore channel.

[0003] The detection of amino acids has always been a hot topic in biological research. The side chain structure of different amino acids determines their chemical properties, such as polarity, hydrophobicity, charge state, etc. These properties affect the three-dimensional structure of proteins and their functions. By studying the position and amount of different amino acids in proteins, we can deeply understand the functional mechanism and structural dynamics of proteins. The difference in amino acids directly affects the function and biological activity of proteins. Therefore, it is necessary to distinguish between different amino acids.

[0004] Chirality is ubiquitous in nature. Countless biological and physiological processes are closely related to the chirality of molecules. Chiral preference is an important secret in studying the origin of life. It can induce the participation of biological molecules and produce different biological effects. It has very important research significance in the occurrence and development of diseases and drug design.

[0005] There have been a lot of studies on the distinction between different amino acids, but the resolution needs to be improved, and few people still focus on different chiral amino acids. The opening current of the anthrax virus antigen protein channel is small, and the resolution can reach the fA level. Using this channel can effectively improve the efficiency of distinguishing different chiral amino acids. Summary of the invention

[0006] In view of this, the object of the present invention is to provide a nanopore electrochemical detection and analysis device and its application in detecting amino acid chiral isomer molecules.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The invention provides an anthrax virus protein nanopore electrochemical detection and analysis device. The anthrax virus protein nanopore electrochemical detection and analysis device comprises anthrax virus protein, a membrane, and an electrode. The anthrax virus protein forms a nanopore embedded in the membrane, the membrane is placed in a resin chamber containing a basic buffer solution, the membrane divides the entire resin chamber into two small chambers, cis and trans, two electrodes are placed in the two small chambers respectively, a power supply, an ammeter and other devices are connected in sequence between the two electrodes to form a current loop, the buffer in the cis chamber is buffer A: the buffer A contains 1-butyl-3-methylimidazole chloride at a concentration of 0.5M, tris(hydroxymethyl)aminomethane at a concentration of 10mM, and pH=5.6, and the buffer in the trans chamber is buffer B: the buffer B contains 1-butyl-3-methylimidazole chloride at a concentration of 0.5M, tris(hydroxymethyl)aminomethane at a concentration of 10mM, and pH=7.6.

[0009] The anthrax virus protein is cleaved by the membrane enzyme of the Furin family from PA83, releasing a 20kDa N-terminal fragment PA20 and a 63kDa C-terminal fragment PA63, and PA63 forms the anthrax virus protein heptamer nanopore.

[0010] The membrane is any one of a Teflon membrane and a silicon nitride solid material.

[0011] The electrode is a silver electrode with silver chloride coated on the surface.

[0012] Furthermore, the application of the anthrax virus protein nanopore electrochemical detection and analysis device in detecting amino acid chiral isomer molecules comprises the following detection steps:

[0013] S1: adding the amino acid solution into the chamber cis of the anthrax virus protein nanopore electrochemical detection and analysis device according to claim 3;

[0014] S2: The anthrax virus protein nanopore electrochemical detection device applies voltage to form an electric field force in the solution, and the amino acid is driven to interact with the nanopore under the action of the electric field force, and the current signal is collected;

[0015] S3: The current signal collected in step S2 is amplified by a low-noise current amplifier, and the amplitude, current blocking time and signal generation frequency of the collected current signal are characteristically analyzed to finally obtain the corresponding amino acid test results.

[0016] The beneficial effects of the present invention are:

[0017] The present invention utilizes the pH asymmetric system in the electrochemical device to achieve the stability of the anthrax virus antigen protein nanopore channel and distinguish amino acids of different chirality; the anthrax virus antigen protein channel used in the present invention has a small opening current and a resolution of fA level. The channel can effectively improve the efficiency of distinguishing between amino acids of different chirality, filling the research gap in this area.

[0018] Other advantages, objectives and features of the present invention will be described in the following description to some extent, and to some extent, will be obvious to those skilled in the art based on the following examination and study, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0020] Figure 1 This is a diagram of the heptamer nanopore formed by anthrax virus protein;

[0021] Figure 2 Figure 2 shows the anthrax virus antigen protein nanopore channels constructed in four buffer systems (AB, BB, BC, BD) and their blank trajectory diagrams under applied voltage. a is system AB, b is system BB, c is system BC, and d is system BD.

