Colletotrichum virus protein nanopore buffer solution system and biomolecule detection application

Abstract

The invention relates to an anthrax virus protein nanopore buffer solution system and biomolecule detection application, and belongs to the field of nanopore electrochemistry. The invention aims to solve the problem that an anthrax virus protein nanopore is unstable in electrochemical detection, and provides an anthrax virus protein nanopore buffer solution system and application thereof. The buffer solution is composed of trihydroxymethyl aminomethane and a compound I composed of specific cations and anions, the compound I comprises the cations such as NH4 < + >, K < + >, Li < + > and Mg < 2 + > and the anions such as Cl <->, HCOO <-> and SO4 < 2->, and the stability of nanopores is achieved by optimizing the concentration and the pH value. The method is applied to high-sensitivity and high-selectivity detection of different charged small molecules. According to the technical scheme, the stability of the anthrax virus protein nanopore is remarkably improved, the application prospect of the anthrax virus protein nanopore in the field of single molecule detection is widened, and a new thought and method are provided for development of a nanopore sensing technology.

Description

Technical Field

[0001] The present invention belongs to the field of nanopore electrochemistry, and relates to an anthrax virus protein nanopore buffer system and its application in biomolecule detection. Background Art

[0002] The Hofmeister series, also known as the solute series, is a classification method based on the ability of ions to salting-out or salting-in proteins. In solution, desolvation is the most crucial factor in maintaining the connection between solute and solvent. Whether cations or anions are attracted to the surface of these protein molecules or excluded from the surface of these protein molecules by cosolutes may have a significant impact on the solution desolvation effect. Ions in the Hofmeister series affect the salting-out process according to factors such as ionic strength and hydration energy, and their effectiveness varies greatly, being divided into chaotropes and kosmotropes. The closer the ionic properties are to chaotropes, the stronger the charge delocalization and the larger the polarizability, that is, the stronger the ability to disrupt the hydrogen bonds between water molecules. The addition of a suitable chaotrope salt can enhance the hydrophobicity of ionic analytes.

[0003] The research on anthrax virus proteins (especially PA63) began in the 1980s, and scientists have gradually revealed their key role in the pathogenic mechanism of anthrax virus. With the rapid development of nanotechnology, anthrax virus proteins have been applied to the field of nanopore single-molecule detection, especially in the study of their interactions with edema factor and lethal factor. In nanopore detection, the differences between individual molecules are mainly reflected by ionic interactions and water molecule hydrogen bonds. The pore diameter of the anthrax virus protein protective antigen nanopore is relatively long, and there are more interaction sites for molecules in the pore, which can better analyze its ionic current. The anthrax virus protein protective antigen nanopore is formed by the self-assembly of the remaining part into a cyclic homoheptameric channel after a small part is cleaved by furin family proteases after the protective antigen binds to cell surface receptors. However, a key problem faced by existing research is that the formed nanopore channels are unstable, which severely limits their application in electrochemical detection.

[0004] CN119269595A provides an anthrax virus protein nanopore electrochemical detection and analysis device to solve the problems of unstable nanopore channels and difficulty in distinguishing amino acid chiral isomers in the prior art. The device mainly includes an anthrax virus protein, a membrane, electrodes, and a resin chamber containing a basic buffer solution. The anthrax virus protein forms nanopores embedded in the membrane, and the membrane divides the entire resin chamber into two small chambers, cis and trans. Each chamber contains a buffer solution with a specific pH, and electrodes are placed respectively to form a current loop.

[0005] The present invention further solves the problems of instability and clamping of the nanopore of the protective antigen of anthrax virus protein, so as to be widely applied in the field of nanopore single molecule detection. And as a nanopore with a relatively long pore diameter and a small opening current, this nanopore has good application prospects for nanopore single molecule detection with higher selectivity, high sensitivity, and high specificity. Summary of the Invention

[0006] In view of this, the present invention analyzes the electrical properties of the nanopore of the protective antigen of anthrax virus protein using the Hofmeister series, and invents an electrolyte buffer system that can stabilize the nanopore of the protective antigen of anthrax virus protein and distinguish charged small molecules. The purpose of the present invention is to provide a buffer system for anthrax virus protein nanopores and its application in biomolecule detection.

