Single molecule detection method of stevioside

Abstract

The invention provides a method for detecting a stevioside single molecule by using an Aerolysin nanopore channel. The method comprises the following multiple steps. The preparation method comprises the following steps: (1) assembling a phospholipid bilayer, and embedding Aerolysin protein to form a single nanopore channel; (2) adding stevioside into a buffer solution of the nanopore detection device, collecting a monomolecular signal of the stevioside, changing voltage to regulate and control electroosmotic flow, and improving the resolution of the nanopore to the stevioside; (3) performing statistical analysis on the stevioside monomolecular signal to obtain a characteristic signal of stevioside, and identifying and distinguishing the stevioside on the basis of the characteristic signal of the stevioside; and (4) establishing linear correlation between the concentration of the stevioside and the event frequency, and realizing quantitative analysis of the stevioside. According to the method, high-resolution recognition can be carried out on various steviosides on the single molecule level, and the method is high in sensitivity, has important significance in determining the effectiveness and safety of the steviosides in food and medicines, and is beneficial to deep research of structure-function relationships and pharmacokinetics.

Description

Technical Field

[0001] This invention relates to the field of biotechnology in life sciences, specifically a method for detecting steviol glycosides at the single-molecule level using Aerolysin nanopores. Background Technology

[0002] Stevioside is a tetracyclic diterpenoid compound with a sweetness 200-300 times that of sucrose and a calorific value only 1 / 300 that of sucrose. It can be used as a natural low-calorie sweetener and a substitute for synthetic sweeteners. There is a large demand for steviol glycoside products in the food and dietary supplement industry. High-purity steviol glycosides have been assessed by various global scientific and regulatory agencies as safe for consumption by the general population. In addition, steviol glycosides have a variety of important biological activities, such as anti-diabetic, anti-hyperglycemic, anti-hypertensive, anti-caries, and anti-tumor activities. Strict quality control is essential to ensure the effectiveness and safety of this substance in food and pharmaceuticals. The structure of steviol glycosides is complex and diverse: (1) a rich variety of sugar groups, such as the difference of only one hydroxyl group, and isomers; (2) a diverse tetracyclic diterpenoid skeleton structure and the connection sites between sugar groups and the tetracyclic diterpenoid skeleton; (3) a branched sugar chain structure and the existence of isomers; and (4) a wide range of molecular weight distribution, with large differences in polarity and content. These complexities make the separation and analysis of steviol glycosides difficult, which restricts further exploration of structure-activity relationships and pharmacokinetic processes. Therefore, there is still an urgent need for more sensitive and low-cost analytical methods to achieve rapid qualitative identification and quantitative analysis of steviol glycosides.

[0003] Nanopores are a modern, emerging single-molecule detection technique for nucleic acids, peptides, and carbohydrates. They offer advantages such as simplicity, speed, label-free operation, high sensitivity, and low cost. Driven by electroosmosis, electrophoresis, or diffusion, molecules pass through nanopores, causing changes in current and generating pulse characteristics closely related to the molecule's shape, size, and charge, which can then be monitored and analyzed. Aerolysin nanopores possess even smaller pore sizes and higher sensitivity. Their positively charged cavities allow for modulation of electroosmotic flow (EOF) and electrophoretic forces by altering the applied voltage, electrolyte type, concentration, and pH, further enhancing recognition capabilities. Nanopore single-molecule detection technology holds great promise for the identification and analysis of steviol glycosides, which possess inherent complexity and diversity, but no reports have yet been published on this technology. Summary of the Invention

[0004] This invention aims to address the aforementioned challenges by providing a method for detecting steviol glycosides based on Aerolysin nanopores. This method enables rapid, low-cost, highly sensitive, and precise qualitative and quantitative analysis of steviol glycosides, providing a potential new industry standard for the quality control of steviol glycosides. To achieve this objective, the invention adopts the following technical solution:

[0005] A single-molecule detection method for steviol glycosides, the main steps of which are as follows:

[0006] (1) Assemble a phospholipid bilayer and embed Aerolysin protein to form a single nanopore. Prepare a phospholipid (diphytylphosphatidylcholine) solution (30 mg / mL) using n-decane as solvent. A phenolic resin detection cell (consisting of a 1.5 mL resin cup inserted into a resin tank, with the remaining tank volume at 1.5 mL) (Warner Corporation, USA) was used. The resin cup served as the CIS cell, and the remaining resin tank space served as the trans cell. A 150 μm pore was located in the 1 mm thick wall of the resin cup. Approximately 2 μL of the phospholipid solution was applied to a 5 mm diameter area around the pore using a No. 00 sable brush and allowed to dry. 1 mL of electrolyte was added to each cell (ensuring the liquid level was above the pore), and a phospholipid bilayer was formed on the pore using a dip-coating method. Then, Aerolysin protein solution was added, and the assembly and embedding of the pores was awaited. Insert Ag / AgCl electrodes into both cells. Connect the trans cell electrode to the positive terminal of the headstage probe of the Axopatch 200B patch clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and connect the cis cell electrode to the negative terminal of the headstage (grounded).

[0007] (2) Dissolve steviol glycosides in water, and inject 1 μL of the steviol glycoside solution into the CIS cell electrolyte. The final concentration of steviol glycosides in the electrolyte is 0.5 μM to 12 μM. Apply a voltage of +40 to +200 mV to the instrument, a sampling rate of 100 to 250 kHz, and a low-pass filter of 5 to 250 kHz. Begin acquiring the blocking current signal of steviol glycosides.

[0008] (3) Analyze the steviol glycoside signal. The recorded blocking current pulse signal can be used to extract the blocking current ratio, blocking time and standard deviation of blocking current fluctuation. A scatter fingerprint of the event can be made based on the blocking current ratio and blocking time, and a scatter plot can be drawn based on the blocking current ratio and standard deviation of blocking current fluctuation.

[0009] (4) Quantify steviol glycosides. Based on the frequency of the pulse and the concentration of steviol glycosides, plot the linear correlation between concentration and event frequency, and perform linear fitting to obtain the quantitative curves or formulas for different steviol glycosides. Substitute the event frequency of the sample to be tested into the quantitative curves or formulas to obtain the concentrations of different steviol glycosides, thus realizing the quantitative analysis of different steviol glycosides.

[0010] The stevioside mentioned in step (2) may be one or more of the following: stevioside (Steb), stevioside (Rub), stevioside (Ste), durcuryl glycoside A (DuA), B (DuB), rebadi glycoside B (RebB), G (RebG), A (RebA), C (RebC), E (RebE), F (RebF), D (RebD), J (RebJ), M (RebM), N (RebN), O (RebO), H (RebH), I (RebI), K (RebK), L (RebL), suavioside B (SuaB), Q1 (SuaQ1), R1 (SuaR1), S1 (SuaS1), C1 (SuaC1), D1 (SuaD1), Q2 (SuaQ2), S2 (SuaS2), C2 (SuaC2), and D2 (SuaD2).

[0011] The electrolyte in step (2) can be one or more of the following buffer solutions: 10–25 mM HEPES (containing 0.5–4 M LiCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 0.5–4 M NaCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 1–4 M KCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 0.5–4 M CaCl2, 1 mM EDTA, pH 3.5–10) buffer, 10 mM Tris-HCl (containing 0.5–4 M KCl, 1 mM EDTA, pH 3.5–10) buffer, or 10 mM Tris-HCl (containing 0.5–4 M NaCl, 1 mM EDTA, pH 3.5–10) buffer. 3.5–10) buffer solution.

