A method for determining the binding constant of a protein to a small molecule

Abstract

The present application relates to a kind of method for determining the binding constant of protein and small molecule, based on the binding constant determination of protein and drug molecule under pseudo-physiological condition can be realized by multi-chamber electrophoresis separation technology and Scatchard model, provide new ideas for the mechanism research of drug molecule in vivo, clinical medical application.The binding constant determination method of the present application has the advantages of high efficiency, high accuracy.

Description

Technical Field

[0001] This invention relates to the field of analytical detection technology, and specifically to a method for determining the binding constant of proteins and small molecules. Background Technology

[0002] The binding constant is an important parameter for determining the interaction between biomolecules and small molecules, reflecting the strength of the interaction between biomolecules and drug molecules, and is of great significance for drug development and clinical medical applications. Currently, common methods for detecting the binding constant include equilibrium dialysis and ultrafiltration, but these methods suffer from low efficiency and low accuracy.

[0003] For example, ultrafiltration primarily utilizes centrifugation and membrane retention to separate free small molecules from protein-drug molecule complexes. The binding constant is then determined by measuring the concentration of free drug molecules in the system. This method allows for the simultaneous separation of multiple samples in a single centrifugation, offering high throughput. However, ultrafiltration membranes exhibit some adsorption of free small molecules, requiring multiple washings to reduce residue within the membrane. Therefore, the measurement process is cumbersome, and the accuracy of the test results is low.

[0004] Therefore, there is a need to develop a new method for determining the binding constant of proteins and small molecules, which can efficiently and accurately determine the binding constant. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for determining the binding constant of proteins and small molecules, which can efficiently and accurately determine the binding constant.

[0006] To achieve the above objectives, the present invention provides the following technical solution.

[0007] A method for determining the binding constant of a protein to a small molecule includes the following steps:

[0008] Provides proteins, small molecules, and buffer solutions;

[0009] Using the buffer solution, prepare n sample solutions containing the protein and the small molecule, where n≥3; in each sample solution, the total protein concentration is the same, but the total small molecule concentration is different;

[0010] Each of the sample solutions was processed as follows:

[0011] The sample solution is incubated to form charged protein-small molecule complexes;

[0012] The protein-small molecule complex was separated using multi-compartment electrophoresis.

[0013] A dissociation agent is added to the separated protein-small molecule complex to dissociate it into protein and small molecules, resulting in a dissociation solution. The concentration of the small molecules in the dissociation solution is measured and recorded as the transmembrane small molecule concentration.

[0014] Based on the Scatchard model, the total protein concentration, the total small molecule concentration in each sample solution, and the transmembrane small molecule concentration in each sample solution, the binding constant K between the protein and the small molecule is calculated.

[0015] To address the issues of low accuracy and efficiency in determining the binding constants of biomolecules and small molecules, this invention proposes a novel method for determining binding constants using the chamber membrane separation concept of multi-chamber electrophoresis, providing a new approach and strategy for the clinical application of drugs. This method offers advantages such as high efficiency and high accuracy.

[0016] This invention utilizes multi-compartment electrophoresis to separate protein-small molecule complexes, requiring a very short separation time, typically less than 20 minutes. Compared to existing separation methods such as equilibrium dialysis and ultrafiltration (which take at least 1 hour), this significantly shortens the separation time, greatly improving separation efficiency and binding constant determination efficiency. The superior separation effect of multi-compartment electrophoresis also contributes to improving the accuracy of binding constant determination.

[0017] Furthermore, the determination method of this invention has universality. While existing fluorescence spectroscopy methods, compared to equilibrium dialysis and ultrafiltration, offer relatively higher accuracy and efficiency, they require at least one of the protein and small molecule to exhibit fluorescence, thus limiting their applicability. In contrast, the determination method of this invention can be used regardless of whether the protein or small molecule exhibits fluorescence, demonstrating strong universality.

[0018] The equations for the Scatchard model are shown below:

[0019] n / [L]=-nK+KN

[0020] in,

[0021] n = Concentration of transmembrane small molecules in each sample solution ÷ Total protein concentration

[0022] [L] represents the concentration of free small molecules in each sample solution, where [L] = total concentration of small molecules in each sample solution - concentration of transmembrane small molecules in each sample solution.

[0023] K: The binding constant between proteins and small molecules.

[0024] N: The number of binding sites between proteins and small molecules.

[0025] Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

[0026] In this invention, "transmembrane small molecules" refers to small molecules that pass through the separation membrane of the multi-chamber electrophoresis separation device from the sample chamber to the receiving chamber.

[0027] In some embodiments, m can be 3-15, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0028] In some embodiments, before calculating the binding constant K, the method of the present invention further includes: testing the optimal migration rate of the protein; and calculating the binding constant K between the protein and the small molecule based on the Scatchard model, the optimal migration rate of the protein, the total concentration of the protein, the total concentration of the small molecule in each sample solution, and the concentration of the transmembrane small molecule in each sample solution.

[0029] The equations for the Scatchard model are shown below:

[0030] n / [L]=-nK+KN

[0031] in,

[0032] n = Concentration of transmembrane small molecules in each sample solution ÷ (Total protein concentration × Optimal protein migration rate),

[0033] [L] represents the concentration of free small molecules in each sample solution, where [L] = total concentration of small molecules in each sample solution - concentration of transmembrane small molecules in each sample solution.

[0034] K: The binding constant between proteins and small molecules.

[0035] N: The number of binding sites between proteins and small molecules.

[0036] Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

[0037] To further improve the accuracy of binding constant determination, this invention conducted a protein migration rate optimization experiment, obtained the optimal protein migration rate, and multiplied the total protein concentration by the optimal protein migration rate to obtain the total amount of protein migrating across the membrane. This eliminated the measurement error caused by some proteins being unable to migrate across the membrane, thus improving the accuracy of the measurement.

[0038] By adjusting the pH of the buffer solution, the electrophoresis voltage, and the electrophoresis time, a migration rate greater than or equal to 90% is achieved. This migration rate can be considered the optimal migration rate for subsequent determination of the binding constant. The main purpose of using the optimal migration rate is to ensure the accuracy of the determination.

[0039] In some embodiments, the applied voltage for the multi-compartment electrophoresis separation method can be 8-100V, for example, 8V, 10V, 15V, 20V, 25V, 30V, 35V, 40V, 45V, 50V, 55V, 60V, 65V, 70V, 75V, 80V, 85V, 90V, 95V, or 100V, preferably 8-60V. Multi-compartment electrophoresis uses relatively low voltage, resulting in high safety. The voltage for multi-compartment electrophoresis can be selected according to the type of protein. Excessive voltage will generate Joule heating during electrophoresis, leading to an increase in system temperature, which may cause changes in the protein or even inactivation. Furthermore, since the binding constant is a function of temperature (temperature affects the magnitude of the binding constant), excessively high temperatures will result in inaccurate measurements of the binding constant (generally, the temperature for determining the equilibrium constant between proteins and small molecules is less than 37 degrees Celsius).