[0007] To achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides a buffer system for anthrax virus protein nanopores, which is composed of tris(hydroxymethyl)aminomethane and Compound I. Compound I includes cations: NH4 + >K + >Li + >Mg 2+ at least one of them, and anions: Cl – >HCOO – >SO4 2- at least one of them. The concentration of tris(hydroxymethyl)aminomethane is 10 mM, the concentration of Compound I is 0.1 - 1.5 M, and the pH of the buffer is 7.6;

[0009] Preferably, Compound I is composed of at least one compound selected from magnesium sulfate, magnesium formate, magnesium chloride, lithium sulfate, lithium formate, lithium chloride, potassium sulfate, potassium formate, potassium chloride, ammonium sulfate, ammonium formate, and ammonium chloride;

[0010] Preferably, Compound I is lithium formate, and the concentration of lithium formate is 0.5 M;

[0011] Furthermore, the application of the buffer system for anthrax virus protein nanopores in an anthrax virus protein nanopore device;

[0012] Furthermore, the application of the buffer system for anthrax virus protein nanopores in an anthrax virus protein nanopore device. The anthrax virus protein nanopore device includes anthrax virus protein, a membrane, electrodes. The anthrax virus protein forms a nanopore and is embedded in the membrane. The membrane is placed in a resin chamber containing the buffer system for anthrax virus protein nanopores. The membrane divides the entire resin chamber into two small chambers, cis and trans. Two electrodes are respectively placed in the two small chambers, and a power supply and an ammeter device are sequentially connected between the two electrodes to form a current loop;

[0013] Preferably, the anthrax virus protein nanopore is a PA63 nanopore. The PA63 nanopore is formed by the intramembrane enzyme of the Furin family cleaving PA83 to release a 20 kDa N-terminal fragment PA20, and the remaining 63 kDa C-terminal fragment PA63 oligomerizes to form a heptameric nanopore.

[0014] Furthermore, for the method of detecting small molecules by the anthrax virus protein nanopore device, the detection steps are as follows:

[0015] S1: Dissolve the small molecule to be detected into the cis chamber filled with the buffer system of the anthrax virus protein nanopore.

[0016] S2: Apply a voltage of -120 mV to -80 mV to the nanopore device.

[0017] S3: Collect the current signal and analyze the amplitude, blocking time, and signal frequency of the current signal.

[0018] Preferably, the voltage is preferably -100 mV.

[0019] The beneficial effects of the present invention are as follows:

[0020] The present invention utilizes the relationship between the electrical properties of the anthrax virus protein protective antigen nanopore and the Hofmeister series to achieve the stability of the anthrax virus protein protective antigen nanopore and distinguish different charged small molecules. As an emerging technology, nanopore detection technology has advantages such as high throughput, low cost, label-free, and high sensitivity, and is widely used in fields such as nucleic acid, protein, and enzymatic reaction monitoring. As an emerging technology, nanopore sensing technology has multiple advantages, can provide important information on the structure and function of nucleic acids and proteins, and can also monitor enzymatic reactions in real time, solving problems in previous traditional detections, such as low selectivity. The pore diameter of the anthrax virus protein protective antigen nanopore is relatively long, and there are more interaction sites for molecules in the pore, which can better analyze its ionic current and achieve the distinction of charged small molecules. The present invention enables the anthrax virus protein protective antigen nanopore to have a wider application prospect in the field of single molecule detection.

[0021] Other advantages, objectives, and features of the present invention will be described to some extent in the subsequent specification, and to some extent, will be obvious to those skilled in the art based on the study of the following text, or can be taught from the practice of the present invention. The objectives and other advantages of the present invention can be achieved and obtained through the following specification. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be described in detail preferably with reference to the accompanying drawings, where:

[0023] Figure 1 is the nanopore electrochemical detection and analysis device used in the embodiments;

[0024] Figure 2 is a schematic diagram of the representative current traces of the anthrax virus protein protective antigen nanopores in different electrolyte buffers in the embodiments and the selected cations and anions in the Hofmeister series;

[0025] Figure 3 is the clamping phenomenon and representative current traces of the anthrax virus protein protective antigen nanopores, and corresponding schematic diagrams of different open-pore currents under clamping and non-clamping phenomena;