[0012] The applied voltage described in step (2) is the main parameter affecting the perforation behavior of steviol glycosides. By adjusting the voltage, i.e., increasing the voltage in increments of 20mV within the range of +40 to +200mV, the driving force of electroosmotic flow on neutral steviol glycosides can be altered. The higher the voltage, the stronger the driving force of electroosmotic flow, and the binding mode of steviol glycosides to nanopores is significantly regulated: when the voltage is below the voltage threshold (the blocking event has a certain voltage dependence; if the blocking time of the blocking event first increases and then decreases with increasing voltage, then the voltage value corresponding to the monotonicity change is the voltage threshold), most of the steviol glycosides are bound to the nanopores without passing through the pores; when the voltage exceeds the voltage threshold, the steviol glycosides will undergo translocation. The voltage threshold varies under different electrolyte conditions (type, concentration, and pH of electrolyte), so increasing the voltage to near the voltage threshold (±20mV) can prolong the blocking time of neutral steviol glycosides inside the pores, increase the blocking depth and event frequency, and improve resolution.

[0013] This invention enables high-resolution identification and quantitative analysis of various steviol glycosides at the single-molecule level. The method is rapid, highly sensitive, and accurate, which is of vital importance for determining the efficacy and safety of steviol glycosides in food and pharmaceuticals. At the same time, the method is simple to operate and low in cost, and is expected to provide a potential new industry standard for the quality control of steviol glycosides, which is conducive to in-depth research on structure-activity relationships and pharmacokinetics. Attached Figure Description

[0014] Figure 1 A schematic diagram illustrating the principle of the method for detecting steviol glycosides based on Aerolysin nanopores in this invention;

[0015] Figure 2 Structural formulas of 15 steviol glycosides;

[0016] Figure 3 .Scatter plot of nanopore ion current blocking events and event distribution in Steb;

[0017] Figure 4 .Rub's nanopore ion current blocking events and event distribution scatter plot;

[0018] Figure 5 Scatter plot of nanopore ion current blocking events and event distribution in Ste;

[0019] Figure 6 .Scatter plot of ion current blocking events and event distribution in the nanopores of DuA;

[0020] Figure 7 .Scatter plot of ion current blocking events and event distribution in nanopores of RebB;

[0021] Figure 8.Scatter plot of ion current blocking events and event distribution in RebG nanopores;

[0022] Figure 9 .Scatter plot of ion current blocking events and event distribution in RebA nanopores;

[0023] Figure 10 .Scatter plot of ion current blocking events and event distribution in RebC nanopores;

[0024] Figure 11 .Scatter plot of ion current blocking events and event distribution in RebE nanopores;

[0025] Figure 12 .Scatter plot of ion current blocking events and event distribution in RebF nanopores;

[0026] Figure 13 .Scatter plot of nanopore ion current blocking events and event distribution in RebD;

[0027] Figure 14 .Scatter plot of nanopore ion current blocking events and event distribution in RebJ;

[0028] Figure 15 .Scatter plot of ion current blocking events and event distribution in RebM nanopores;

[0029] Figure 16 .Scatter plot of ion current blocking events and event distribution in RebN nanopores;

[0030] Figure 17 .Scatter plot of ion current blocking events and event distribution in the nanopores of RebO;

[0031] Figure 18 Scatter plot of the blocking current ratio and standard deviation of blocking current fluctuation for 15 steviol glycosides;

[0032] Figure 19 .Scatter plots of nanopore ion current blocking events, event distributions, and histograms of blocking current ratios for equimolar mixtures of RebA and RebC at voltages of 60 mV and 160 mV, respectively.

[0033] Figure 20 Linear correlation plot of RebA concentration and event frequency;

[0034] Note: Figure 2 The 15 steviol glycosides corresponding to Example 2; Figure 3-18 , Figure 19 and Figure 20 The detection results correspond to Examples 2, 3, and 4, respectively. Detailed Implementation

[0035] To make the content, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments and accompanying drawings. These embodiments are only used to illustrate the present invention, and the present invention is not limited to the following embodiments.

