Nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers

Abstract

The present invention relates to a nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers, wherein the method comprises the following steps: constructing a nanopore single-point mutation homology model with wild-type aerolysin as a template; establishing a mutant nanopore-phospholipid bilayer composite system of the homology model in a neutral electrolyte environment; performing energy minimization and equilibrium simulation processing on the composite system to achieve water relaxation and the balance of the entire system; analyzing the characteristic parameters of the nano-mutation pore and its detection of polyfluorocarboxylic acid for the homology model described currently; and verifying the detection performance of the nano-mutation pore for polyfluorocarboxylic acid isomers. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers of the present invention is adopted, and the influence of the interface design on the detection performance of the nanopore is predicted by using the simulation results, and the feasibility of guiding the nanopore interface design by simulation means is verified by experiments.

Description

Technical Field

[0001] The invention relates to the technical field of environmental analysis, and in particular to a nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers. Background Art

[0002] Per- and polyfluorinated carboxylic acids (PFCAs) are important raw materials for fine chemicals, and their corresponding isomers are usually produced as by-products along with the products, and eventually enter the water or soil. Due to their difficulty in degradation, PFCAs are easily enriched in organisms, and their toxic effects are amplified through the food chain. However, the health risks of PFCAs isomers vary greatly. Straight-chain PFCAs are more difficult to be metabolized by the human body than branched forms and have higher toxicity. Therefore, a more comprehensive screening of PFCAs and their isomers is needed.

[0003] The mainstream detection methods for PFCAs are high performance liquid chromatography-tandem mass spectrometry (HPLC-MS / MS) and gas chromatography-mass spectrometry (GC-MS). The chromatographic methods involved in the front end of these two methods are highly dependent on standards, and the number of standards synthesized so far is less than 1% of the number of PFCAs included, which is not conducive to the existing detection methods to achieve high-throughput screening of PFCAs.

[0004] The single-molecule detection method based on nanopore electrochemistry has potential application prospects in accurately identifying the presence of PFCA isomers in complex environmental media due to its ultra-high spatial resolution. By constructing a linear response relationship between the blocking current and the molecular volume of PFCAs, qualitative analysis of PFCAs without standards can be achieved. However, the fixed sensing area volume of the nanopore limits the range of the single-molecule detection method. Based on the total number of PFCAs isomers that need to be detected, in order to achieve comprehensive screening of PFCAs, the statistical error of the blocking current corresponding to PFCAs in the single-molecule detection method should be further reduced. Therefore, it is urgent to optimize the single-molecule detection method to further improve the resolution of PFCAs. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art and provide a method for the simulation and screening of nanopore interface design for single-molecule detection of polyfluorocarboxylic acid isomers. The method simulates single-point mutations in wild-type aerolysin nanopores, screens potential sites with improved single-molecule detection performance by comparing the nanopore diameter, electrostatic potential, and the force conditions of PFCAs in the pores, and can verify the accuracy of the simulation screening through experiments.

[0006] In order to achieve the above-mentioned object, the nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers of the present invention is as follows:

[0007] The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers is mainly characterized in that the method comprises the following steps:

[0008] (1) Construction of a single-point mutation homology model of the nanopore using wild-type aerolysin as a template;

[0009] (2) establishing a mutant nanopore-phospholipid bilayer composite system of the homology model in a neutral electrolyte environment;

[0010] (3) performing energy minimization and equilibrium simulation processing on the composite system to achieve relaxation of water and equilibrium of the entire system;

[0011] (4) Analyze the characteristic parameters of the nano-mutation pore and its detection of polyfluorocarboxylic acids based on the homology model described above;

[0012] (5) Verify the detection performance of the nano-mutation pore for polyfluorocarboxylic acid isomers.

[0013] Preferably, the nanopore is formed by homopolymerization of 7 aerolysin monomers, and the homology model is constructed according to the following steps:

[0014] (1.1) Obtaining the amino acid sequence of aerolysin monomer from the protein data bank and pruning it, retaining the sequence length as the shortest length that maintains the internal structure of the nanopore;

[0015] (1.2) Using the pruned aerolysin sequence as a template, the amino acid abbreviations at the target position were changed, and the matching nanopore structure and the number of polymers were searched for using the Swiss-Model platform and the structure file was written, including the name, coordinate position, occupancy, and temperature factor of each atom in the nanopore, so as to construct the homology model.

