Three-stage translocation model for revealing Aerolysin nanopore fingerprint characteristics

Abstract

The invention provides a three-stage translocation model for revealing Aerolysin nanopore fingerprint characteristics, a mathematical framework related to the nanopore fingerprint characteristics (charge-to-mass ratio, resistance-to-mass ratio and other parameters) is established by decomposing a charged single-molecule translocation total process into three stages of pore entry, perforation and pore exit, and a single-molecule-level fingerprint characteristic extraction technology is established. And explaining the translocation attribute of the single molecule by using fingerprint characteristics. Theoretical and experimental analysis proves that the molecular charge-to-mass ratio is positively correlated with the metathesis speed, the molecular metathesis speed is dominated by the charge-to-mass ratio during hole entering, and the acceleration phenomenon of molecules in the hole exiting stage is predicted. The invention provides a framework for quantitative characterization, modeling and prediction of nanopore fingerprints in biosensing applications.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical fields of biotechnology and signal processing, and particularly relates to a three-stage translocation model for revealing the fingerprint characteristics of an Aerolysin nanopore. BACKGROUND

[0002] Nanopore sensing technology is currently widely used in the fields of DNA / RNA sequencing, protein analysis, chemical small molecule detection and nanoparticle characterization. The translocation of molecules in a confined nanopore is the basis for the application of these technologies. When a single molecule in a liquid passes through a nanopore under the driving force of an electric field, the ion current used to monitor the translocation of the molecule will form a blockage signal. The degree and time characteristics of the blockage are directly related to the translocation speed of the molecule in the pore, and the difference in translocation speed is derived from the differences between the size, shape, mass and molecule-nanopore interaction of the molecules. Therefore, understanding the underlying physical mechanism of molecular translocation is of great significance for the modeling and identification of nanopore molecular signals.

[0003] The actual physical phenomena and force processes of molecular translocation observed in pores of different structures and properties often differ to varying degrees. In order to reveal the molecular translocation process, a large number of experimental, theoretical and simulation studies have focused on the relationship between translocation time and molecular length, pore length and other factors. When the length of the molecule is greater than the length of the pore, its translocation time is proportional to the chain length, and for shorter polymers, a steeper dependence is observed. However, there is still little known about the properties of single molecule translocation when the chain length is less than the length of the pore.

[0004] Aerolysin nanopores have the characteristics of ultra-narrow diameter and long lumen structure, with a diameter of about 1 (nm) and a length of about 10 (nm). They have high sensitivity for single molecule detection of length differences, single amino acid differences, direction dependence and charge differences. Using the sensing strategy of Aerolysin pores, molecules with 3' or 5' end modified phosphate groups of single-strand DNA (ssDNA) can be identified. When the end carrying a higher charge of phosphate groups enters the pore first, it produces a faster translocation speed than the end without phosphate groups. However, the dominant role of the charge effect in the molecular translocation process still needs further theoretical and experimental research. SUMMARY

[0005] To solve the above problems, the present application provides a three-stage translocation model for revealing the fingerprint characteristics of an Aerolysin nanopore (attached Figure 1 ), which comprises the following steps:

[0006] Step 1: According to the force difference of charged molecules in the channel, the total process of translocation is divided into three stages of entering the hole, perforating the hole and exiting the hole, and the corresponding displacement and velocity equations are established, which are functions of three physical fingerprint characteristics of the nanopore channel, which are the ratio of the linear charge density of the molecule to the mass (λ / m), the ratio of the channel resistance coefficient to the mass (α / m), and the ratio of the liquid viscous resistance coefficient to the mass (η / m);

[0007] Step 2: A method for extracting physical fingerprint characteristics of a nanopore at a single molecule level is established, which uses four inflection points of the blocking current generated when the molecule perforates the hole to determine the time and boundary values of the three stages, substitutes into the corresponding displacement and velocity equations, thereby establishing a nonlinear equation group, and obtains the estimated values of the nanopore fingerprint parameters related to λ / m, α / m and η / m by using the trust region algorithm to solve the nonlinear equation group;

[0008] Step 3: By substituting the fingerprint parameters extracted from the single molecule translocation signal in step 2 and the time boundary values of the three stages into the velocity equations of entering the hole, perforating the hole and exiting the hole, the velocity estimates of the single molecule in entering the hole, perforating the hole and exiting the hole (v ENT , v TRAV and v EXIT ) can be obtained, and by analyzing the statistical properties of v ENT , v TRAV and v EXIT , the velocity variation properties of the molecule in the channel are revealed.