[0036] Example 1. Assembly of Aerolysin nanopores

[0037] (1) Assembly of Aerolysin nanopores Reference: Cao, C., Liao, DF., Yu, J. et al. Construction of an aerolysin nanopore in a lipid bilayer for single-oligonucleotide analysis. Nat. Protoc. 12, 1901–1911 (2017). Specifically, a phospholipid (diphytylphosphatidylcholine) solution (30 mg / mL) was prepared using n-decane as the solvent. A phenolic resin detection cell (consisting of a resin cup with an open top inserted into a resin tank with an open top, wherein the cup has a volume of 1.5 mL and the remaining volume of the resin tank after filling the cup is 1.5 mL) (Warner Corporation, USA) was used. The inner cavity of the resin cup served as the CIS cell, and the remaining cavity in the resin tank after filling the cup served as the trans cell. There was a small hole with a diameter of 150 μm in the lower middle part of the 1 mm thick resin cup wall. The surface of the 5 mm diameter circular area (centered on the center of the hole) on both sides of the small hole on the cup wall was coated with phospholipid solution using a No. 00 sable brush (2 μL was used on both sides) and dried. 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution was added to each of the two cells (CIS cell and trans cell) as electrolyte (the liquid level in both cells was ensured to be higher than the position of the small hole). Insert Ag / AgCl electrodes into both cells. Connect the trans cell electrode to the positive terminal of the headstage probe of an Axopatch 200B patch-clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the cis cell electrode to the negative terminal (grounded) of the headstage. Turn on the Axopatch 200B patch-clamp amplifier and the Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA). Insert a 1mL syringe into the bottom of the cis cell and slowly draw out and then slowly inject the drawn-out solution to form a phospholipid bilayer on the pore (observe the current; the current becomes 0pA at the instant of film formation). Then, 0.5 μL of Aerolysin protein solution (20 μg / mL) was added to the CIS cell near the pore (for details on the expression and purification of Aerolysin protein, please refer to the literature Li, M., Xiong, Y., Cao, Y. et al. Identification of tagged glycans with a protein nanopore. Nat Commun 14, 1737 (2023)). A voltage of +200 mV was applied between the positive and negative electrodes to wait for the pores to assemble and embed. Once a single Aerolysin nanopore was assembled, the observed ion current immediately changed from 0 pA to about 200 pA.

[0038] Example 2. Detection of steviol glycosides using Aerolysin nanopores

[0039] (1) Stevioside samples were dissolved in water at a concentration of 4 mM. 1 μL of the steviol glycoside solution was added to the cis cell electrolyte (same as described in Example 1, containing 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer) of the Aerolysin nanopore detection cell prepared in Example 1. Another trans cell contained 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer. Ag / AgCl electrodes were inserted into both cells. The trans cell electrode was connected to the positive terminal of the headstage probe of the Axopatch200B patch-clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the cis cell electrode was connected to the negative terminal (grounded) of the headstage. The Axopatch200B patch-clamp amplifier and the Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA) were turned on. The Axopatch 200B patch-clamp amplifier applies a voltage of +60mV between the positive and negative terminals, a sampling rate of 100kHz, and a low-pass filter of 5kHz to begin acquiring the blocking current signal of steviol glycosides.

[0040] (2) The blocking current pulse signals recorded by the Axopatch 200B patch-clamp amplifier and Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA) were used to extract the pulse current magnitude and time, and obtain the blocking current ratio (Ig). b / I0, where I b I0 is the blocking current, I0 is the open-circuit current (i.e., the constant baseline current obtained under the applied voltage before the addition of the steviol glycoside sample) and the blocking time (T0). d The standard deviation (SD) of the blocking current fluctuation was calculated, and then a scatter plot was plotted with the blocking current ratio as the x-axis and the logarithm of the blocking time as the y-axis. For the blocking current ratio and blocking time, Gaussian distribution fitting and single exponential fitting were performed using OriginPro 2021 (OriginLab, Northampton, Massachusetts, USA) to obtain the average blocking current and average blocking time specific to steviol glycosides, respectively. The standard deviation of the blocking current fluctuation was obtained using the single-channel search function of Clampfit 11.2 software. Among these:

[0041] The SteB's characteristic blocking current ratio averages 0.797, and the blocking time averages 5.03ms.