[0016] Preferably, the step (2) specifically comprises the following steps:

[0017] (2.1) Introduction of phospholipid bilayer:

[0018] A phospholipid bilayer is inserted into the transduction region of the aerolysin nanopore, wherein the same phospholipid type is selected for the upper and lower layers, with a ratio of 1:1; and the inside and around the nanopore The phospholipid molecules that overlap the phospholipid bilayer will be removed;

[0019] (2.2) Configure the electrolyte environment:

[0020] The nanopores inserted into the phospholipid bilayer are solvated with water molecules, and some of the water molecules are replaced by anions and cations in the electrolyte to simulate the actual detection environment until the electrolyte reaches the target concentration, that is, the saturation concentration of the electrolyte solution; depending on the number of effective charges of the mutant nanopores, the number of anions and cations is increased to maintain the electrical neutrality of the composite system.

[0021] Preferably, the step (3) is specifically as follows:

[0022] Gromacs was used for energy minimization and equilibrium simulation, and CHARMM36 force field calculation was selected to realize the relaxation of electrolyte, phospholipid bilayer and nanopore in the composite system in turn.

[0023] Preferably, the step (3) specifically comprises the following steps:

[0024] (3.1) In the operating parameter file of Gromacs, position constraints are added to the hydrophilic ends of the nanopore and the phospholipid molecules for pre-processing, and the energy of the composite system is minimized to achieve water relaxation;

[0025] (3.2) by deleting or reducing the position constraints on each part of the composite system in the operation parameter file, the constraints on the amino acid side chains in the phospholipid bilayer and the nanopore are released, so as to perform step-by-step pre-equilibrium on the composite system, thereby achieving relaxation of the phospholipid bilayer;

[0026] (3.3) By adding a Z-axis uniform electric field, a negative voltage is applied to the composite system and the position restriction is released at the same time, thereby achieving balance of the entire composite system.

[0027] Preferably, the step (4) specifically comprises the following steps:

[0028] (4.1) The trajectory file obtained in step (3) is visualized using VMD software, and the average protein coordinate position of the last 10 frames is extracted as the input file for mutant nanopore parameter analysis;

[0029] (4.2) calculating the equivalent pore size of the mutant nanopore by combining the atomic coordinates and the corresponding van der Waals radius, setting the maximum pore size according to the structure of the nanopore itself, and obtaining a pore size result with a lower resolution by interpolation;

[0030] (4.3) Combined with pH conditions and atomic coordinates, the Charmm36 force field is used to obtain atomic charge information, the Poisson-Boltzmann equation is used to solve the electrostatic potential, and the corresponding ionic strength and Pauling radius are set according to the type and concentration of the electrolyte;

[0031] (4.4) Visualize the electrostatic potential dx file calculated using the Poisson-Boltzmann equation and convert the values ​​into color blocks, where the x or y position corresponding to the maximum grayscale value is the coordinate of the central axis of the nanopore;

[0032] (4.5) Search for the slice in the electrostatic potential result file that is closest to the aperture result file, retain the corresponding result in the aperture result file and the value of the grid point in the corresponding electrostatic potential result file, and set the rest to 0;

[0033] (4.6) averaging the non-zero values ​​on each slice of the electrostatic potential result file, and superimposing a linearly decreasing external potential on the result to obtain the true electrostatic potential in the mutant nanopore;

[0034] (4.7) Extract the distribution of electrostatic potential on the z-axis in the grid file, and superimpose the linearly decreasing external potential to obtain the actual electrostatic potential result inside the mutant nanopore;

[0035] (4.8) A perfluorocarboxylic acid model was established by Chem3D, whose structure included a positively charged guide chain and a molecule to be tested, wherein the N-terminus of the guide chain was connected to the C-terminus of the molecule to be tested, and the guide chain was protonated according to the actual pH value of the electrolyte, and the GenFF force field was used to generate a topology file that could be recognized by Gromacs;

[0036] (4.9) An imaginary external force is applied to the C-terminus of the analyte bound to the guide chain with a harmonic potential to ensure that it moves at a constant speed in the nanopore and achieves translocation, so as to obtain the force conditions of the analyte at different positions.