[0040] In some embodiments, the electrophoresis time of the multi-compartment electrophoresis separation method can be less than 20 minutes, for example, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes, preferably 2-20 minutes. The separation time of the present invention is very short, and separation can be completed even within a few minutes. The shortened separation time saves the total time of the entire determination process, thereby improving the testing efficiency.

[0041] In some embodiments, the pH of the buffer solution may be 4-9, for example, 4, 5, 6, 7, 8, or 9. Controlling the pH of the buffer solution within the above range is beneficial for maximally restoring the physiological structure of proteins and small drug molecules, and improving the accuracy of binding constant determination.

[0042] In some embodiments, the protein is trypsin, human serum albumin, bovine serum albumin, or α-glucosidase, etc. Of course, the proteins used in this application are not limited to these listed proteins. The method of this application is universal and can determine the binding constant of any protein to a small molecule, without particularly limiting the type of protein.

[0043] In some embodiments, the small molecule is a drug small molecule, which may be matrine, indomethacin, baicalin, or wogonin, etc.

[0044] In some embodiments, the dissociation agent is one or more selected from methanol, ethanol, acetone, formaldehyde, and isopropanol. Of course, the small molecules used in this application are not limited to those listed. The method of this application is universal and can determine the binding constant of any small molecule to a protein, without particular limitation on the type of small molecule.

[0045] When the protein is trypsin, the concentration of the protein in the sample solution can be 70-80 μmol / L. -1 For example, 70 μmol L -1 71 μmol L -1 72 μmol L -1 73 μmol L -1 74 μmol L -1 75 μmol L -1 75.3 μmol / L -1 76 μmol L -1 77 μmol L -1 78 μmol L -1 79 μmol L -1 Or 80 μmol L -1 .

[0046] When the small molecule is matrine, the concentration of matrine in the sample solution can be 20-1250 μmol / L. -1 40-810 μmol L can be selected. -1 For example, it could be 20.13 μmol L. -1 40.26 μmol L -1 120.79 μmol L -1 201.31 μmol L -1 301.97 μmol L -1 402.63 μmol L -1 483.15 μmol L -1 603.94 μmol L -1 805.25 μmol L -1 Or 1207.88 μmol L -1 When calculating the binding constant using the Scatchard model, a series of sample solutions are required, with identical protein solutions but varying small molecule concentrations. The concentration of matrine should be controlled within the aforementioned range. A standard curve for matrine (e.g., ...) is needed. Figure 6 The linear relationship is good, and the curve equation is Y = 366X + 29538, R 2 =0.9950, where X is the concentration of matrine, Y is the chromatographic peak area, and R... 2The correlation coefficient is generally required to be greater than 0.99. The closer the value is to 1, the higher the linear correlation between the two variables. In other words, the more accurate the concentration calculated based on this standard curve is.

[0047] Determining the binding constants of trypsin and matrine within the above-mentioned protein and matrine concentration ranges is beneficial for improving test accuracy.

[0048] When the protein is trypsin, the applied voltage for the multi-compartment electrophoresis separation method can be 9-12V (e.g., 9V, 10V, 11V, or 12V), and the time can be 8-15min (e.g., 8min, 9min, 10min, 11min, 12min, 13min, 14min, or 15min). The pH of the buffer solution can be 7.0-8.2 (e.g., 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, or 8.2). Under these conditions, trypsin exhibits optimal migration. Performing multi-compartment electrophoresis under these conditions maximizes protein transmembrane migration and improves the accuracy of binding constant determination.

[0049] When the protein is human serum albumin, the applied voltage for the multi-compartment electrophoresis separation method can be 58-62V (e.g., 58V, 59V, 60V, 61V, or 62V), and the time can be 8-12min (e.g., 8min, 9min, 10min, 11min, or 12min). The pH of the buffer solution can be 7.8-8.2 (e.g., 7.8, 7.9, 8.0, 8.1, or 8.2). Under these conditions, human serum albumin exhibits optimal migration. Performing multi-compartment electrophoresis under these conditions maximizes protein transmembrane migration and improves the accuracy of binding constant determination.

[0050] In some embodiments, the separation is performed in a multi-chamber electrophoretic separation apparatus. The separation membrane of the multi-chamber electrophoretic separation apparatus is a dual-channel separation membrane. The dual-channel separation membrane comprises a dual-channel base membrane and a hydrophobic material. The channels of the dual-channel base membrane have a through-pore structure. The hydrophobic material is modified on the upper and lower surfaces and the surface of the channels of the dual-channel base membrane.

[0051] This invention utilizes a hydrophobic solution to modify a double-pass membrane, giving its upper and lower surfaces and pore surfaces a low surface free energy similar to that of the hydrophobic substance. This reduces protein adsorption on the membrane surface and pores, thereby decreasing protein residue, increasing protein permeability, and ultimately improving protein migration. In the determination of the binding constant between proteins and small drug molecules, protein migration directly affects the accuracy of the determination. This invention, by using a hydrophobic double-pass membrane, improves protein migration, thus enhancing the accuracy of binding constant determination.

[0052] The dual-channel base film of the present invention is a dual-channel film, characterized by: short-range highly ordered pore arrangement, all pores being through-hole structures, non-intersecting internal pores with uniform diameter, adjustable from tens to hundreds of nanometers.

[0053] In some embodiments, the dual-passage base film may be a porous alumina (AAO) dual-passage film. The porous alumina dual-passage film is made of Al2O3, possessing high-temperature resistance and hydrophilic properties. The pore size of the porous alumina film can be 200-300 nm (e.g., 200 nm, 210 nm, 220 nm, 230 nm, 240 nm, 250 nm, 260 nm, 270 nm, 280 nm, 290 nm, or 300 nm), the pore spacing can be 400-500 nm (e.g., 400 nm, 410 nm, 420 nm, 430 nm, 440 nm, 450 nm, 460 nm, 470 nm, 480 nm, 490 nm, or 500 nm), and the thickness can be 40-60 μm (e.g., 40 μm, 45 μm, 50 μm, 55 μm, or 60 μm). Compared with existing cellulose acetate membranes used in multi-chamber electrophoresis technology (such as...) Figure 1 Compared to (as shown), the AAO membrane pore structure (such as...) Figure 2 (As shown) makes it easier for molecules to pass through. Multi-compartment electrophoresis using an AAO double-pass membrane as the protein separation membrane can efficiently reduce the residual amount of protein-drug small molecule complexes on the membrane, thereby improving the accuracy of measuring protein migration and binding constants.

[0054] When using cellulose acetate membranes as separation membranes to determine binding constants, the membranes' multilayered, interlaced arrangement, varying pore sizes, and hydrophilicity lead to high protein residue and reduced accuracy. Furthermore, cellulose acetate membranes are limited by pH and temperature, making them non-reusable. In contrast, the hydrophobic AAO dual-channel membrane used in this invention features a short-range, highly ordered, through-pore structure with no internal cross-linking, tightly packed hexagonal arrangement, and uniform pore size adjustable from tens to hundreds of nanometers. Compared to cellulose acetate membranes, its straight-channel structure results in lower molecular resistance, facilitating protein passage, reducing membrane residue, and increasing protein migration, thus improving the accuracy of binding constant determination. It also eliminates the need for soaking, saving time; is unaffected by experimental temperature and pH; and is reusable.