[0022] Figure 3 a is a comparison chart of the discrete constant σ, conductivity, opening current and clamping frequency of four buffer systems (AB, BB, BC, BD). Figure 3 b is the IV curves of four buffer systems (AB, BB, BC, and BD) and the Gaussian fitting curve of the conductivity of the AB system;

[0023] Figure 4 Characteristic electrical signals of the charged type, polarity and non-polarity of 20 L-amino acids (4a) and 20 D-amino acids (4b) after classification;

[0024] Figure 5 Violin plots of 20 L-amino acids (5a) and 20 D-amino acids (5b) classified into charged type, polar and non-polar types according to their properties;

[0025] Figure 6 The two-dimensional graphs of specific peaks and amino acid volumes and specific peaks and pore-passing time for 20 L-amino acids (6a) and 20 D-amino acids (6b) divided into charged type, polar and non-polar types according to their properties;

[0026] Figure 7The difference analysis matrix diagram of 20 L-amino acids (7a) and 20 D-amino acids (7b) compared with their amino acid molecules of the same chirality but different types;

[0027] Figure 8 , Figure 8 a is a difference analysis matrix diagram of the structural formulas of 8 non-polar L-amino acids and 8 non-polar D-amino acids and the amino acid molecules of the same type with different chirality. Figure 8 b is the structural formula of 4 kinds of charged L-amino acids and 4 kinds of charged D-amino acids and the difference analysis matrix diagram of the amino acid molecules of the same type and different chirality. Figure 8 c is a difference analysis matrix diagram of the structural formulas of 6 polar L-amino acids and 6 polar D-amino acids and their comparison with amino acid molecules of the same type but different chirality. DETAILED DESCRIPTION

[0028] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0029] Among them, the drawings are only used for illustrative explanations, and they only represent schematic diagrams rather than actual pictures, and should not be understood as limitations on the present invention. In order to better illustrate the embodiments of the present invention, some parts of the drawings may be omitted, enlarged or reduced, and do not represent the size of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions in the drawings may be omitted.

[0030] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if the terms "upper", "lower", "left", "right", "front", "rear", etc. indicate the orientation or position relationship, they are based on the orientation or position relationship shown in the drawings, which is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operate in a specific orientation. Therefore, the terms describing the position relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0031] The amino acid samples used in the following examples are taken from the 20 common amino acids and the corresponding amino acids of different chirality. When the amino acid is left-handed, it is called L-amino acid for short, and when the amino acid is right-handed, it is called D-amino acid for short.

[0032] Example 1

[0033] 1. Prepare solution:

[0034] Prepare a variety of basic buffer solutions: dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (hydroxymethylaminomethane) (Tris) in deionized water, and adjust the pH to form different basic buffer solutions containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (Tris) (buffer A has a concentration of 0.5 M butyl-3-methylimidazolium chloride, a concentration of 10 mM tris, and a pH of 5.6; buffer B has a concentration of 0.5 M butyl-3-methylimidazolium chloride, a concentration of 10 mM tris, and a pH of 7.6; buffer C has a concentration of 1 M butyl-3-methylimidazolium chloride, a concentration of 10 mM tris, and a pH of 7.6; buffer D has a concentration of 1 M butyl-3-methylimidazolium chloride, a concentration of 10 mM tris, and a pH of 5.6). Finally, filter it with a 0.22μM filter before use;

[0035] 2. Construction of nanopore electrochemical detection and analysis device:

[0036] The anthrax virus protein biological nanopore is embedded in a membrane (the membrane selected here is a Teflon membrane), the membrane is placed in a resin chamber containing a basic buffer solution, the entire resin chamber is divided into two small chambers by the membrane, two electrodes (the two electrodes are silver electrode materials with silver chloride on the surface) are placed in the two small chambers respectively, and a power supply, an ammeter and other devices are connected in sequence between the two electrodes to form a current loop, that is, a nanopore electrochemical detection and analysis device (such as Figure 2 shown).

[0037] 3. Detect the stability of anthrax virus protein channels in different systems:

[0038] (1) The prepared buffer solution is grouped into two groups according to AB, BB, BC and BD, and added into the two chambers divided into two by the Teflon membrane. A voltage is applied to the device to form an electric field force in the solution. No sample to be tested is added, and a blank current signal is collected to analyze the stability of the anthrax virus protein nanopore channel under different systems;

[0039] (2) The current signal collected in step (1) is amplified by a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current blocking time and signal generation frequency of the collected current signal are analyzed (the collected current signal file is imported into the clampfit software to select the characteristic current signal and calculate its σ value. In physics, σ usually represents the standard deviation (Standard Deviation) is a statistic used to measure the deviation of the distribution of data points in a data set from the mean. The physical meaning of the standard deviation σ can be interpreted as the degree of distribution of data points around the mean. Specifically, the standard deviation describes the average deviation of each data point in the data set from the mean. The larger the standard deviation, the wider the distribution of data points relative to the mean; the smaller the standard deviation, the denser the distribution of data points relative to the mean. In physics applications, standard deviation can help scientists and engineers understand the reliability and stability of experimental data or measurement results. A small standard deviation means that the data points are closer to the mean, reflecting the stability of the data. Therefore, the smaller the σ value obtained in the clampfit software, the more stable the anthrax virus protein nanopore channel in this system. At the same time, the smoothness of the IV curve is also one of the evaluation criteria. The closer the IV curve is to a straight line, the more stable the channel is.