[0026] Figure 4 is a bar chart and Gaussian fitting curve of the open-pore currents and open-pore current dispersion constant σ of the anthrax virus protein protective antigen nanopores at different concentrations in 12 electrolyte buffers;

[0027] Figure 5 is an exponential fitting curve and comparison chart of the noise frequency and clamping frequency of the anthrax virus protein protective antigen nanopores at different concentrations in 12 electrolyte buffers;

[0028] Figure 6 is a network diagram of the electrical properties of the anthrax virus protein protective antigen nanopores in electrolyte buffers containing magnesium ions or lithium ions;

[0029] Figure 7 is a network diagram of the electrical properties of the anthrax virus protein protective antigen nanopores in electrolyte buffers containing potassium ions or ammonium ions;

[0030] Figure 8 is an analysis of the specific peak differences and voltage-dependence test chart of 4 charged small molecules;

[0031] Figure 9 is an analysis of the differences in translocation times and voltage-dependence test chart of 4 charged small molecules; Detailed implementation manners

[0032] The following specific examples illustrate the implementation manners of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation manners. Various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the diagrams provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner. Without conflict, the following embodiments and the features in the embodiments can be combined with each other.

[0033] Among them, the attached drawings are only for illustrative purposes, showing only schematic diagrams rather than physical diagrams, and should not be construed as limiting the present invention; in order to better illustrate the embodiments of the present invention, some components in the attached drawings will be omitted, enlarged or reduced, which does not represent the size of the actual product; for those skilled in the art, it is understandable that some well-known structures and their descriptions in the attached drawings may be omitted.

[0034] In the attached drawings of the embodiments of the present invention, the same or similar reference numerals correspond to the same or similar components; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "rear", etc. indicating the orientation or positional relationship, they are based on the orientation or positional relationship shown in the attached drawings. This 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 operated in a specific orientation. Therefore, the terms describing the positional relationship in the attached drawings are only for illustrative purposes and should not be construed as limiting the present invention. For those of ordinary skill in the art, the specific meanings of the above terms can be understood according to specific circumstances.

[0035] Embodiment 1

[0036] Construct a biological nanopore channel of anthrax virus protein protective antigen, specifically as follows:

[0037] 1. Prepare the solution:

[0038] Prepare multiple basic buffer solutions: Dissolve magnesium sulfate, magnesium formate, magnesium chloride, lithium sulfate, lithium formate, lithium chloride, potassium sulfate, potassium formate, potassium chloride, ammonium sulfate, ammonium formate, and ammonium chloride separately with tris(hydroxymethyl)aminomethane (Tris) in deionized water, and adjust the pH to form 12 different basic buffer solutions (where buffer A: the concentration of magnesium sulfate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer B: the concentration of magnesium formate ranges from 0.1 - 0.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer C: the concentration of magnesium chloride ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer D: the concentration of lithium sulfate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer E: the concentration of lithium formate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer F: the concentration of lithium chloride ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer G: the concentration of potassium sulfate ranges from 0.1 - 0.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer H: the concentration of potassium formate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer I: the concentration of potassium chloride ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer J: the concentration of ammonium sulfate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer K: the concentration of ammonium formate ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6; buffer L: the concentration of ammonium chloride ranges from 0.1 - 1.5 M, the concentration of tris(hydroxymethyl)aminomethane is 10 mM, pH = 7.6;). Finally, filter through a 0.22 μM filter before use.

[0039] 2. Construct a nanopore electrochemical detection and analysis device:

[0040] Place a support film (the support film is a Teflon film) containing the biological nanopore of the protective antigen of anthrax virus protein in a solution chamber containing an electrolyte (the chamber material is resin), divide the entire chamber into two small chambers, and then place two electrodes (both of the two electrodes are silver electrode materials coated with silver chloride) in the two small chambers respectively. Connect a power supply, an ammeter, etc. in sequence between the two electrodes to form a nanopore electrochemical detection and analysis device (as Figure 1 shown).

[0041] Example 2

[0042] 1. Detect the stability of the nanopore channels of the protective antigen of anthrax virus protein under different electrolytes:

[0043] (1) Add the configured buffer solution into the two chambers separated by the Teflon membrane mentioned above, apply a voltage to the device to form an electric field force in the solution, without adding the sample to be measured, collect the blank current signal, and analyze the stability of the nanopore channels of the protective antigen of anthrax virus protein under different electrolytes.