[0037] Preferably, the step (5) comprises:

[0038] The mutant nanopore is used to detect the pure or mixed polyfluorocarboxylic acid isomers, and the original current signal within the detection time is recorded to obtain a histogram of the blocking current and the residence time; wherein the blocking current is the ratio of the difference between the residual current and the open pore current to the open pore current, and the residence time is the duration of the blocking event.

[0039] Preferably, the step (5) further comprises:

[0040] The blocking current is fitted using a Gaussian distribution model to obtain the average blocking current and corresponding statistical error of each of the polyfluorocarboxylic acid isomers.

[0041] Preferably, the step (5) further comprises:

[0042] The separation of the two polyfluorocarboxylic acid isomers on the blocking current was obtained using the peak position spacing and the respective statistical errors, and the relative detection performance between different nanopores was compared based on this.

[0043] Preferably, the step (5) further comprises:

[0044] The classifier is trained using pure sample signals of polyfluorocarboxylic acid isomers to obtain the resolution accuracy of several polyfluorocarboxylic acid isomers on the blocking current, and the detection performance improvement effect of the mutant nanopore is quantitatively calculated.

[0045] The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers of the present invention is adopted. By formulating a nanopore interface design screening process based on molecular dynamics simulation, the simulation results are used to predict the influence of interface design on nanopore detection performance, and the feasibility of guiding nanopore interface design by simulation means is verified by experiments. Since interface design of nanopores is the main means to optimize their single-molecule detection performance, interface design can unify the behavior of PFCAs in nanopores, generate higher quality current signals, and improve the signal-to-noise ratio, thereby improving the accuracy of single-molecule detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1a This is the first schematic diagram of the molecular dynamics simulation model of the single molecule detection system of the present invention.

[0047] Figure 1b This is the second schematic diagram of the molecular dynamics simulation model of the single molecule detection system of the present invention.

[0048] Figure 2a This is the first schematic diagram showing the differences in characteristic parameters of different nanopores according to the present invention.

[0049] Figure 2b This is a second schematic diagram showing the differences in characteristic parameters of different nanopores according to the present invention.

[0050] Figure 2c This is a third schematic diagram showing the differences in characteristic parameters of different nanopores according to the present invention.

[0051] Figure 3a This is the first schematic diagram for comparing the detection performance of different nanopores in the present invention.

[0052] Figure 3b This is the second schematic diagram for comparing the detection performance of different nanopores in the present invention.

[0053] Figure 3c This is the third schematic diagram for comparing the detection performance of different nanopores in the present invention.

[0054] Figure 3d This is a fourth schematic diagram for comparing the detection performance of different nanopores according to the present invention. DETAILED DESCRIPTION

[0055] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.

[0056] Before describing in detail embodiments according to the present invention, it should be noted that, hereinafter, the terms "comprises", "includes" or any other variations are intended to cover non-exclusive inclusion, whereby a process, method, article or apparatus comprising a series of elements includes not only these elements, but also other elements not explicitly listed or inherent to such process, method, article or apparatus.

[0057] The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers comprises the following steps:

[0058] (1) Construction of a single-point mutation homology model of the nanopore using wild-type aerolysin as a template;

[0059] (2) establishing a mutant nanopore-phospholipid bilayer composite system of the homology model in a neutral electrolyte environment;

[0060] (3) performing energy minimization and equilibrium simulation processing on the composite system to achieve relaxation of water and equilibrium of the entire system;

[0061] (4) Analyze the characteristic parameters of the nano-mutation pore and its detection of polyfluorocarboxylic acids based on the homology model described above;

[0062] (5) Verify the detection performance of the nano-mutation pore for polyfluorocarboxylic acid isomers.

[0063] As a preferred embodiment of the present invention, in the step (1), the nanopore is formed by homopolymerization of 7 aerolysin monomers, and the nanopore model is simplified during the simulation process to improve the calculation efficiency.

[0064] The method for simplifying the nanopore model is specifically as follows:

[0065] The amino acid sequence of aerolysin monomer is obtained from the protein data bank and pruned, and the retained sequence length is the shortest length that can maintain the internal structure of the nanopore;

[0066] The homology modeling method is specifically:

[0067] Using the pruned aerolysin sequence as a template, the amino acid abbreviations at the target position were changed, and the nanopore structure and polymer number with the highest matching rate were searched using the Swiss-Model platform and the structural file was compiled.