[0055] In some embodiments, the hydrophobic material may be one or more of polytetrafluoroethylene, polydimethylsiloxane, polymethyl methacrylate, polystyrene, polypropylene, and polyvinyl chloride.

[0056] For example, polytetrafluoroethylene (PTFE) has a very low surface free energy, resulting in low adhesion and a low coefficient of friction, making it a commonly used hydrophobic modification material. This invention combines the ease of surface modification of PTFE and AAO double-pass membranes, and evaluates the hydrophobic state and separation performance of the modified AAO membrane, laying a solid theoretical foundation for improving membrane separation performance.

[0057] In some embodiments, the contact angle of the dual-channel separation membrane can be 50°-120°, for example, 50°, 60°, 70°, 80°, 90°, 100°, 110°, or 120°, preferably 90°-120°. By controlling the contact angle of the dual-channel separation membrane within the above range, this invention helps to reduce the amount of protein residue on the membrane and improve protein migration. When the contact angle is too small, the hydrophobicity of the dual-channel separation membrane is poor, resulting in strong protein adsorption and a high amount of protein residue on the membrane. When the contact angle is too large, the hydrophobicity of the dual-channel separation membrane is too high, causing the solution to mainly form spherical shapes in the chamber, affecting the uniformity of protein stress and making it difficult for the solution to uniformly fill the chamber, which is detrimental to protein migration.

[0058] The above-mentioned dual-pass separation membrane can be prepared by a method including the following steps:

[0059] The double-channel base membrane is immersed in a hydrophobic substance solution, wherein the channels of the double-channel base membrane are through-pore structures;

[0060] After removing the double-channel base film, the hydrophobic substance solution is dropped onto it and allowed to flow through the channels to reach the bottom of the double-channel base film, thereby making full contact with the channel surface;

[0061] By heating, the hydrophobic material is fixed on the upper and lower surfaces and the pore surface of the double-channel base membrane to obtain a hydrophobic double-channel separation membrane.

[0062] The separation membrane obtained by the method of the present invention has a low protein residue, and the method is simple to operate, low in cost, and suitable for large-scale industrial production.

[0063] In some embodiments, the soaking includes: immersing the dual-pass base membrane in the hydrophobic substance solution for 10-20 minutes (e.g., 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min), then turning it over and soaking it again for 10-20 minutes (e.g., 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min, or 20 min). Optionally, during the soaking process, the hydrophobic substance solution is stirred every few minutes (e.g., every 4-6 minutes, e.g., 5 minutes) to ensure uniform solute distribution in the solution.

[0064] In some embodiments, the hydrophobic substance solution is a polytetrafluoroethylene (PTFE) solution. The preparation of the PTFE solution includes: shaking the purchased PTFE solution well and diluting it with water until it reaches a non-viscous state, avoiding clogging the pores of the dual-channel base film.

[0065] In some embodiments, the mass percentage concentration of the hydrophobic substance solution can be 0.004%-0.04%, for example, 0.004%, 0.006%, 0.008%, 0.01%, 0.015%, 0.02%, 0.025%, 0.03%, 0.035%, or 0.04%, preferably 0.01%-0.04%. Controlling the concentration of the hydrophobic substance solution within the above range is beneficial for optimizing the contact angle of the dual-channel separation membrane. If the concentration is too low, the improvement in the contact angle of the dual-channel base membrane is limited, resulting in poor hydrophobicity of the dual-channel base membrane. If the concentration is too high, there is significant polymer on the surface and cross-section of the dual-channel base membrane, the polymer distribution is uneven, and soaking the dual-channel base membrane at high concentrations can also cause polymer blockage of the pores, affecting protein passage.

[0066] In some embodiments, the heating temperature is greater than the melting point of the hydrophobic material and less than its thermal decomposition temperature. At the heating temperature, the hydrophobic material melts and solidifies, fixing itself onto the upper and lower surfaces and the pore surfaces of the dual-channel base film. Excessively high temperatures can cause thermal decomposition of the hydrophobic material, while excessively low temperatures prevent it from fully melting and solidifying, resulting in poor fixation.

[0067] In some specific embodiments, the heating temperature can be 350-400℃, for example, 350℃, 355℃, 360℃, 365℃, 370℃, 375℃, 380℃, 385℃, 390℃, 395℃, or 400℃. The heating time can be 20-40 minutes, for example, 20 minutes, 22 minutes, 24 minutes, 26 minutes, 28 minutes, 30 minutes, 32 minutes, 34 minutes, 36 minutes, 38 minutes, or 40 minutes.

[0068] In some specific embodiments, the heating is performed in a muffle furnace. Specifically, after the hydrophobic substance solution flows through the channels to the area below the double-pass base film, the double-pass base film is placed on a tin foil support and positioned horizontally to prevent uneven modification.

[0069] In some embodiments, the hydrophobic solution can be drawn through the pores and reach the underside of the double-pass membrane by vacuum extraction or filtration, ensuring that the pores are modified with solution. Since the pores of the double-pass membrane are very narrow, it is difficult for the hydrophobic solution to flow through the pores using only gravity. This invention utilizes vacuum extraction and filtration to effectively draw the hydrophobic solution through the pores and reach the underside of the double-pass membrane. During this process, it can be clearly observed that the liquid flows from above the membrane through the pores to below the membrane.

[0070] In some specific embodiments, the filtration time can be 10-20 min, for example, 10 min, 11 min, 12 min, 13 min, 14 min, 15 min, 16 min, 17 min, 18 min, 19 min or 20 min.

[0071] In some specific embodiments, the dual-channel base film is placed in a vacuum oven for vacuum extraction. The vacuum extraction time can be 10-20 minutes, for example, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, or 20 minutes.

[0072] In some embodiments, after the vacuum extraction or filtration is completed, the double-pass membrane is flipped over, and the hydrophobic substance solution is repeatedly dropped onto the double-pass membrane. Then, vacuum extraction or filtration is performed to allow the hydrophobic substance solution to flow through the pores, thereby ensuring sufficient contact with the pore surface. Repeatedly flowing the hydrophobic substance solution through the pores helps to better modify the pore surface, improve its hydrophobicity, and reduce protein adsorption on the pore surface.

[0073] In some embodiments, the volume of the hydrophobic substance solution dropped onto the double-channel base membrane can be determined according to the size of the double-channel base membrane, as long as it can cover the entire upper surface of the double-channel base membrane to ensure that the solution flows through all channels.

[0074] In some embodiments, the multi-chamber electrophoretic separation device includes a positive receiving chamber, a sample chamber, and a negative receiving chamber, wherein the sample chamber is separated from the positive receiving chamber and the sample chamber is separated from the negative receiving chamber by a separation membrane.