[0040] Compare the blank trajectory diagrams under different systems, such as Figure 2 As shown, a is a pH asymmetric and concentration symmetric system of buffer AB; b is a pH symmetric and concentration symmetric system of buffer BB; c is a pH symmetric and concentration asymmetric system of buffer BC; d is a pH and concentration asymmetric system of buffer BD. Figure 2 The trajectory diagram shows that the anthrax virus antigen nanopore channel is the most stable in the buffer AB system among the four systems. Figure 3 As shown in a, the σ value is the smallest in the buffer AB system. The smaller the value, the closer the opening current is to the average level, and the more stable it is. Figure 3 As shown in b, the IV curve of the buffer AB system is also closest to a straight line, indicating that this channel is the most stable.

[0041] Example 2

[0042] 1. Prepare solution:

[0043] (1) Prepare the basic buffer solution: dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (hydroxymethyl)aminomethane (Tris) in deionized water to form a basic buffer solution containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (hydroxymethyl)aminomethane (Tris) (the concentration of 1-butyl-3-methylimidazolium chloride in buffer A is 0.5 M, the concentration of tris is 10 mM, and the pH is 5.6; the concentration of 1-butyl-3-methylimidazolium chloride in buffer B is 0.5 M, the concentration of tris is 10 mM, and the pH is 7.6). Finally, filter with a 0.22uM filter before use;

[0044] (2) Preparation of amino acid solution: Dissolve various amino acid solids in deionized water to form an amino acid solution with a concentration of 10 mM.

[0045] 2. Construction of nanopore electrochemical detection and analysis device:

[0046] The anthrax virus protein biological nanopore is embedded in a membrane (the membrane selected here is a silicon nitride solid material), the membrane is placed in a resin chamber containing a basic buffer solution, the entire resin chamber is divided into two small chambers cis and trans by the membrane, buffer solutions A and B are added to the two small chambers respectively, and two electrodes (the two electrodes are silver electrode materials with silver chloride on the surface) are placed in the two small chambers respectively, and a power supply, an ammeter and other devices are connected in sequence between the two electrodes to form a current loop, thus forming the nanopore electrochemical detection and analysis device.

[0047] 3. Detection of amino acids with different chirality:

[0048] (1) adding the prepared amino acid solution to the chamber cis of the nanopore electrochemical detection device constructed above, using the basic buffer solutions A and B as electrolytes in the device, applying voltage to the device to form an electric field force in the solution, and promoting the amino acid to interact with the nanopore under the action of the electric field force, and collecting current signals;

[0049] (2) The current signal collected in step (1) is amplified using a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current blocking time and signal generation frequency of the collected current signal are subjected to characteristic analysis (the collected current signal file is imported into the clampfit software to select the characteristic current signal, and the selected current signal data is imported into the origin software for statistical analysis and a mathematical statistical graph of the signal is drawn), and finally the corresponding amino acid sample statistical graph is obtained.

[0050] The 20 L-amino acids and 20 D-amino acids were divided into three categories according to their properties: charged type, polarity and non-polarity. The differences of the 20 L-amino acids and 20 D-amino acids were analyzed and compared in terms of signal morphology, specific peak, pore time, amino acid volume, etc. using violin plots, scatter plots, matrix plots and other forms of expression. Figure 5 As shown in Figure 2, the 20 L-amino acids and 20 D-amino acids differ in signal morphology. Figure 6 As shown, it can be seen that there are obvious differences between the three types of L-amino acids and D-amino acids. Figure 7 As shown in Figure 2, the differences between different amino acids can also be clearly seen from the two-dimensional aspects of volume and specific peak and transit time and specific peak. Figure 8 As shown in the figure, 20 L-amino acids and 20 D-amino acids were analyzed for differences, and according to the obtained P values, it can be seen that there are significant differences between different amino acids. The above results show that the anthrax virus antigen protein biological nanopore can achieve high-resolution distinction between 20 L-amino acids and 20 D-amino acids.

[0051] Example 3

[0052] 1. Prepare solution:

[0053] (1) Prepare the basic buffer solution: dissolve 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (hydroxymethyl)aminomethane (Tris) in deionized water to form a basic buffer solution containing 1-butyl-3-methylimidazolium chloride (BMIMCL) and tris (hydroxymethyl)aminomethane (Tris) (the concentration of 1-butyl-3-methylimidazolium chloride in buffer A is 0.5 M, the concentration of tris is 10 mM, and the pH is 5.6; the concentration of 1-butyl-3-methylimidazolium chloride in buffer B is 0.5 M, the concentration of tris is 10 mM, and the pH is 7.6). Finally, filter with a 0.22uM filter before use;

[0054] (2) Prepare amino acid solution: Dissolve various amino acid solids in deionized water to form an amino acid solution with a concentration of 10 mM.