[0009] In step 1, according to the force difference of charged molecules in the channel, the total process of translocation is divided into three stages of entering the hole, perforating the hole and exiting the hole, and the corresponding displacement and velocity equations are established, which are functions of three physical fingerprint characteristics of the nanopore channel, which are the ratio of the linear charge density of the molecule to the mass (λ / m), the ratio of the channel resistance coefficient to the mass (α / m), and the ratio of the liquid viscous resistance coefficient to the mass (η / m), and the specific method is as follows:

[0010] A single molecule with a length of d and a mass of m is regarded as a linear long rigid body, which carries a charge Ze and is uniformly distributed on the long line segment, and the charge-to-mass ratio of the molecule is defined as the ratio of the charge carried per unit length λ to the mass m:

[0011]

[0012] The unit is C·nm -1 ·M -1 (M represents kg), Z inside is the number of net charges carried by a single molecule in the channel sensing region, d insideis the length of the channel occupying in the sensing region. The length of the channel is L, the applied voltage is V, and when the length of the channel is much larger than its diameter, the electric field strength E≈V / L can be approximated as symmetric and uniformly distributed. The single molecule in the electrolyte is captured by translocation from the cis side to the trans side of the channel, and the translocation speed is v. The origin O of the one-dimensional global coordinate system is defined at the cis side of the channel, and the OX axis points to the trans side.

[0013] According to the force difference of the charged molecule in the channel in step 1, wherein the molecule is decelerated into the hole by the action of the electric field force and the molecule-hole interaction resistance in the hole-in phase, the molecule is uniformly accelerated through the hole by the force balance of the electric field force and the molecule-hole interaction resistance in the hole-through phase, and the molecule is accelerated out of the hole by the action of the electric field force and the liquid viscosity resistance outside the membrane in the hole-out phase. The specific method is as follows:

[0014] The three-stage translocation model mainly considers the acceleration generated by three forces: (1) the acceleration a E generated by the electric field force F E ; (2) the acceleration a INT generated by the molecule-hole interaction resistance F INT ; and (3) the acceleration a viscous generated by the liquid viscosity resistance F VISCOUS outside the membrane. The change of the force acting on the whole molecule leads to the speed change process of the molecule in three different stages:

[0015] (1) Hole-in phase: the partial volume of the molecule enters the channel sensing region (0<x<d), and the degree of current blockage is highly related to the volume of the molecule occupying in the channel, so the start of the hole-in of the molecule is represented by the start of the decline of the blocked current (point 1: t=t1, x=0). The force acting on the molecule in this stage mainly has F E (F E =λE) and F INT (F INT =αv, assuming that α is a constant resistance coefficient). According to Newton’s second law, the motion equation of the hole-in phase is shown in formula (2):

[0016]

[0017] Formula (2) is a second-order ordinary differential equation, and the analytical solution of the equation is the displacement function x(t), and the velocity v(t) is the derivative of the displacement function with respect to time, as shown in formula (3) and formula (4):

[0018]

[0019] where a1 and a2 are arbitrary constants, and a1 + a2 = 0, which value depends on the initial velocity of the molecule entering the pore. According to equation (4), when t→ +∞, v→ λE / α, which means that λ, E and α together determine the minimum velocity v required for a single molecule to complete the translocation process min , v min may be further expressed as:

[0020]

[0021] According to equation (5), when E and α remain constant, the translocation velocity of the molecule is proportional to the charge-to-mass ratio λ|m of the molecule entering the pore.

[0022] (2) Perforation phase: the molecule sharply changes its velocity due to the pore resistance when entering the pore, and quickly reaches a balance with the electric field force, thereby quickly entering the uniform perforation phase. The volume of the molecule completely enters the sensing area of the pore (d≤x≤L), and since the degree of blocking of the translocation current is highly related to the translocation velocity of the molecule occupying the volume in the pore, when the blocking current signal begins to remain relatively stable (point 2: t=t2, x=d), it can be approximately considered that the molecule enters the uniform perforation phase. F E and F INT are balanced, the molecule translocates to the trans side at a velocity v a , and the displacement equation can be expressed as:

[0023] x(t) = d + v a (t-t d ) (6)

[0024] The velocity equation is:

[0025]

[0026] (3) Exit phase: the molecule exits the pore from the trans side, and part of the volume is discharged outside the membrane (L E and F viscous (F viscous = η v , η is the liquid viscosity resistance coefficient), the liquid resistance coefficient η and the pore resistance coefficient α are different, breaking the force balance of the molecule, the molecule changes its velocity and exits the pore, until it is completely discharged outside the pore (point 4: t=t4, x=L+d). The velocity and displacement equations of the exit phase are similar to those of the entry phase, which are represented by equations (8) and (9):

[0027]

[0028] where c1 and c2 are arbitrary constants whose values depend on the position and velocity of the molecule at the beginning of the pore exit.