[0068] The structure file should at least contain the information of the name, coordinate position, occupancy rate and temperature factor of each atom in the nanopore.

[0069] As a preferred embodiment of the present invention, in the step (2), the nanopore, the phospholipid bilayer and the electrolyte coexist in the composite system, and no two of them overlap with each other.

[0070] The specific method for establishing the composite system is:

[0071] Introduction of phospholipid bilayer;

[0072] A phospholipid bilayer was inserted into the transduction region of the aerolysin nanopore, and the same phospholipid type was selected for the upper and lower layers, with a ratio of 1:1. Phospholipid molecules that overlap the phospholipid bilayer should be removed.

[0073] Configure the electrolyte environment;

[0074] The nanopores inserted into the phospholipid bilayer are solvated with water molecules, and some water molecules are replaced by electrolyte anions and cations to simulate the actual detection environment until the electrolyte reaches the target concentration. Depending on the number of effective charges of the nanopores, the number of anions (cations) is increased to maintain the electrical neutrality of the composite system. The target concentration of the electrolyte is the concentration required to eliminate the charge effect in the nanopores, which is usually the saturation concentration of the electrolyte solution.

[0075] As a preferred embodiment of the present invention, the specific parameters of system energy minimization and equilibrium simulation are set as follows:

[0076] Energy minimization and equilibrium simulation were performed using Gromacs. Using Gromacs, CHARMM36 force field calculation was selected, LINCS algorithm was used to constrain covalent hydrogen bonds, PME method was used to calculate long-range electrostatic forces, and double-range cutoff method was used to calculate van der Waals forces and short-range electrostatic forces.

[0077] The specific process of system energy minimization and equilibrium simulation is as follows:

[0078] In the running parameter file of Gromacs, position constraints were added to the hydrophilic ends of the nanopores and phospholipid molecules for pretreatment, and the system was energy minimized to achieve water relaxation.

[0079] By deleting or reducing the position constraints on each part of the composite system in the operating parameter file, the constraints on the amino acid side chains in the phospholipid bilayer and the nanopore are released, so that the composite system is pre-balanced in steps to achieve relaxation of the phospholipid bilayer.

[0080] By adding a uniform electric field on the Z axis, a negative voltage is applied to the composite system, and at the same time, the position restriction is released to achieve balance of the entire system.

[0081] As a preferred embodiment of the present invention, in the step (4), the nano-mutation pore and its characteristic parameter analysis method for detecting perfluorocarboxylic acid are specifically as follows:

[0082] Extract input files for result analysis;

[0083] The trajectory file obtained in step (3) above was visualized using VMD software, and the average protein coordinate position of the last 10 frames was extracted as the input file for mutant nanopore parameter analysis.

[0084] The equivalent pore size of the mutant nanopore is calculated by combining the atomic coordinates and the corresponding van der Waals radius. The maximum pore size is set according to the structure of the nanopore itself, and the pore size result with lower resolution is obtained by interpolation.

[0085] Combine pH conditions and atomic coordinates to obtain atomic charge information using the Charmm36 force field, and solve the electrostatic potential using the Poisson-Boltzmann equation. Set the corresponding ionic strength and Pauling radius according to the type and concentration of the electrolyte. The dielectric constant of the nanopore and the phospholipid bilayer is 2, and the dielectric constant of the solvent is 78. The ambient temperature depends on the actual detection temperature. Set an equal number of xyz grid points and calculate the specific value based on the model size and target resolution.

[0086] Determination of the central axis of the nanopore;

[0087] The electrostatic potential dx file calculated using the Poisson-Boltzmann equation is visualized, the values ​​are converted into color blocks, and the x or y position corresponding to the maximum grayscale value is the coordinate of the central axis of the nanopore.

[0088] Matching screening of pore size and electrostatic potential distribution;

[0089] Search for the slice in the electrostatic potential result file that is closest to the aperture result file, retain the corresponding result in the aperture file and the value of the grid point in the corresponding electrostatic potential result file, and set the rest to 0;

[0090] Calculation of mean electrostatic potential;

[0091] The non-zero values ​​on each slice of the electrostatic potential file are statistically averaged, and the linearly decreasing external potential is superimposed on the result to obtain the true electrostatic potential inside the mutant nanopore.