[0042] Rub's characteristic blocking current ratio is 0.827, and its blocking time is 3.94ms.

[0043] Ste's characteristic blocking current ratio averages 0.756, and blocking time averages 4.71ms;

[0044] The average blocking current ratio of DuA is 0.767, and the average blocking time is 4.21ms.

[0045] The RebB characteristic blocking current ratio is 0.739 on average, and the blocking time is 28.3ms on average.

[0046] The RebG's characteristic blocking current ratio averages 0.710, and the blocking time averages 12.3ms.

[0047] The RebA's characteristic blocking current ratio averages 0.660, and the blocking time averages 41.4 ms.

[0048] The RebC's characteristic blocking current ratio averages 0.646, and its blocking time averages 40.0 ms.

[0049] The RebE has a characteristic blocking current ratio of 0.725 and a blocking time of 6.23ms.

[0050] The RebF's characteristic blocking current ratio averages 0.699, and the blocking time averages 27.9 ms.

[0051] The RebD's characteristic blocking current ratio averages 0.600, and the blocking time averages 24.8ms.

[0052] The RebJ's characteristic blocking current ratio is 0.632, and the blocking time is 16.3ms.

[0053] The RebM's characteristic blocking current ratio is 0.405, and the blocking time is 49.2ms.

[0054] The RebN's characteristic blocking current ratio averages 0.541, and the blocking time averages 35.8ms.

[0055] The RebO's characteristic blocking current ratio averages 0.476, and the blocking time averages 45.6 ms.

[0056] Example 3. Improving the resolution of steviol glycoside detection using Aerolysin nanopores by adjusting voltage.

[0057] (1) RebA and RebC samples were dissolved in water at a concentration of 4 mM. 1 μL of each steviol glycoside solution was added to the cis cell electrolyte of the Aerolysin nanopore detection cell prepared in Example 1 (containing 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer as described in Example 1). Another trans cell contained 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer. Ag / AgCl electrodes were inserted into both cells. The trans cell electrode was connected to the positive terminal of the headstage probe of an Axopatch 200B patch-clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the cis cell electrode was connected to the negative terminal (grounded) of the headstage. The Axopatch 200B patch-clamp amplifier and the Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA) were turned on. The Axopatch200B patch-clamp amplifier applies a voltage of +160mV between the positive and negative terminals, with a sampling rate of 100kHz and a low-pass filter of 5kHz, to begin acquiring the blocking current signal of an equimolar mixture of steviol glycosides.

[0058] (2) (The process and conditions are the same as step (2) in Example 2). Wherein:

[0059] The RebA has a characteristic blocking current ratio of 0.653 and a blocking time of 376ms at 160mV.

[0060] The RebC has a characteristic blocking current ratio of 0.636 and a blocking time of 286ms at 160mV.

[0061] With 1 mL of 25 mM HEPES buffer (containing 4 M LiCl, 1 mM EDTA, pH 7.5) as the electrolyte, the voltage threshold for steviol glycoside translocation behavior was ~160 mV. Compared to a low voltage of 60 mV (as shown in Example 2), at a high voltage of 160 mV, the electroosmotic flow increased, the blocking time of RebA and RebC increased (from 41.4 ms to 376 ms and 40.0 ms to 286 ms, respectively), and the blocking current ratio decreased (from 0.660 to 0.653 and 0.646 to 0.636, respectively). Therefore, increasing the voltage to the voltage threshold of 160 mV significantly improved the resolution.