[0075] The separation includes: adding the incubated sample solution to the sample chamber, adding the buffer solution to the positive receiving chamber and the negative receiving chamber respectively; performing electrophoresis to allow the protein-small molecule complex to migrate through the separation membrane to the positive receiving chamber or the negative receiving chamber; and removing the solution in the positive receiving chamber or the negative receiving chamber containing the migrated protein-small molecule complex.

[0076] The dissociation includes: if the protein cannot precipitate in the dissociation solution, drying the taken solution to obtain a protein-small molecule complex solid; adding the dissociation agent to dissociate; centrifuging the dissociation solution after standing, and taking the supernatant; or, if the protein can precipitate in the dissociation solution, adding the dissociation agent to the taken solution to dissociate, centrifuging the dissociation solution after standing, and taking the supernatant.

[0077] The detection includes: using HPLC-MS / MS to detect the concentration of the small molecules in the supernatant, which is denoted as the transmembrane small molecule concentration.

[0078] In some embodiments, the method of the present invention further includes:

[0079] Prepare m control solutions containing the small molecule using the buffer solution, where m ≥ 3;

[0080] The control solution is subjected to the same treatment process as the sample solution.

[0081] The concentration of the small molecules in the resulting dissociated solution is recorded as the concentration of the diffused small molecules;

[0082] Based on the Scatchard model, the optimal protein migration rate, the total protein concentration, the total concentration of small molecules in each sample solution, the concentration of transmembrane small molecules in each sample solution, and the concentration of diffusing small molecules in each control solution, the binding constant K between the protein and the small molecules is calculated.

[0083] The equations for the Scatchard model are shown below:

[0084] n / [L]-nK+KN

[0085] in,

[0086] n = (concentration of transmembrane small molecules in each sample solution - concentration of diffusing small molecules in each control solution) ÷ (total protein concentration × optimal protein migration rate),

[0087] [L] represents the concentration of free small molecules in each sample solution, where [L] = total concentration of small molecules in each sample solution - (concentration of transmembrane small molecules in each sample solution - concentration of diffusing small molecules in each control solution).

[0088] K: The binding constant between proteins and small molecules.

[0089] N: The number of binding sites between proteins and small molecules.

[0090] Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

[0091] To further improve the accuracy of binding constant determination, this invention designs a control solution. The control solution contains no protein, and the concentration of small molecules in m portions of the control solution corresponds one-to-one with the concentration of small molecules in m portions of the sample solution. This invention measures the concentration of small molecules that cross the membrane to reach the receiving chamber through free diffusion. The concentration of small molecules across the membrane minus the concentration of diffused small molecules equals the concentration of small molecules actually bound to the protein (referred to as the bound small molecule concentration). By eliminating the influence of freely diffused small molecules, the accuracy of binding constant determination can be improved.

[0092] In some embodiments, m can be 3-15, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0093] refer to Figure 3-4The multi-chamber electrophoretic separation device used in this invention typically includes a buffer solution circulation system 1, a separation system 3, a condensation system 4 for cooling the separation system, and a power supply system 5. The separation system 3 consists of five chambers in sequence: a positive electrode chamber 7, a positive receiving chamber 8, a sample chamber 9, a negative receiving chamber 10, and a negative electrode chamber 11. The sample chamber 9 is separated from the positive receiving chamber 8 by a double-pass separation membrane 12, and the sample chamber 9 is separated from the negative receiving chamber 10 by a retention membrane 13. A buffer solution inlet 15 is located on the lower side of each of the positive and negative electrode chambers 7 and 11, and a buffer solution outlet 16 is located on the upper side of the corresponding side for buffer solution circulation. A platinum positive electrode 14a and a platinum negative electrode 14b, connected to the power supply system, are respectively placed in the positive electrode chamber 7 and the negative electrode chamber 11. Each of the positive electrode chamber 7 and the negative electrode chamber 11 has an organic glass clamp 6 on its outer side, and the two organic glass clamps 6 are connected by screws 17 on both sides and fixed by nuts 18. The buffer solution circulation system 1 consists of a container containing buffer solution, circulation pipes, and a pump 2 installed on the pipes, and is connected to the buffer solution inlet 15 and buffer solution outlet 16 of the separation system 3 to form a loop. The multi-chamber electrophoretic separation device used in this invention is described in detail in the applicant's previous patent application CN201410611855.8, and the disclosure of the multi-chamber electrophoretic separation device in patent CN201410611855.8 can be referred to.

[0094] Retention membranes (ultrafiltration membranes) can be used to retain biomolecules and small molecules, allowing them to remain in the positive and negative receiving chambers. Dual-pass separation membranes allow charged proteins to pass through. By adjusting the pH of the system, the applied voltage, the electrode distance, the membrane thickness, and the pore size, transmembrane migration of biomolecules can be achieved, while simultaneously carrying small molecules to migrate.

[0095] In some embodiments, a flowchart for determining the binding constant of proteins and small molecules is shown below. Figure 5 As shown.

[0096] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0097] 1. This invention utilizes the chamber membrane separation concept of multi-chamber electrophoresis technology to propose a novel method for determining the binding constant. This method enables the determination of the binding constant of proteins and drug molecules under simulated physiological conditions, providing new insights for the study of the mechanism of action of drug molecules in vivo and their clinical applications. The binding constant determination method of this invention has the advantages of high efficiency and high accuracy.

[0098] 2. This invention utilizes a hydrophobic double-pass separation membrane to separate protein-small molecule complexes, reducing the amount of protein residue on the membrane, increasing protein permeability, thereby improving protein migration rate and the accuracy of binding constant determination. Attached Figure Description

[0099] Figure 1 This is a SEM image of a cellulose acetate membrane.

[0100] Figure 2 This is a SEM image of the AAO dual-channel base film.

[0101] Figure 3 This is a schematic diagram of a multi-chamber electrophoretic separation device.

[0102] Figure 4 This is a schematic diagram of the separation system in a multi-chamber electrophoretic separation device.

[0103] Figure 5 This is a flowchart illustrating the determination of the binding constants of proteins and small molecules according to an embodiment of the present invention.

[0104] Figure 6 This is a standard curve diagram of the drug molecule matrine.

[0105] Figure 7 SEM images of the surface and cross-section of the double-pass base film before and after modification with different concentrations of PTFE (scale bar in the figure is 3 μm): (a) Unmodified; (b) 40% PTFE modified (Comparative Example 1); (c) 4% PTFE modified (Comparative Example 2); (d) 0.04% PTFE modified (Example 2); (e) 0.02% PTFE modified (Example 1); (f) 0.004% PTFE modified (Example 3).

[0106] Figure 8 SEM images of the surface and cross-section of the double-pass base film before and after modification with different concentrations of PTFE (scale bar in the figure is 10 μm): (a) Unmodified; (b) 40% PTFE modified (Comparative Example 1); (c) 4% PTFE modified (Comparative Example 2); (d) 0.04% PTFE modified (Example 2); (e) 0.02% PTFE modified (Example 1); (f) 0.004% PTFE modified (Example 3).

[0107] Figure 9 Infrared spectra of double-pass base films before and after modification with different concentrations of PTFE.