[0092] The distribution of the electrostatic potential on the z-axis in the grid file is extracted, and the linearly decreasing external potential is superimposed to obtain the actual electrostatic potential result inside the mutant nanopore.

[0093] Build a perfluorocarboxylic acid model to obtain a profile;

[0094] The perfluorocarboxylic acid model was drawn by Chem3D. The structure included a positively charged guide chain and a molecule to be tested. The N-terminus of the guide chain was connected to the C-terminus of the analyte. The guide chain was protonated according to the actual pH value of the electrolyte, and the GenFF force field was used to generate a topology file that could be recognized by Gromacs.

[0095] An imaginary external force is applied to the C-terminus of the analyte bound to the guide chain with a harmonic potential to ensure that it moves at a constant speed in the nanopore and achieves translocation, so as to obtain the force conditions of the analyte at different positions.

[0096] As a preferred embodiment of the present invention, in the step (5), the specific method of experimental verification is:

[0097] The mutant nanopore was used to detect pure or mixed polyfluorocarboxylic acid isomers, and the original current signal during the detection time was recorded to obtain a histogram of the blocking current and the residence time. The blocking current is the ratio of the difference between the residual current and the open pore current to the open pore current, and the residence time is the duration of the blocking event.

[0098] The blocking current is fitted using a Gaussian distribution model to obtain the average blocking current and corresponding statistical error of each polyfluorocarboxylic acid isomer, specifically, the Gaussian fitting average value of the characteristic peak generated by each polyfluorocarboxylic acid isomer in the mixed sample or pure sample.

[0099] The peak position spacing and their respective statistical errors were used to obtain the separation of the two polyfluorocarboxylic acid isomers in blocking current and compare the relative detection performance between different nanopores. Specifically, after the above average values ​​are arranged in absolute numerical values, the peak spacing refers to the difference between two adjacent average values, and the statistical error refers to the standard deviation corresponding to the above two average values.

[0100] The classifier was trained using pure sample signals of polyfluorocarboxylic acid isomers to obtain the resolution accuracy of several polyfluorocarboxylic acid isomers in blocking current, and the detection performance improvement effect of the mutant nanopore was quantitatively calculated.

[0101] In order to more clearly describe the technical content of the present invention, further description is given below in conjunction with specific embodiments.

[0102] Example 1

[0103] A simulation method for a single point mutation at position A260 of the aerolysin nanopore is used to verify the detection performance by identifying two isomers of the C5H5F3O2 molecule, comprising the following steps:

[0104] (1) Construction of a single-point mutation homology model of the nanopore using wild-type aerolysin as a template;

[0105] ① Obtain the amino acid sequence of the aerolysin monomer from the protein data bank. The abbreviation corresponding to the nanopore is 5JZT and the sequence length is 424.

[0106] ② On the basis of ensuring the pore-forming activity of aerolysin, the monomer was pruned, retaining only 125 sequence lengths, with the corresponding amino acids starting from 190 as the statistical position.

[0107] ③ Using the pruned aerolysin sequence as a template, the amino acid at position 71 was changed to lysine, and the aerolysin heptamer was searched and the structure file was compiled using the Swiss-Model platform. The results of the structure file visualization by Pymol are as follows: Figure 1a shown.

[0108] (2) Establishing a mutant nanopore-phospholipid bilayer composite system in a neutral electrolyte environment;

[0109] ①Insert a phospholipid bilayer into the transduction region of the mutant aerolysin nanopore. The phospholipid types of the upper and lower layers are 4ME16:0, with a ratio of 1:1, and remove the inside and around the nanopore. Phospholipid molecules that overlap with the phospholipid bilayer.

[0110] ② If Figure 1b As shown, water molecules are solvated around the mutant aerolysin nanopores and the phospholipid bilayer, and some water molecules are replaced by chloride ions and potassium ions until the electrolyte concentration is increased to 4 mol / L, and an additional 7 chloride ions are introduced to maintain the system neutrality.