[0062] Example 4. Quantification of steviol glycosides using Aerolysin nanopores

[0063] (1) RebA samples were dissolved in 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution at concentrations of 0.5, 1, 2, 4, 6, 8, 10, 12, and 14 μM. 10 mL of this solution was used to replace 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution in the CIS cell of the Aerolysin nanopore detection cell prepared in Example 1 10 times (1 mL of sample solution was used each time, for a total of 10 mL). The replacement was performed by simultaneously drawing solution from the detection cell with a syringe and adding 1 mL of solution with a pipette, ensuring the liquid level in the detection cell was higher than the pores to maintain the stability of the Aerolysin nanopores and phospholipid membrane. The final CIS cell contained 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution with concentrations of 0.5, 1, 2, 4, 6, 8, 10, 12, and 14 μM RebA. One chamber contained EDTA (pH 7.5) buffer, and the other trans chamber contained 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer. Ag / AgCl electrodes were inserted into both chambers. The trans chamber electrode was connected to the positive terminal of the headstage probe of an Axopatch 200B patch-clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the cis chamber electrode was connected to the negative terminal (grounded) of the headstage. The Axopatch 200B patch-clamp amplifier and the Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA) were turned on. A voltage of +60 mV was applied between the positive and negative terminals of the Axopatch 200B patch-clamp amplifier, with a sampling rate of 100 kHz and a low-pass filter of 5 kHz, to begin acquiring blocking current signals of different concentrations of RebA.

[0064] (2) Based on the pulse frequency and RebA concentration, a linear correlation graph was plotted with concentration on the x-axis and event frequency (i.e., the number of steviol glycoside blocking events with a blocking time greater than 1 ms per unit time) on the y-axis. A standard curve was obtained by linear fitting. The event frequency was obtained using the event analysis window of the threshold retrieval function in Clampfit 11.2 software.

[0065] (3) The RebA sample was dissolved in 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution, with a concentration of 3 mM. 1 μL was added to the cis cell electrolyte of the Aerolysin nanopore detection cell prepared in Example 1 (containing 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution as described in Example 1). Another trans cell contained 1 mL of 25 mM HEPES (containing 4 M LiCl, 1 mM EDTA, pH 7.5) buffer solution. Ag / AgCl electrodes were inserted into both cells. The trans cell electrode was connected to the positive terminal of the headstage probe of the Axopatch 200B patch-clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the cis cell electrode was connected to the negative terminal (grounded) of the headstage probe. The Axopatch 200B patch-clamp amplifier and the Axon Digidata 1550B analog-to-digital converter (Molecular Instruments, Sunnyvale, California, USA) were turned on. A voltage of +60mV was applied between the positive and negative terminals of the Axopatch 200B patch-clamp amplifier, with a sampling rate of 100kHz and a low-pass filter of 5kHz, to begin acquiring the RebA blocking current signal. The event frequency was set to 0.56s. -1 Substituting the values ​​into the standard curve, the final concentration of Reb A was measured to be 2.9 mM, with a recovery rate of 96.67%.

[0066] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A single-molecule detection method for steviol glycosides, characterized in that: (1) Assemble a phospholipid bilayer on the connecting pores of the two cells of the detection cell and embed Aerolysin protein to form a single nanopore; (2) Add steviol glycosides to the electrolyte of the nanopore detection cell, connect the patch clamp instrument, insert electrodes in the two cells and apply voltage to collect the single molecule current signal of steviol glycosides. (3) a. Statistical analysis of single-molecule signals of steviol glycosides was performed to obtain characteristic signals of steviol glycosides, and based on this, different steviol glycosides were identified and distinguished. And / or, b. Establish a linear correlation between the concentration of steviol glycosides and the event frequency to achieve quantitative analysis of steviol glycosides.

2. The method for single-molecule detection of steviol glycosides according to claim 1, characterized in that: The phospholipid used in step (1) is diphytylphosphatidylcholine, and the protein channels used are assembled from Aerolysin.

3. The method for single-molecule detection of steviol glycosides according to claim 1, characterized in that: The stevioside mentioned in step (2) may be one or more of the following: stevioside (SteB), stevioside (Rub), stevioside (Ste), durcuryl glycoside A (DuA), B (DuB), rebaudioside B (RebB), G (RebG), A (RebA), C (RebC), E (RebE), F (RebF), D (RebD), J (RebJ), M (RebM), N (RebN), O (RebO), H (RebH), I (RebI), K (RebK), L (RebL), suavioside B (SuaB), Q1 (SuaQ1), R1 (SuaR1), S1 (SuaS1), C1 (SuaC1), D1 (SuaD1), Q2 (SuaQ2), S2 (SuaS2), C2 (SuaC2), and D2 (SuaD2).