[0029] In step 2, the method of extracting the single-molecule level nanopore physical fingerprint characteristics is established, and the time and boundary values of the three stages are determined by using the four inflection points of the blocking current generated when the molecule perforates. The nonlinear equation set is established by combining the displacement equation and the velocity equation established in step 1, and the estimation values of the nanopore fingerprint parameters λ / m, α / m and η / m are obtained by solving the nonlinear equation set using the trust region algorithm.

[0030] When a single molecule translocates in a nanopore, an ion blocking current is formed, with a blocking amplitude ΔI and a blocking time τ. The four inflection points (t1, t2, t3, t4) in the blocking current curve define the start and end time points of the three translocation stages: the pore entry stage [t1, t2], the pore perforation stage [t2, t3] and the pore exit stage [t3, t4]. The start time and end time of the blocking current signal are defined as t1 = 0 and t4 = τ, and the slope s down The values of t1 and t2 can be obtained, and the slope s up The values of t3 and t4 can be obtained:

[0031]

[0032] Let Q z = λ / m, Q α = α / m, Q η = η / m, and the displacement and velocity equations of the three stages are combined to form the following nonlinear equation set:

[0033]

[0034] In formula (12), the parameter Q α = λ / m is a physical parameter related to the charge-to-mass ratio of the molecule, Q α = β / m, and Q η = η / m is a physical parameter that integrates the mass of the molecule and the properties of the pore.

[0035] The nonlinear differential equation set of formula (12) is a positive equation set composed of six unknown variables and six equations, which is converted into an unconstrained minimization problem where X = (d, a2, Q z , Q α , c2, Q β ), and the equation set is solved by the trust region algorithm. The algorithm establishes a trust region centered on the current iteration point, approximately solves the objective function in the trust region, and then enlarges or reduces the trust region according to the change of the objective function value, thereby obtaining the estimation values of the nanopore fingerprint parameters λ / m, α / m and η / m.

[0036] Step 3: By substituting the fingerprint parameters and the time boundary values of the three stages extracted from the single-molecule translocation signal in step 2 into the velocity equations of the entry hole, the perforation hole and the exit hole, the velocity estimates of the single molecule in the entry hole, the perforation hole and the exit hole (v ENT , v TRAV and v EXIT ) can be obtained, and by analyzing the statistical properties of v ENT , v TRAV and v EXIT , the velocity variation properties of the molecule in the pore can be revealed, and the specific method is as follows:

[0037] Step 2: The solving method of the nonlinear equation group established for the single-molecule translocation signal can approximately solve the key parameters d, a1, λ / m, α / m, η / m, c2 involved in the velocity equation, and the boundary time points t1, t2, t3 and t4 determined according to the shape of the single-molecule blocking current, so that the velocity estimates of the single molecule in the entry hole, the perforation hole and the exit hole (v ENT , v TRAV and v EXIT ) can be obtained, and by analyzing the distribution of v ENT , v TRAV and v EXIT of the molecule, the velocity variation trend of the molecule in the pore can be reflected.

[0038]

[0039]

[0040] By using the above method, step 1 realizes the mathematical description of the single-molecule translocation process with a chain length less than the length of the pore, step 2 realizes the extraction of the fingerprint characteristics of the nanopore at the single-molecule level, and step 3 uses the fingerprint characteristics to reveal the influence mechanism of the translocation velocity of the molecule, so as to realize the prediction of the velocity variation trend of the molecule in the pore.