[0111] (3) Conduct energy minimization and equilibrium simulations of complex systems;

[0112] ① Use Gromacs for energy minimization and equilibrium simulation. Use Gromacs, select CHARMM36 force field calculation, LINCS algorithm to constrain covalent hydrogen bonds, PME method to calculate long-range electrostatic forces, and the grid size is The two-way cutoff method calculates the van der Waals force and the short-range electrostatic force. The cutoff distance of the neighbor list is The cutoff distance of VdW is The simulation temperature is 293.15K.

[0113] ② Use the steepest descent method to minimize the energy of the system for 5000 steps, constrain the nanopores and the heads of the phospholipid molecules, and relax the water.

[0114] ③ The composite system was pre-equilibrated in steps for a total of 2 ns using the leapfrog algorithm, which released the restrictions on the phospholipid and nanopore side chains.

[0115] ④ The equilibrium simulation is carried out at constant temperature and pressure, with a step size of 2fs, an equilibrium time of 200ns, and all atoms are unconstrained. A uniform electric field is set in the Z-axis direction, the electric field unit is V / nm, and the total applied potential is -50mV.

[0116] (4) Analysis of nano-mutated pores and their characteristic parameters for detecting perfluorocarboxylic acids;

[0117] Analyze the pore size of the aerolysin mutant nanopores;

[0118] like Figure 2a As shown, the average protein coordinate position in the last 0.1 ns is input to calculate the equivalent pore size of the mutant nanopore, and the maximum pore size is limited to The resolution of the z-axis radius is obtained as The spacing is obtained by interpolation. The aperture results.

[0119] Analysis of the electrostatic potential of the aerolysin mutant nanopore;

[0120] ① Such as Figure 2b As shown, the average coordinate position of the protein in the last 0.1ns is input, the Charmm36 force field is used to obtain the atomic charge information in combination with the pH condition, and the Poisson-Boltzmann equation is used to solve the electrostatic potential. The positive monovalent cation strength is set to 4, the Pauling radius is 1.3, the negative monovalent ion strength is set to 4, and the Pauling radius is 1.8. The dielectric constant of the nanopore and the phospholipid bilayer is 2, and the dielectric constant of the solvent is 78. The ambient temperature is 293.15K. The number of grid points is 225×225×225.

[0121] ② Extract the distribution of electrostatic potential on the z-axis in the grid file, and superimpose the linearly decreasing -50mV potential to obtain the actual electrostatic potential result inside the mutant nanopore.

[0122] Analyze the forces on perfluorocarboxylic acids in mutant nanopores;

[0123] ① The perfluorocarboxylic acid model was drawn by Chem3D. The structure contained a positively charged arginine guide chain and fully charged propionic acid. The N-terminus of the guide chain was connected to the C-terminus of the analyte, and the GenFF force field was used to generate a topology file that could be recognized by Gromacs.

[0124] ② If Figure 2c The imaginary external force is applied in the aerolysin mutant nanopore. The object was moved at a constant speed and the simulation calculation was performed for 1.6 ns to obtain the force conditions of the object under test at different positions in the mutant nanopore.

[0125] (5) Verify the detection performance of the nano-mutation pore for polyfluorocarboxylic acid isomers;

[0126] ① Prepare pure samples of 4,5,5-trifluoro-4-pentenoic acid and 3-trifluoromethylbutenoic acid of the same concentration and add them into the single molecule detection system respectively.

[0127] ② Count the blocking current signals generated when the aerolysin mutant nanopore detects polyfluorocarboxylic acid isomers, and draw the corresponding frequency histogram.

[0128] ③If Figure 3a-3b As shown, the obtained histogram was fitted with a Gaussian model to obtain the blocking current expectation and statistical error corresponding to the pure sample of polyfluorocarboxylic acid isomers. According to the calculation formula of the separation degree, that is, the ratio of the peak spacing to the sum of 2 times the half-peak width, the detection performance of the mutant nanopore and the wild-type nanopore for polyfluorocarboxylic acid isomers was compared.

[0129] ④ Such as Figure 3c~3d As shown, the statistically obtained blocking current results of pure samples of polyfluorocarboxylic acid isomers are input into the classifier for training, the resolution on the blocking current is calculated, and the improvement effect of the mutant nanopore relative to the wild-type nanopore is calculated.