4. The method for single-molecule detection of steviol glycosides according to claim 1, characterized in that: In step (2), the Ag / AgCl electrode in the trans cell electrolyte of the test device is connected to the positive electrode of the headstage probe of the Axopatch 200B patch clamp amplifier (Molecular Instruments, Sunnyvale, California, USA), and the Ag / AgCl electrode in the cis cell electrolyte is connected to the ground terminal; steviol glycoside molecules are added to the cis cell; the instrument applies a voltage of +40 to +200mV, a sampling rate of 100 to 250kHz, and a low-pass filter of 5 to 10kHz.

5. The method for single-molecule detection of steviol glycosides according to claim 1 or 4, characterized in that: The electrolyte may be one or more of the following buffer solutions; the buffer solutions include: 10–25 mM HEPES (containing 0.5–4 M LiCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 0.5–4 M NaCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 0.5–4 M KCl, 1 mM EDTA, pH 3.5–10) buffer, 10–25 mM HEPES (containing 0.5–4 M CaCl2, 1 mM EDTA, pH 3.5–10) buffer, 10 mM Tris-HCl (containing 0.5–4 M KCl, 1 mM EDTA, pH 3.5–10) buffer, or 10 mM Tris-HCl (containing 0.5–4 M NaCl, 1 mM EDTA, pH 3.5–10) buffer.

6. The method for single-molecule detection of steviol glycosides according to claims 1 and 4, characterized in that: In step (2), electrodes are inserted into the two cells and voltage is applied to collect the single-molecule current signal of steviol glycoside. In the range of +40 to +200mV, the voltage is gradually increased by a voltage difference of 20mV to regulate the electroosmotic flow in order to improve the resolution of steviol glycoside in the nanopores. The applied voltage is the main parameter affecting the pore-perforation behavior of steviol glycosides; By adjusting the voltage, the driving force of electroosmotic flow on neutral steviol glycosides can be altered. The higher the voltage, the stronger the driving force of electroosmotic flow, and the binding mode of steviol glycosides to nanopores is significantly regulated: when the voltage is below the voltage threshold (blockage events have a certain voltage dependence; if the blocking time of the blocking event first increases and then decreases with increasing voltage, then the voltage value corresponding to the monotonic change is the voltage threshold), most of the steviol glycosides are bound to the nanopores without passing through the pores; when the voltage exceeds the voltage threshold, the steviol glycosides will undergo translocation, i.e., pass through the pores. The voltage threshold varies under different electrolyte conditions (type, concentration, and pH of electrolyte), so increasing the voltage to near the voltage threshold (±20mV) can prolong the blocking time of neutral steviol glycosides inside the pores, increase the blocking depth and event frequency, and improve resolution.

7. The method for single-molecule detection of steviol glycosides according to claim 1, characterized in that, In step (3)a, the steviol glycoside single-molecule signal includes the blocking current, blocking time, and standard deviation of the blocking current fluctuation, wherein different steviol glycosides correspond to different blocking characteristic signals.

8. The method for single-molecule detection of steviol glycosides according to claim 1, characterized in that, In step (3)b, the event frequency is the number of times a steviol glycoside blocking event with a blocking time greater than 1ms occurs per unit time. A linear correlation graph of concentration and event frequency is plotted with the concentration of steviol glycoside as the abscissa and the event frequency as the ordinate. Linear fitting is then performed to obtain the quantitative curve or formula for different steviol glycosides. By substituting the event frequency of the sample to be tested into the quantitative curve or formula, the concentration of different steviol glycosides can be obtained, thus realizing the quantitative analysis of different steviol glycosides.

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