[0108] Figure 10 Surface mapping diagrams of the double-pass base film before and after modification with different concentrations of PTFE: (a, a') unmodified; (b, b') 40% PTFE modification (Comparative Example 1); (c, c') 4% PTFE modification (Comparative Example 2); (d, d') 0.04% PTFE modification (Example 2); (e, e') 0.02% PTFE modification (Example 1); (f, f') 0.004% PTFE modification (Example 3).

[0109] Figure 11 Cross-sectional mapping diagrams of the double-pass base film before and after modification with different concentrations of PTFE: (a, a') unmodified; (b, b') 40% PTFE modification (Comparative Example 1); (c, c') 4% PTFE modification (Comparative Example 2); (d, d') 0.04% PTFE modification (Example 2); (e, e') 0.02% PTFE modification (Example 1); (f, f') 0.004% PTFE modification (Example 3).

[0110] Figure 12 The contact angles of the double-pass base film before and after modification with different concentrations of PTFE are shown: (a) unmodified; (b) 0.02% PTFE modified (Example 1); (c) 0.004% PTFE modified (Example 3); (d) 0.04% PTFE modified (Example 2).

[0111] Figure 13 The protein migration rates of the double-pass membrane before and after modification with different concentrations of PTFE are shown.

[0112] Figure 14 The images show the hydrophobic state of the AAO double-channel base film before and after hydrophobic modification, in a water droplet experiment (left) and an immersion experiment (right).

[0113] Figure 15 SEM images of the surface and cross-section of the dual-channel separation membrane of Example 1 before and after use (scale bar is 3 μm): (a) before use; (b) after use.

[0114] Figure 16 Mapping diagrams of the surface and cross-section of the dual-channel separation membrane in Example 1 before and after use.

[0115] Figure 17 To characterize the contact angle of the AAO dual-channel base membrane before and after hydrophobic modification in a dry state: (a) AAO dual-channel base membrane before modification, (b) dual-channel separation membrane of Example 1.

[0116] Figure 18 To characterize the contact angle of the AAO dual-channel base membrane before and after hydrophobic modification in a wetted state: (a) AAO dual-channel base membrane before modification, (b) dual-channel separation membrane of Example 1.

[0117] Figure 19 This is a comparison chart showing the residual amounts of protein on the cellulose acetate membrane and the double-pass separation membrane of Example 1 after electrophoresis.

[0118] Figure 20 This is a comparison of RSDs of electrophoresis performed using the same cellulose acetate membrane and the same double-pass separation membrane from Example 1.

[0119] Figure 21 RSD comparison diagrams of electrophoresis using different cellulose acetate membranes and different double-pass separation membranes of Example 1.

[0120] Figure 22 Comparison of protein migration rates under different electrophoresis conditions: (a) pH of buffer solution; (b) voltage; (c) electrophoresis time.

[0121] Figure 23 Scatchard curves for determining the binding constants of trypsin and matrine using multicompartment electrophoresis.

[0122] Figure 24 The images show the fluorescence spectra of trypsin at different drug concentrations.

[0123] Figure 25 Scatchard curves for determining the binding constant of trypsin and matrine by fluorescence method.

[0124] Figure 26 This is a standard curve diagram of the drug molecule indomethacin.

[0125] Figure 27 Scatchard curves for determining the binding constant of human serum albumin to indomethacin using multicompartment electrophoresis.

[0126] Figure 28 The fluorescence spectra of human serum albumin at different drug concentrations are shown.

[0127] Figure 29 Scatchard curve for determining the binding constant of human serum albumin to indomethacin using a fluorescence method.

[0128] Explanation of reference numerals in the attached figures

[0129] 1. Buffer solution circulation system; 2. Pump; 3. Separation system; 4. Condensation system; 5. Power supply system; 6. Acrylic glass clamp; 7. Positive electrode chamber; 8. Positive receiving chamber; 9. Sample chamber; 10. Negative receiving chamber; 11. Negative electrode chamber; 12. Dual-pass separation membrane; 13. Retention membrane; 14a. Platinum sheet positive electrode; 14b. Platinum sheet negative electrode; 15. Buffer solution inlet; 16. Buffer solution outlet; 17. Screw; 18. Nut. Detailed Implementation

[0130] To make the technical problem to be solved, the technical solution, and the beneficial effects of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are merely for explaining the present invention and are not intended to limit the present invention.

[0131] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained commercially or through existing methods; unless otherwise specified, the amounts of experimental reagents used are the amounts used in conventional experimental operations; unless otherwise specified, the experimental methods are all conventional methods.

[0132] In this embodiment, the migration rate can be calculated using the formula: Protein migration rate = Solution peak area in the protein receiving chamber after electrophoresis ÷ Solution peak area in the sample chamber before electrophoresis. Proteins carrying a positive charge will migrate to the negative receiving chamber under the influence of an electric field; proteins carrying a negative charge will migrate to the positive receiving chamber under the influence of an electric field.

[0133] Preparation of dual-pass separation membrane

[0134] Example 1

[0135] The experimental flowchart based on the hydrophobic modification of AAO double-pass membrane with polytetrafluoroethylene solution is as follows: Figure 6 As shown.

[0136] First, shake the purchased polytetrafluoroethylene (PTFE) solution well and dilute it with ultrapure water in a gradient to 30%, 15%, 5%, 0.5%, and 0.02% (mass percentage concentration) to dilute the solution to a non-viscous state and avoid clogging the pores. Add 20 mL of 0.02% PTFE solution to a petri dish. Use tweezers to place the purchased AAO double-pass membrane (i.e., double-pass base membrane) into the petri dish containing the 0.02% PTFE solution. Immerse for 15 minutes, then turn it over and immerse it again for 15 minutes to ensure that the membrane surface is fully in contact with the PTFE solution. During the immersion process, stir the liquid in the petri dish every 5 minutes to prevent uneven distribution of solute in the solution. The soaked AAO double-pass membrane was removed and placed on a support in a vacuum oven, allowing it to float vertically. 400 μL of polytetrafluoroethylene (PTFE) solution was dropped onto the membrane, and vacuum was applied for 15 minutes. This vacuum process allowed the liquid to flow through the pores, ensuring full contact between the membrane pores and the PTFE solution. After 15 minutes, the membrane was flipped over, and another 400 μL of PTFE solution was dropped onto it. Vacuum was then applied for another 15 minutes. During this process, the liquid was clearly observed flowing from the top of the membrane through the pores to the bottom. After vacuuming, the AAO double-pass membrane was removed and placed on a horizontal support to prevent uneven modification. It was then placed in a muffle furnace for high-temperature curing at 380°C for 30 minutes to fix the PTFE solution onto the membrane and pore surfaces, thus obtaining the hydrophobic AAO double-pass separation membrane (Hydrophobic-AAO).

[0137] Example 2

[0138] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.04%.

[0139] Example 3

[0140] The hydrophobic AAO dual-pass separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.004%.

[0141] Example 4

[0142] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 0.01%.

[0143] Example 5

[0144] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 350°C.

[0145] Example 6

[0146] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 400°C.