[0041] Compared with the prior art, the present application has the following beneficial effects:

[0042] (1) The three-stage translocation model provided by the present application divides the translocation process into three stages of entry hole, perforation hole and exit hole, and is used to reveal the velocity distribution of the single molecule with a chain length less than the length of the nanopore, so as to provide a simple and effective analysis framework for understanding the complex nonlinear motion of the single molecule passing through the nanopore;

[0043] (2) The fingerprint characteristic extraction technology of the nanopore provided by the present application can reveal the influence mechanism of the physical properties such as the mass-to-charge ratio of the molecule on the translocation velocity in combination with experiments;

[0044] (3) The speed formula of the three-stage translocation model provided by the application can predict and calculate the speed change trend of a single molecule during translocation, which helps to further clarify the translocation dynamics of charged biomolecules;

[0045] (4) The real-time single molecule fingerprint extraction strategy provided by the application has great potential in improving the sensitivity and accuracy of nanopore-based biosensing when combined with advanced artificial intelligence methods. BRIEF DESCRIPTION OF DRAWINGS

[0046] Figure 1 A three-stage translocation model for revealing the fingerprint characteristics of an Aerolysin nanopore

[0047] Figure 2 A set of typical poly(A)4 molecule translocation signals

[0048] Figure 3 Poly(A)3, poly(A)4 and poly(A)5 molecule translocation experiments with uniform distribution of charge-to-mass ratio

[0049] Figure 4 Direction-dependent ssRNA molecule translocation experiments DETAILED DESCRIPTION

[0050] In order to make the purpose, technical scheme and advantages of the application more clear, the application is further described in detail below with reference to the specific examples and the accompanying drawings.

[0051] A short molecule translocation model based on a T274L / N226Q / S228K Aerolysin nanopore, the specific steps are as follows:

[0052] (1) According to the force difference of the charged molecule in the pore, the total translocation process is divided into three stages of entering the pore, perforating the pore and exiting the pore, and the corresponding displacement and speed equations are established, the displacement and speed equations are functions of three physical fingerprint characteristics of the nanopore, the three physical fingerprint characteristics are the linear charge density to mass ratio (λ / m) of the molecule, the pore resistance coefficient to mass ratio (α / m), and the liquid viscous resistance coefficient to mass ratio (η / m). In order to prove the rationality of the translocation model framework, a set of poly(A)3 translocation current signals collected by a typical Aerolysin pore were analyzed (Appendix Figure 2 ). Under the assumption that the pore is a uniform electric field, the instantaneous current value represents the local displacement of the molecule carrying electric charge in the local time. By integrating the current value in the local time, the displacement of the molecule in the pore with time is obtained, and the relative displacement change curve x rel(0 represents the beginning of the molecule entering the pore, and 1 represents the completion of the molecule exiting the pore). The slope change process of the relative displacement trajectory can reflect the speed change of the molecule translocation. When the molecule enters the pore, the blocked current drops by about 20 pA, and the corresponding displacement rate changes small in a very short time, indicating that the molecule is decelerated when entering the pore (a < 0); when the blocked current is stable, the molecule completely enters the pore, and the corresponding displacement slope change is very small, which is an approximate uniform motion (a ≈ 0); when the blocked current begins to rise, the molecule begins to exit the pore from the trans side, and at this time the corresponding displacement slope increases sharply, indicating that the molecule is accelerated when exiting the pore (a > 0). This set of experimental data shows that there are three different speed change processes in the translocation process of the single molecule, proving that it is reasonable to describe the single molecule translocation process in stages.

[0053] (2) The method for extracting the physical fingerprint characteristics of the nanopore at the single molecule level described in step 2, which determines the time and boundary values of the three stages by using the four inflection points of the blocked current generated when the molecule penetrates the pore, substitutes into the corresponding displacement and speed equations, thereby establishing a nonlinear equation set, and solves the nonlinear equation set by using the trust region algorithm to obtain the estimated values of the nanopore fingerprint parameters related to λ|m, α / m and η / m. This step is described in detail using the translocation signal of a single molecule in the Aerolysin pore (Appendix 2). Figure 1 b), the ion blocking current formed when the single molecule translocates in the nanopore has a blocking amplitude of ΔI and a blocking duration of T, and the four inflection points (t1, t2, t3, t4) in the blocked current curve define the start and end time points of the three translocation stages: the pore entry stage is [t1, t2], the pore penetration stage is [t2, t3], and the pore exit stage is [t3, t4]. The input parameters of the method for extracting the physical fingerprint characteristics of the nanopore at the single molecule level are t2, t3, τ, L, E, V a , the output parameters are d, a1, Q z , Q α , Q η , and c2 (Table 1).