[0055] 2. Construction of nanopore electrochemical detection and analysis device:

[0056] The anthrax virus antigen protein biological nanopore is embedded in a membrane (the membrane selected here is a Teflon membrane), the membrane is placed in a resin chamber containing a basic buffer solution, the entire resin chamber is divided into two small chambers cis and trans by the membrane, buffer solutions A and B are added into the two small chambers respectively, and two electrodes (the two electrodes are silver electrode materials with silver chloride on the surface) are placed in the two small chambers respectively, and a power supply, an ammeter and other devices are connected in sequence between the two electrodes to form a current loop, thus forming the nanopore electrochemical detection and analysis device.

[0057] 3. Detection of amino acids with different chirality:

[0058] (1) adding the prepared amino acid solution to the chamber cis of the nanopore electrochemical detection device constructed above, using the basic buffer solutions A and B as electrolytes in the device, applying voltage to the device to form an electric field force in the solution, and promoting the amino acid to interact with the nanopore under the action of the electric field force, and collecting current signals;

[0059] (2) The current signal collected in step (1) is amplified using a low-noise current amplifier (Axon Axopatch 200B), and the amplitude, current blocking time and signal generation frequency of the collected current signal are subjected to characteristic analysis (the collected current signal file is imported into the clampfit software to select the characteristic current signal, and the selected current signal data is imported into the origin software for statistical analysis and a mathematical statistical graph of the signal is drawn), and finally the corresponding amino acid sample statistical graph is obtained.

[0060] The statistical diagrams of different chiral amino acids were analyzed and compared, such as Figure 8 As shown in the figure, it can be found that there are significant differences between amino acids of different chirality. The above results indicate that in response to changes in chirality, the stable anthrax virus antigen protein nanopore exhibits selective detection of amino acids of different chirality with high resolution, reflecting the multi-dimensional advantages of the nanopore, which can successfully distinguish between left-handed and right-handed amino acids.

[0061] In summary: The present invention designs a stable nanopore channel based on anthrax virus antigen protein, and uses the channel to detect and distinguish 20 kinds of amino acid molecules and their chiral isomers. This method significantly improves the stability of the anthrax virus antigen protein channel by constructing a pH asymmetric system, so that it can be put into the next step of detection. The anthrax virus antigen protein channel has the characteristics of small opening current and high resolution, so that it can distinguish 20 kinds of amino acids and their chiral isomers with high resolution.

[0062] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. An anthrax virus protein nanopore electrochemical detection and analysis device, characterized in that: The anthrax virus protein nanopore electrochemical detection and analysis device comprises anthrax virus protein, a membrane, and an electrode. The anthrax virus protein forms a nanopore embedded in the membrane, the membrane is placed in a resin chamber containing a basic buffer solution, the membrane divides the entire resin chamber into two small chambers, cis and trans, two electrodes are placed in the two small chambers respectively, a power supply and an ammeter are connected in sequence between the two electrodes to form a current loop, the buffer in the cis chamber is buffer A: the buffer A contains 1-butyl-3-methylimidazole chloride at a concentration of 0.5M, tris(hydroxymethyl)aminomethane at a concentration of 10mM, and pH=5.6, the buffer in the trans chamber is buffer B: the buffer B contains 1-butyl-3-methylimidazole chloride at a concentration of 0.5M, tris(hydroxymethyl)aminomethane at a concentration of 10mM, and pH=7.6, The anthrax virus protein is cleaved by the membrane enzyme of the Furin family from PA83, releasing a 20kDa N-terminal fragment PA20 and a 63kDa C-terminal fragment PA63, and PA63 forms the anthrax virus protein heptamer nanopore.

2. The anthrax virus protein nanopore electrochemical detection and analysis device according to claim 1, characterized in that: The membrane is any one of a Teflon membrane and a silicon nitride solid material.

3. The anthrax virus protein nanopore electrochemical detection and analysis device according to claim 1, wherein the electrode is a silver electrode with silver chloride coated on the surface.

4. Application of the anthrax virus protein nanopore electrochemical detection and analysis device according to claim 3 in detecting amino acid chiral isomer molecules, characterized in that: The detection steps are as follows: S1: adding the amino acid solution into the chamber cis of the anthrax virus protein nanopore electrochemical detection and analysis device according to claim 3; S2: The anthrax virus protein nanopore electrochemical detection device applies voltage to form an electric field force in the solution, and the amino acid is driven to interact with the nanopore under the action of the electric field force, and the current signal is collected; S3: The current signal collected in step S2 is amplified by a low-noise current amplifier, and the amplitude, current blocking time and signal generation frequency of the collected current signal are characteristically analyzed to finally obtain the corresponding amino acid test results.

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