[0147] Comparative Example 1

[0148] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 40%.

[0149] Comparative Example 2

[0150] The hydrophobic AAO dual-pass separation membrane was prepared according to the method described in Example 1, except that the concentration of the polytetrafluoroethylene solution added to the petri dish was 4%.

[0151] Comparative Example 3

[0152] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 300°C.

[0153] Comparative Example 4

[0154] The hydrophobic AAO dual-channel separation membrane was prepared according to the method described in Example 1, except that the high-temperature curing temperature was 500°C.

[0155] Characterization analysis of dual-pass separation membrane

[0156] 1. Characterization of double-pass base films before and after modification with different concentrations of PTFE

[0157] The surfaces and cross sections of the unmodified double-pass base membrane and the double-pass separation membranes prepared in Examples 1-3 and Comparative Examples 1-2 were characterized by scanning electron microscopy (SEM), X-ray energy dispersive spectroscopy (EDS), Fourier transform infrared spectroscopy (FT-IR), and contact angle.

[0158] (1) SEM characterization

[0159] From the 3μm scale ( Figure 7 It was clearly observed that, compared with the unmodified double-pass membrane, the double-pass membrane modified with high concentrations (40%, 4% PTFE) showed obvious polymers on both the surface and cross-section. Furthermore, at higher concentrations, the polymers easily blocked the pores, affecting protein passage. In contrast, the double-pass membranes modified with low concentrations (0.04%, 0.02%, 0.004% PTFE) showed no obvious polymers on the surface. Compared with the unmodified double-pass membrane, the surface smoothness was slightly altered, but no obvious polymers were observed, and the cross-sectional pores remained unobstructed without significant blockage.

[0160] From a 10μm scale ( Figure 8 Observing the surface and cross-section of the membrane, compared with the unmodified dual-channel base membrane, the polymer distribution on the surface and cross-section of the AAO membrane modified with high concentration (40%, 4% PTFE) was uneven, while no obvious polymer was observed on the surface of the dual-channel base membrane modified with low concentration (0.04%, 0.02%, 0.004% PTFE).

[0161] (2) FT-IR characterization

[0162] Compared with the unmodified double-pass film, the infrared spectrum of the modified double-pass film ( Figure 9 The presence of absorption peaks at 1215 cm⁻¹ and 1155 cm⁻¹, which are -CF₂ stretching vibration peaks, confirms successful modification of polytetrafluoroethylene (PTFE). As the modification concentration decreases from high (40%, 4%) PTFE to low (0.04%, 0.02%, 0.004%) PTFE, the two characteristic absorption peaks gradually become rounded and turn into single peaks. This may be because as the concentration of PTFE decreases, the vibrational freedom of PTFE molecules is significantly reduced due to the influence of the double-pass membrane substrate.

[0163] (3) EDS characterization

[0164] The changes in F and Al elemental content on the membrane surface and cross-section were analyzed, and the results are as follows: Figure 10 , Figure 11 As shown.

[0165] from Figure 10 It is evident that as the modification concentration decreases, the content of F and Al elements on the membrane surface gradually increases.

[0166] Figure 11 The results showed that as the modification concentration decreased, the Al content in the membrane cross section gradually increased, while the F content gradually decreased. Moreover, the F content in the cross section was higher than that on the membrane surface, proving that the modification effect inside the pores was better than that on the membrane surface. The hydrophobicity inside the membrane pores was greater than that on the membrane surface, making it less likely for proteins to remain inside the pores.

[0167] according to Figure 10 and 11 and combined with characterization Figure 7-8 Under high concentration (40%, 4% PTFE) modification, the distribution of fluorine element is uneven, and a large number of pores are blocked, making it difficult for substances to pass through. However, under low concentration (0.04%, 0.02%, 0.004% PTFE) modification, the distribution of fluorine element is more uniform. Therefore, modification is preferably carried out under low concentration (0.04%, 0.02%, 0.004% PTFE).

[0168] (4) Contact angle characterization

[0169] In a dry state, the contact angles of the double-pass base film before and after modification with different concentrations of PTFE are as follows: Figure 12 As shown.

[0170] Hydrophobic modification of the double-pass membrane was performed using PTFE solutions at concentrations of 0.004%, 0.01%, 0.02%, and 0.04%. Under dry conditions, the contact angles of the resulting double-pass membranes were 51.2°, 100.3°, 111.1°, and 117.3°, respectively. Compared to the contact angle of the unmodified double-pass membrane (29.3°), these angles increased by 21.9°, 71°, 81.8°, and 88°, respectively. The differences in hydrophobicity resulted in different protein migration rates, consistent with the following values ​​under PTFE concentrations: 28.08%, 50.60%, 61.43%, and 52.80%, respectively. It can be seen that as the contact angle increases, the protein migration rate first increases and then decreases, that is, the amount of protein residue on the membrane first decreases and then increases.

[0171] 2. Optimization of the concentration of hydrophobic modification of polytetrafluoroethylene

[0172] The protein migration rates of the double-pass membrane before and after modification with polytetrafluoroethylene (PTFE) solutions of different concentrations were measured, and the results are as follows: Figure 13 As shown.

[0173] Compared to the unmodified dual-channel membrane, the protein migration rate of the dual-channel separation membrane of the present invention is significantly improved. Both excessively high and low concentrations of the modification solution have a significant impact on protein migration rate. When the modification solution concentration is too low, polytetrafluoroethylene (PTFE) is not uniformly modified onto the membrane surface and within the pores, resulting in poor hydrophobicity. When the modification solution concentration is too high, excessive PTFE deposits on the membrane surface and within the pores, causing blockage and hindering material passage. Therefore, both excessively high and low modification concentrations affect the improvement of protein migration rate. When the PTFE solution concentration is 0.02%, the protein migration rate reaches a maximum of 61.43%. The protein migration rates of the unmodified dual-channel membrane and the dual-channel membranes modified with 0.004% PTFE, 0.01% PTFE, and 0.04% PTFE are 23.53%, 28.08%, 50.60%, and 52.80%, respectively. Therefore, the most preferred modification concentration is 0.02%.

[0174] 3. Temperature optimization during high-temperature curing

[0175] The contact angle and migration rate of the dual-channel separation membrane after different curing temperatures were measured, as shown in Table 1 below.

[0176] Table 1

[0177] Example 1 380℃ 111.1° 61.43％ Example 5 350℃ 105° 58％ Example 6 400℃ 95° 60％ Comparative Example 3 300℃ 80° 35％ Comparative Example 4 500℃ 45° 30％

[0178] As can be seen from Table 1, both excessively high and excessively low curing temperatures are detrimental to improving the migration rate.

[0179] Verification of the hydrophobic effect of the dual-channel separation membrane

[0180] To further verify the hydrophobic effect of the AAO dual-channel base membrane modification and ensure low protein residue, drop and immersion experiments were conducted before and after the AAO dual-channel base membrane modification, and the dual-channel separation membrane was characterized by SEM and EDS before and after use.