[0054] Table 1 Explanation of input and output parameters of the method for extracting the physical fingerprint characteristics of the nanopore at the single molecule level

[0055]

[0056]

[0057] (3) The speed estimation values of the single molecule in the pore entry, pore penetration and pore exit (v ENT , V TRAV and v EXIT ) can be obtained by substituting the fingerprint parameters and the time boundary values of the three stages extracted from the single molecule translocation signal in step 2 into the speed equations of the pore entry, pore penetration and pore exit, respectively. By analyzing vENT v TRAV and v EXIT The statistical properties of three velocities reveal the velocity variation properties of molecules within the pores. To illustrate this step, translocation signals of three sets of uniformly distributed poly(A)3, poly(A)4, and poly(A)5 molecules with λ / m were collected using T274L / N226Q / S228K Aerolysin nanopores, and feature extraction was performed on the samples (see attached). Figure 3 a). The theoretical lengths of A3, A4, and A5 are 0.99, 1.32, and 1.65 (nm), respectively, carrying 2, 3, and 4 negative charges, and have molecular weights of 923.65, 1251.8, and 1580.1 (Da), respectively. The theoretical λ / m values ​​of A3, A4, and A5 calculated according to formula (1) are 3.40 × 10⁻⁶. -22 2.90×10 -22 and 2.45×10 -22 (C·nm -1 ·[Da] -1 The average translocation velocities of A3, A4, and A5 were 1.49±2.57, 2.04±0.53, and 0.10±0.15 (nm / ms), respectively. (See attached figure) Figure 3 (b) A3 has the fastest translocation speed, followed by A4 and A5. The center positions of the Gaussian fitting curves for the λ / m distribution of A3, A4, and A5 are 8.98 × 10⁻⁶ respectively. -29 2.90×10 -30 and 5.66×10 -31 (C·nm -1 ·[M] -1 (Appendix) Figure 3 The experimental values ​​of λ / m show good linear fit with the theoretical values ​​on the log scale (see appendix). Figure 3 f) indicates that this parameter reflects the actual charge-to-mass ratio of the molecule. This set of translocation experimental data proves that the λ / m of the molecule is proportional to the translocation velocity.

[0058] To further verify the minimum velocity v required for molecules to complete the translocation process. min The displacement was proportional to the charge-to-mass ratio λ / m at the time of entry into the pore. Further direction-dependent translocation experiments were conducted using T274L / N226Q / S228K Aerolysin nanopores (see attached). Figure 4a-b). The phosphate group modified at 3' or 5' end of A4 molecule (5'-A4-3'-P and P-5'-A4-3') has a non-uniform linear charge density. When the phosphate group carrying molecule with high charge amount enters the pore first, it has a higher initial λ / m value due to the increased charge amount per unit length. The translocation signals of these phosphate first-in-pore molecules (PI(3') of 5'-A4-3'-P and PII(5') of P-5'-A4-3') show a shorter translocation time in the translocation experiment. By comparing the full width at half maximum (FWHM) values of λ / m of A4, 5'-A4-3'-P and P-5'-A4-3' molecules, it is found that only the samples of phosphate first-in-pore translocation signals have the largest FWHM value, while the samples of deoxyribonucleotide first-in-pore signals remain at a very low level regardless of whether the phosphate group is modified at 3' or 5' end (Fig. 2). Figure 3 e, Fig. 2 Figure 4 c-d). This indicates that the FWHM of λ / m parameter distribution is highly dependent on the initial λ / m value of the molecule entering the pore. The broadening of λ / m distribution also means that the λ / m attribute of the phosphate end first-in-pore molecule fluctuates more complexly with time during the translocation process. This set of experimental data proves that the λ / m value of the molecule when entering the pore dominates the translocation speed of the molecule.

[0059] According to the speed prediction method described in step 3, the speed changes of A4, 5'-A4-3'-P and P-5'-A4-3' molecules in translocation are predicted (Table 1). The penetration speeds of the three molecules all show a trend of continuous acceleration from entering the pore to exiting the pore, and the speed in the exit stage is about 1.19-1.61 times that in the entry stage. It is worth noting that the PI(3') direction of P-5'-A4-3' molecule exhibits the largest acceleration factor, which can be attributed to the fact that the λ / m of the phosphate group carrying molecule instantaneously increases when the molecule locally exits the pore, thereby being expelled from the pore with a greater electric field force.