[0044] (2) Amplify the current signal collected in step (1) using a low-noise current amplifier (Axon Axopatch 200B), and perform feature analysis on the amplitude, current blocking time, and signal generation frequency of the collected current signal (import the collected current signal file into clampfit software to select the characteristic current signal and calculate its σ value. In physics, σ usually represents the standard deviation, which is a statistic used to measure the deviation of the data points in a data set from the average value. The physical meaning of the standard deviation σ can be interpreted as the degree of distribution of the data points around the average value. Specifically, the standard deviation describes the average deviation degree of each data point in the data set relative to the average value. The larger the standard deviation, the wider the distribution of the data points relative to the average value; the smaller the standard deviation, the more concentrated the distribution of the data points relative to the average value. In the application in physics, the 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 average value, reflecting the stability of the data. Therefore, the smaller the σ value obtained in the clampfit software, the more stable the nanopore channels of the anthrax virus antigen protein under the electrolyte.

[0045] Compare the opening current and the dispersion constant σ under different concentrations and different electrolytes, as Figure 4 shown, where a is the opening current of the nanopore of the protective antigen of anthrax virus protein under 12 different electrolytes at different concentrations, and b is the dispersion constant σ of the nanopore of the protective antigen of anthrax virus protein under 12 different electrolytes at different concentrations. It can be seen from Figure 4 this that the opening current of the nanopore of the protective antigen of anthrax virus protein is dominated by cations in the electrolyte, and the increasing trend of the opening current is consistent with the order of the 4 cations in the Hofmeister series; while the dispersion constant σ of the opening current of the nanopore of the protective antigen of anthrax virus protein is dominated by anions in the electrolyte, and the increasing trend of the dispersion constant σ is opposite to the order of the 4 cations in the Hofmeister series. At the same time, the dispersion constant σ of the opening current of the nanopore of the protective antigen of anthrax virus protein shows a Gaussian distribution as the electrolyte concentration increases. There is a clamping phenomenon in the nanopore of the protective antigen of anthrax virus protein, as Figure 3 shown, and its clamping frequency decreases as the concentration increases, as Figure 5As shown, the noise frequency of the anthrax virus protein protective antigen nanopore increases exponentially with the increase in the electrolyte concentration. Based on the detection of the anthrax virus protein protective antigen nanopore in various electrical properties, we conducted a comprehensive analysis, such as Figure 6 , 7 shown. In the network diagram, the closer each electrical property is to the inner side of the dotted line, the more stable the anthrax virus protein protective antigen nanopore is in the corresponding electrolyte, that is, the anthrax virus protein protective antigen nanopore is the most stable in the lithium formate electrolyte.

[0046] Example 3

[0047] The nanopore electrochemical method was used to detect different charged small molecules. The specific detection process is as follows:

[0048] 1. Prepare the solution:

[0049] (1) Prepare the basic buffer solution: Dissolve lithium formate and tris(hydroxymethyl)aminomethane (Tris) in deionized water and adjust the pH = 7.6 to form an electrolyte buffer solution containing lithium formate and tris(hydroxymethyl)aminomethane (Tris) (where the concentration of lithium formate is 0.5 M and the concentration of tris(hydroxymethyl)aminomethane is 10 mM). Finally, filter it with a 0.22 μM filter before use.

[0050] (2) Prepare the sample solution:

[0051] Dissolve various sample solids or solutions in deionized water to form a solution with a concentration of 10 mM.

[0052] 2. Construct the nanopore electrochemical detection and analysis device:

[0053] Place the support film containing the anthrax virus protein protective antigen biological nanopore (where the support film is a Teflon film) in a solution chamber containing an electrolyte (the chamber material is resin), divide the entire chamber into two small chambers, and then place two electrodes (both of the two electrodes are silver electrode materials coated with silver chloride) in the two small chambers respectively. Connect a power supply and an ammeter in sequence between the two electrodes to form a nanopore electrochemical detection and analysis device (as Figure 1 shown).