[0181] (1) A water droplet test was performed on the AAO double-channel base film before and after hydrophobic modification. Figure 14 (Left) and immersion test ( Figure 14 (Right) Observe the hydrophobic state of the membrane.

[0182] Droplet test: After adding buffer solution to the membrane surface and letting it stand for 30 minutes, it can be clearly observed that, compared with before modification, the droplets on the surface of the dual-channel separation membrane in Example 1 are spherical, while the droplets on the surface of the unmodified AAO dual-channel base membrane are scattered.

[0183] Hydrophobicity test: Unmodified and modified AAO dual-channel membranes were placed in a buffer solution and allowed to stand for 30 minutes. The dual-channel separation membrane of Example 1 floated on the liquid surface, while the unmodified AAO dual-channel membrane sank to the bottom of the liquid. This is because polytetrafluoroethylene has a smaller surface free energy, while water has a larger surface free energy and cannot remain on a surface with a smaller surface free energy, thus exhibiting hydrophobic properties.

[0184] (2) SEM characterization of the dual-channel separation membrane before and after use

[0185] The surface and cross-section of the dual-channel separation membrane of Example 1 before and after use were characterized by SEM. Figure 15 It can be observed that after 20 uses, the dual-channel separation membrane showed no obvious pore blockage, no significant change in pore cross-section, and the pores were smooth and unobstructed with no residue. This proves that the dual-channel separation membrane of the present invention can be reused multiple times.

[0186] (3) EDS characterization of the dual-channel separation membrane before and after use

[0187] The surface and cross-section of the dual-channel separation membrane from Example 1 before and after use were characterized by EDS to investigate the changes in F and Al elemental content on the membrane surface and cross-section. Figure 16 Compared with the original dual-channel separation membrane, the F element content on the surface and cross-section of the dual-channel separation membrane after 20 uses was slightly reduced, while the Al element content did not change significantly, proving that the dual-channel separation membrane of the present invention can be reused.

[0188] (4) Contact angle characterization experiment:

[0189] The contact angles of the AAO dual-channel base film surface before and after hydrophobic modification were measured under both dry and wet conditions. Measurement parameters: measurement range 0–180°, measurement accuracy ±0.1°, resolution ±0.01°.

[0190] In a dry state ( Figure 17 The contact angle of the unmodified AAO dual-channel membrane was 29.3°, while the contact angle of the dual-channel separation membrane in Example 1 was 111.1°. This is because the surface free energy of the AAO dual-channel membrane was reduced after polytetrafluoroethylene was applied to the membrane surface, preventing the solution from spreading on the surface with low surface energy, thus exhibiting hydrophobic properties. This proves that the hydrophobic modification effect of the dual-channel separation membrane in Example 1 is good.

[0191] Since the experiment requires the dual-channel separation membrane to be immersed in solution to separate protein-drug molecule complexes, the unmodified and hydrophobically modified AAO dual-channel base membranes were first soaked in distilled water for 5 minutes before contact angle characterization was performed. Figure 18Compared to the dry state, the surface contact angle of the dual-channel separation membrane of Example 1 in the wet state decreased, but was still greater than 90°, further demonstrating that the AAO dual-channel base membrane was transformed from hydrophilic to hydrophobic by polytetrafluoroethylene modification.

[0192] The above experiments have all demonstrated that the modified AAO double-pass membrane has good hydrophobicity, low protein residue, and can be reused.

[0193] Application of dual-pass separation membrane

[0194] The dual-pass separation membrane prepared in Example 1 was used as the separation membrane in a multi-chamber electrophoresis separation device to determine protein migration.

[0195] The separation performance of the dual-channel separation membrane of the present invention was evaluated based on protein migration rate, considering protein residue, reusability, and reproducibility, as well as the protein mobility, of the dual-channel separation membrane and cellulose acetate membrane used in Example 1. The specific experimental steps are as follows:

[0196] Solution preparation:

[0197] Buffer solution: First prepare 10 mmol / L -1 The pH of the ammonium acetate solution was adjusted to 6.80 by adding sodium hydroxide solution.

[0198] Protein solution: Weigh a certain amount of human serum albumin powder using an analytical balance, dissolve it in the prepared buffer solution, and the protein concentration is 5 mg / mL. -1 Mix at a low speed to prevent the formation of air bubbles.

[0199] Electrophoresis: Add 100 μL of the prepared buffer solution to the positive and negative receiving chambers of the multi-chamber electrophoresis apparatus, respectively. Add 100 μL of the prepared protein solution to the sample chamber. Turn on the instrument's condenser circulation system at a flow rate of 5 mL / min. -1 A voltage of 12V was applied, and electrophoresis was performed for 10 minutes. After 10 minutes, the samples from each chamber were removed into centrifuge tubes. Among them, human serum albumin pI = 4.7. In the pH = 6.80 buffer solution, the protein carries a negative charge and migrates to the positive receiving chamber.

[0200] (1) Residual content test

[0201] Protein electrophoresis experiments were performed using a cellulose acetate membrane and the double-pass separation membrane from Example 1, under conditions of 12V voltage, 10min electrophoresis time, and ammonium acetate buffer solution pH=6.80. Each electrophoresis experiment was performed in triplicate. After electrophoresis, samples from each chamber were removed and analyzed using an HPLC-UV instrument (mobile phase: pH=6.80 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The sum of the peak areas of each chamber sample was compared with the protein solution detection results before electrophoresis to obtain the residual protein amount.

[0202] (2) Reusability test

[0203] Reusability refers to the ability to reuse the same double-pass separation membrane multiple times, and the reusability of the double-pass separation membrane is evaluated based on protein migration rate.

[0204] Under conditions of 12V voltage, 10min electrophoresis time, and ammonium acetate buffer solution pH=8.00, protein electrophoresis experiments were repeated 5 times using the same cellulose acetate membrane and the same double-pass separation membrane from Example 1. After electrophoresis, samples from each chamber were removed and analyzed using HPLC-UV instrument (mobile phase: pH=8.00 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The relative standard deviation (RSD) of protein migration rates of the two separation membranes was compared; the smaller the RSD, the better the reusability.

[0205] (3) Reproducibility

[0206] Reproducibility is evaluated by conducting parallel experiments using different double-pass separation membranes, with protein migration rate as the basis for assessing the reproducibility of the double-pass separation membrane.

[0207] Protein electrophoresis experiments were conducted using a cellulose acetate membrane and the double-pass separation membrane from Example 1, under conditions of 12V voltage, 10min electrophoresis time, and pH 6.80 ammonium acetate buffer solution. After each electrophoresis, the separation membrane was replaced, and the electrophoresis was performed three times in parallel. After electrophoresis, the samples from each chamber were removed and analyzed using an HPLC-UV instrument (mobile phase: pH 6.80 ammonium acetate buffer solution, injection volume: 5μL, UV detection wavelength: 280nm). The relative standard deviation (RSD) of protein migration rates of the two separation membranes was compared; the smaller the RSD, the better the reproducibility.