[0060] Table 2 Average predicted speed of A4, 5'-A4-3'-P and P-5'-A4-3' molecules in the entry, penetration and exit stages

[0061]

[0062] The above description is only one embodiment of the present application and is not intended to limit the present application. It should be noted that several improvements and modifications can be made without departing from the technical scope of the present application, and these improvements and modifications should also be considered as the protection scope of the present application.

Claims

1. A three-stage translocation model for revealing the fingerprint characteristics of Aerolysin nanopores, characterized in that, Includes the following steps: Step 1: Based on the difference in force experienced by charged molecules within the pores, the overall process of their displacement is divided into three stages: entry into the pore, perforation, and exit from the pore, and corresponding displacement and velocity equations are established. The displacement and velocity equations are functions of three physical fingerprint characteristics of the nanopores. The three physical fingerprint characteristics are the ratio of molecular linear charge density to mass (λ / m), the ratio of pore resistance coefficient to mass (α / m), and the ratio of liquid viscous resistance coefficient to mass (η / m). Step 2: Establish a method for extracting the physical fingerprint characteristics of nanopores at the single-molecule level. This method uses the four inflection points of the blocking current generated during molecular perforation to determine the time and boundary values ​​of the three stages. Substitute these values ​​into the corresponding displacement and velocity equations to establish a nonlinear equation set. Solve the nonlinear equation set using the trust region algorithm to obtain the estimated values ​​of λ / m, α / m, and η / m related to the nanopore fingerprint parameters. Step 3: Substituting the fingerprint parameters extracted from the single-molecule translocation signal in Step 2 and the time boundary values ​​of the three stages into the velocity equations for entry, perforation, and exit, respectively, we can obtain the estimated velocity values ​​(v) of the single molecule at entry, perforation, and exit. ENT v TRAV and v EXIT ), through analysis of v ENT v TRAV and v EXIT The statistical properties of the three velocities reveal the velocity variation properties of molecules within the pores.

2. The three-stage translocation model for revealing the fingerprint characteristics of Aerolysin nanopores according to claim 1, characterized in that, Step 1 describes the difference in force experienced by charged molecules within the pore. Specifically, during the pore-entry stage, the molecule is slowed down by the electric field force and the resistance of the molecule-pore interaction. During the pore-piercing stage, the electric field force and the resistance of the molecule-pore interaction are balanced, and the molecule pierces the pore at a constant speed. During the pore exit phase, molecules are subjected to two forces: electric field force and viscous resistance of the external liquid, which accelerates the molecules out of the pore.

3. The three-stage translocation model for revealing the fingerprint characteristics of Aerolysin nanopores according to claim 1, characterized in that, In step 2, the time and boundary values ​​of the three stages are determined by using the four inflection points of the blocking current generated during molecular perforation. The total translocation time of the blocking current generated by single-molecule translocation is τ. The four inflection points (t1, t2, t3, t4) in the blocking current curve define the start and end time points of the three translocation stages: the entry stage is [t1, t2], the perforation stage is [t2, t3], and the exit stage is [t3, t4]. Here, t1 is the starting point of the falling edge of the blocking current, t2 is the ending point of the falling edge of the blocking current, t3 is the starting point of the rising edge of the blocking current, and t4 is the ending point of the rising edge of the blocking current.

4. The three-stage translocation model for revealing the fingerprint characteristics of Aerolysin nanopores according to claim 1, characterized in that, The nonlinear equation system established in step 2 consists of a positive definite equation system composed of six unknown variables and six equations. The system is then solved using a trust region algorithm. This algorithm establishes a trust region centered on the current iteration point, approximates the objective function within the trust region, and then expands or shrinks the trust region based on the change in the objective function value, thereby obtaining estimates of the nanopore fingerprint parameters λ / m, α / m, and η / m.

5. A three-stage translocation model for revealing the fingerprint characteristics of Aerolysin nanopores according to claim 1, characterized in that, In step 3, the fingerprint parameters extracted from the single-molecule translocation signal in step 2 and the time boundary values ​​of the three stages are substituted into the velocity equations for entry, perforation, and exit, respectively, to obtain the estimated velocity values ​​(v) of the single molecule at entry, perforation, and exit. ENT v TRAV and v EXIT ), through analysis of v ENT v TRAV and v EXIT The statistical properties of the three velocities reveal the velocity variation properties of molecules within the pores, v ENT v TRAV and v EXIT The calculation method is as follows and Q z =λ / m, Q α =α / m, Q η =η / m.

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