[0130] Any process or method description in a flowchart or otherwise described herein may be understood to represent a module, segment or portion of code that includes one or more executable instructions for implementing the steps of a specific logical function or process, and the scope of the preferred embodiments of the present invention includes alternative implementations in which functions may not be performed in the order shown or discussed, including performing functions in a substantially simultaneous manner or in the reverse order depending on the functions involved, which should be understood by those skilled in the art to which the embodiments of the present invention belong.

[0131] It should be understood that each part of the present invention can be implemented by hardware, software, firmware or a combination thereof. In the above embodiments, multiple steps or methods can be implemented by software or firmware stored in a memory and executed by a suitable instruction execution device.

[0132] A person of ordinary skill in the art may understand that all or part of the steps of the method for implementing the above-mentioned embodiment may be completed by instructing the relevant hardware through a program, and the program may be stored in a computer-readable storage medium, which, when executed, includes one of the steps of the method embodiment or a combination thereof.

[0133] The storage medium mentioned above can be a read-only memory, a magnetic disk or an optical disk, etc.

[0134] In the description of this specification, the description with reference to the terms "an embodiment", "some embodiments", "example", "specific example", or "embodiment" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0135] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

[0136] The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers of the present invention is adopted. By formulating a nanopore interface design screening process based on molecular dynamics simulation, the simulation results are used to predict the influence of interface design on nanopore detection performance, and the feasibility of guiding nanopore interface design by simulation means is verified by experiments. Since interface design of nanopores is the main means to optimize their single-molecule detection performance, interface design can unify the behavior of PFCAs in nanopores, generate higher quality current signals, and improve the signal-to-noise ratio, thereby improving the accuracy of single-molecule detection.

[0137] In this specification, the present invention has been described with reference to specific embodiments thereof. However, it is apparent that various modifications and variations may be made without departing from the spirit and scope of the present invention. Therefore, the specification and drawings should be regarded as illustrative rather than restrictive.

Claims

1. A nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers, characterized in that: The method comprises the following steps: (1) Construction of a single-point mutation homology model of the nanopore using wild-type aerolysin as a template; (2) establishing a mutant nanopore-phospholipid bilayer composite system of the homology model in a neutral electrolyte environment; (3) performing energy minimization and equilibrium simulation on the composite system to achieve relaxation of water and equilibrium of the entire system; (4) Analyze the characteristic parameters of the nano-mutation pore and its detection of polyfluorocarboxylic acid isomers based on the homology model described so far; (5) Verifying the detection performance of the nano-mutation pore for polyfluorocarboxylic acid isomers; The nanopore is formed by homopolymerization of 7 aerolysin monomers, and the homology model is constructed according to the following steps: (1.1) Obtain the amino acid sequence of aerolysin monomer from the protein data bank and prune it, retaining the shortest length of the sequence while maintaining the internal structure of the nanopore; (1.2) Using the pruned aerolysin sequence as a template, the amino acid abbreviations at the target position were changed, and the matching nanopore structure and the number of polymers were searched for using the Swiss-Model platform and the structure file was written, including the name, coordinate position, occupancy, and temperature factor of each atom in the nanopore, so as to construct the homology model.

2. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 1, characterized in that: The step (2) specifically includes the following steps: (2.1) Introduction of phospholipid bilayer: Insert a phospholipid bilayer into the transduction region of the aerolysin nanopore, where the same phospholipid species are selected for the upper and lower layers in a 1:1 ratio; and the phospholipid molecules that overlap with the phospholipid bilayer within and around the nanopore within 2 Å will be removed; (2.2) Configure the electrolyte environment: The nanopores inserted into the phospholipid bilayer are solvated with water molecules, and some of the water molecules are replaced by anions and cations in the electrolyte to simulate the actual detection environment until the electrolyte reaches the target concentration, that is, the saturation concentration of the electrolyte solution; depending on the number of effective charges of the mutant nanopores, the number of anions and cations is increased to maintain the electrical neutrality of the composite system.

3. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 2, characterized in that: The step (3) is specifically as follows: Gromacs was used for energy minimization and equilibrium simulation, and CHARMM36 force field calculation was selected to realize the relaxation of electrolyte, phospholipid bilayer and nanopore in the composite system in turn.

4. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 3, characterized in that: The step (3) specifically includes the following steps: (3.1) In the operating parameter file of Gromacs, position constraints are added to the hydrophilic ends of the nanopore and the phospholipid molecules for pre-processing, and the energy of the composite system is minimized to achieve water relaxation; (3.2) removing or reducing the position constraints on each part of the composite system in the operating parameter file, releasing the constraints on the amino acid side chains in the phospholipid bilayer and the nanopore, thereby performing step-by-step pre-balancing on the composite system, thereby achieving relaxation of the phospholipid bilayer; (3.3) By adding a Z-axis uniform electric field, a negative voltage is applied to the composite system and the position restriction is released, thereby achieving balance of the entire composite system.

5. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 4, characterized in that: The step (4) specifically includes the following steps: (4.1) The trajectory file obtained in step (3) was visualized using VMD software, and the average protein coordinate position of the last 10 frames was extracted as the input file for mutant nanopore parameter analysis; (4.2) Calculating the equivalent pore size of the mutant nanopore by combining the atomic coordinates and the corresponding van der Waals radius, setting the maximum pore size according to the structure of the nanopore itself, and obtaining a pore size result with lower resolution by interpolation; (4.3) Combined with pH conditions and atomic coordinates, the Charmm36 force field is used to obtain atomic charge information, the Poisson-Boltzmann equation is used to solve the electrostatic potential, and the corresponding ionic strength and Pauling radius are set according to the type and concentration of the electrolyte; (4.4) Visualize the electrostatic potential dx file calculated using the Poisson-Boltzmann equation and convert the values ​​into color blocks, where the x or y position corresponding to the maximum grayscale value is the coordinate of the central axis of the nanopore; (4.5) Search for the slice in the electrostatic potential result file that is closest to the aperture result file, retain the corresponding result in the aperture result file and the value of the grid point in the corresponding electrostatic potential result file, and set the rest to 0; (4.6) averaging the non-zero values ​​on each slice of the electrostatic potential result file, and superimposing a linearly decreasing external potential on the result to obtain the true electrostatic potential in the mutant nanopore; (4.7) Extract the distribution of electrostatic potential on the z-axis in the grid file, and superimpose the linearly decreasing external potential to obtain the actual electrostatic potential result inside the mutant nanopore; (4.8) A polyfluorocarboxylic acid model was established using Chem3D. Its structure includes a positively charged guide chain and a molecule to be tested. The N-terminus of the guide chain is connected to the C-terminus of the molecule to be tested. The guide chain is protonated according to the actual pH value of the electrolyte. The CGenFF force field is used to generate a topology file that can be recognized by Gromacs. (4.9) Apply an imaginary external force to the C-terminus of the analyte bound to the guide chain with a harmonic potential to ensure that it moves at a constant speed in the nanopore and achieves translocation, so as to obtain the force conditions of the analyte at different positions.

6. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 5, characterized in that: The step (5) comprises: The mutant nanopore is used to detect the pure or mixed polyfluorocarboxylic acid isomers, and the original current signal within the detection time is recorded to obtain a histogram of the blocking current and the residence time; wherein the blocking current is the ratio of the difference between the residual current and the open pore current to the open pore current, and the residence time is the duration of the blocking event.

7. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 6, characterized in that: The step (5) further includes: The blocking current is fitted using a Gaussian distribution model to obtain the average blocking current and corresponding statistical error of each polyfluorocarboxylic acid isomer in the mixed sample or pure sample.

8. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 7, characterized in that: The step (5) further includes: After arranging the obtained several average blocking currents according to their absolute numerical values, the separation of the two polyfluorocarboxylic acid isomers in the blocking current is obtained by using the peak position spacing of two adjacent average blocking current values ​​and the statistical errors corresponding to the two adjacent average blocking current values, and the relative detection performance between different nanopores is compared based on this.

9. The nanopore interface design simulation screening method for single-molecule detection of polyfluorocarboxylic acid isomers according to claim 6, characterized in that: The step (5) further includes: The classifier is trained using pure sample signals of polyfluorocarboxylic acid isomers to obtain the resolution accuracy of several polyfluorocarboxylic acid isomers on the blocking current, and the detection performance improvement effect of the mutant nanopore is quantitatively calculated.

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