[0054] 3. Detect different charged small molecules

[0055] (1) Add the prepared sample solution to the cis end of the above - constructed nanopore electrochemical detection device. Use the above - mentioned basic buffer solution as the electrolyte in the device, apply a voltage to the device to form an electric field force in the solution, and under the action of the electric field force, promote the interaction between the sample and the nanopore, and collect the current signal.

[0056] (2) Use a low-noise current amplifier (Axon Axopatch 200B) to amplify the current signal collected in step (1). Select the characteristic signal from the collected electrical signal file through Clamfit software, and then perform statistical analysis in Origin software to analyze the characteristics of the current amplitude of the electrical signal, and obtain the corresponding sample characteristic diagram.

[0057] Perform a differential analysis on the specific peaks of different charged small molecules. For example, Figure 8 As shown, it can be found from the figure that there are significant differences in the specific peaks between different charged small molecules. At the same time, a differential analysis was performed on the pore-passing time of different charged small molecules. For example, Figure 9 As shown, it can be found from the figure that there are significant differences in the pore-passing time between different charged small molecules. The above results indicate that for the changes in charge and type, the stable anthrax virus protein protective antigen nanopore shows selective detection of different charged small molecules with high resolution.

[0058] In summary, the present invention designs a method to explore the relationship between the electrical properties of the anthrax virus protein protective antigen nanopore and the Hofmeister series, and designs a nanopore channel that can stabilize the anthrax virus protein protective antigen nanopore. At the same time, the detection and differentiation of different charged small molecules are completed using this channel, enabling it to be applied to the next step of detection.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit them. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be modified or equivalently replaced without departing from the purpose and scope of the present technical solution, and they should all be covered by the scope of the claims of the present invention.

Claims

1. Anthrax virus protein nanopore buffer system, characterized in that: The buffer solution is composed of tris(hydroxymethyl)aminomethane and Compound I. Compound I includes at least one of the cations: NH4 + >K + >Li + >Mg 2+ and at least one of the anions: Cl – >HCOO – >SO4 2- The concentration of tris(hydroxymethyl)aminomethane is 10 mM, the concentration of Compound I is 0.1 - 1.5 M, and the pH of the buffer solution is 7.

6.

2. The anthrax virus protein nanopore buffer system according to claim 1, wherein: The compound I is composed of at least one compound selected from magnesium sulfate, magnesium formate, magnesium chloride, lithium sulfate, lithium formate, lithium chloride, potassium sulfate, potassium formate, potassium chloride, ammonium sulfate, ammonium formate, and ammonium chloride.

3. The anthrax virus protein nanopore buffer system according to claim 2, wherein the compound I is lithium formate and the concentration of lithium formate is 0.5 M.

4. Use of the anthrax virus protein nanopore buffer system according to any one of claims 1-3 in an anthrax virus protein nanopore device.

5. Use of the anthrax virus protein nanopore buffer system according to claim 4 in an anthrax virus protein nanopore device, characterized in that: The anthrax virus protein nanopore device includes an anthrax virus protein, a membrane, and electrodes. The anthrax virus protein forms an anthrax virus protein nanopore embedded in the membrane. The membrane is placed in a resin chamber containing the buffer according to any one of claims 1-3. The membrane divides the entire resin chamber into two small chambers, cis and trans. Two electrodes are respectively placed in the two small chambers, and a power supply and an ammeter device are sequentially connected between the two electrodes to form a current loop.

6. Use of the anthrax virus protein nanopore buffer system according to claim 5 in an anthrax virus protein nanopore device, characterized in that: The anthrax virus protein nanopore is a PA63 nanopore. The PA63 nanopore is formed by the membrane-bound enzyme of the Furin family cleaving PA83 to release a 20 kDa N-terminal fragment, PA20, and the remaining 63 kDa C-terminal fragment, PA63, oligomerizing to form a heptameric nanopore.

7. Method for detecting small molecules by anthrax virus protein nanopore device, characterized in that, The detection steps are as follows: S1: Dissolve the small molecule to be detected into the cis chamber filled with the anthrax virus protein nanopore buffer system according to any one of claims 1-3. S2: Apply a voltage of -120 mV to -80 mV to the nanopore device. S3: Collect the current signal and analyze the amplitude, blocking time, and signal frequency of the current signal.

8. The method for detecting small molecules by the anthrax virus protein nanopore device according to claim 7, wherein, The voltage is preferably -100 mV.

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