[0208] Test results:

[0209] Compared to the cellulose acetate membrane, the protein residue of the dual-pass separation membrane in Example 1 was significantly reduced, from 35.17% (cellulose acetate membrane) to 5.80% (dual-pass separation membrane of Example 1). Figure 19 ).

[0210] Compared to the cellulose acetate membrane, the average protein migration rate of the dual-pass separation membrane in Example 1 was significantly improved, increasing from 39.14% (cellulose acetate membrane) to 66.51% (dual-pass separation membrane of Example 1). Furthermore, the reusability of the dual-pass separation membrane of Example 1 (RSD = 1.90%) was superior to that of the cellulose acetate membrane (RSD = 19.81%). Figure 20 This further demonstrates that the dual-channel separation membrane of the present invention has good reusability.

[0211] Compared to cellulose acetate membranes, protein electrophoresis experiments using the double-pass separation membranes of Example 1 with different tensions showed significantly improved protein migration reproducibility, with the RSD decreasing from 11.87% (cellulose acetate membrane) to 1.92% (double-pass separation membrane of Example 1). Figure 21 This demonstrates that the individual differences between the dual-channel separation membranes in Example 1 are small, further proving that the separation performance of the dual-channel separation membrane of the present invention has good reproducibility.

[0212] The dual-pass separation membrane of Example 1 was installed in a multi-chamber electrophoresis separation device, and the multi-chamber electrophoresis separation device was used to optimize protein migration and separate proteins. The binding constants of proteins and small molecules were calculated based on the Scatchard model.

[0213] The protein in the following examples is trypsin, and the small molecule is matrine.

[0214] Trypsin migration optimization experiment

[0215] To obtain the optimal migration rate of trypsin, this experiment optimized the trypsin migration rate under the following electrophoresis conditions: at a voltage of 12V and an electrophoresis time of 10min, the migration rate of 75.3 μmol / L trypsin was measured at pH = 7.00, 8.00, and 9.00. -1 Trypsin migration rate; under conditions of buffer solution pH = 8.00 and electrophoresis time of 10 min, the migration rate of 75.3 μmol L⁻¹ was measured at applied voltages of 8, 12, 20, 40, and 60 V. -1 Trypsin migration rate; under conditions of buffer solution pH = 8.00 and voltage 12V, electrophoresis times of 75.3 μmol L⁻¹ were measured at 1, 3, 5, 10, and 15 min. -1 Trypsin migration rate. All experiments were performed in triplicate. After the experiments, the solution sample from the negative receiving chamber was removed and analyzed by HPLC-UV under the following conditions: detection wavelength 280 nm, injection volume 5 μL, and mobile phase 100% buffer solution. The experimental results are as follows: Figure 22As shown, based on protein migration rate, the optimal migration conditions for trypsin were finally determined to be: buffer solution pH = 8.00, voltage 12V, and electrophoresis time 10min, under which the optimal migration rate of trypsin was 90%.

[0216] Example 7

[0217] Solution preparation: Prepare an ammonium acetate buffer solution with pH = 8.00, and use the buffer solution to prepare a solution with a concentration of 79.3 μmol / L. -1 Prepare a trypsin solution and mix slowly to avoid generating bubbles. Prepare stock solutions of matrine at different concentrations: dissolve matrine in dimethyl sulfoxide to concentrations of 4026.20 μmol / L. -1 6039.40 μmol L -1 8052.60 μmol L -1 9663.00 μmol L -1 12078.80 μmol L -1 .

[0218] Preparation of experimental group sample solutions: Add 5 μL of matrine stock solution of different concentrations to 95 μL of protein solution to prepare 100 μL of experimental group sample solution. At this time, the protein concentration in 100 μL sample solution is approximately 75.3 μmol / L. -1 The concentrations of matrine were 201.31 μmol / L. -1 301.97 μmol L -1 402.63 μmol L -1 483.15 μmol L -1 603.94 μmol L -1 These are denoted as total protein concentration and total small molecule concentration, respectively.

[0219] Control solution: 100 μL of the control solution contained no protein, and the concentration of matrine was the same as that of the experimental group, at 201.31 μmol / L. -1 301.97 μmol L -1 402.63 μmol L -1 483.15 μmol L -1 603.94 μmol / L -1 .

[0220] Incubation: The sample solutions of each experimental group were incubated in a water bath at 37°C for 30 min to obtain trypsin-matrine complex.

[0221] Multi-chamber electrophoresis separation: After installing the separation chambers, add 100 μL of ammonium acetate buffer solution with pH=8.00 to the positive receiving chamber and the negative receiving chamber respectively, and add 100 μL of the experimental group sample solution to the sample chamber respectively. Set the voltage to 12V and the electrophoresis time to 10min.

[0222] Dissociation: Take out all the solution sample from the negative receiving chamber and put it into a centrifuge tube. Since trypsin cannot denature and precipitate after being dissolved in water and then added to methanol, use nitrogen blowing to dry the sample solution first to obtain trypsin solid. Then add 100 μL of methanol (dissociation agent) (trypsin solid is insoluble in methanol), mix well with a mixer, and let stand for more than 2.5 h to completely dissolve the matrine bound to the protein in the methanol solution to obtain the dissociated solution.

[0223] Centrifugation: Centrifuge the dissociated solution after standing for 15 minutes at 12,000 rpm. After centrifugation, take the supernatant for testing. The supernatant contains matrine. If the volume of the supernatant is less than 100 μL, add methanol to make up to 100 μL.

[0224] Determination: The concentration of matrine in the supernatant was determined by HPLC-MS / MS and recorded as the transmembrane small molecule concentration.

[0225] Chromatographic conditions: C18 analytical column (150 mm × 4.6 mm × 5 μm); column temperature: 25 °C; flow rate: 0.5 mL / min -1 The injection volume was 1 μL; the mobile phase was methanol with 0.1% formic acid added.

[0226] Mass spectrometry conditions: Electrospray ionization (ESI); MRM detection mode; precursor ion m / z: 249.2; daughter ion m / z: 148.1; capillary voltage: 4 kV; collision voltage: 110 V; nebulizer gas (N2) flow rate: 13.0 L / min -1 Spray pressure: 30 psi; Dry gas temperature: 300℃; Positive ion mode.

[0227] All experiments were performed in triplicate.

[0228] The control solution underwent the same treatment process as the experimental solution, namely, incubation, electrophoresis, dissociation, and centrifugation. The concentration of matrine in the supernatant was detected by HPLC-MS / MS and recorded as the diffusion small molecule concentration.

[0229] Standard curve of the drug molecule matrine ( Figure 6 The standard curve equation is Y = 366X + 29538, R. 2 =0.9950, proving that the drug molecule matrine is present at 20.13 μmol L. -1 ~1207.88 μmol L-1 The linear relationship is good within this range, so subsequent experiments will be conducted within this concentration range.

[0230] Based on the following Scatchard model, calculate the binding constant K between trypsin and matrine:

[0231] n / [L]=-nK+KN

[0232] in,

[0233] n = (Concentration of transmembrane small molecules - Concentration of diffusing small molecules) ÷ (Total protein concentration × Optimal protein migration rate),

[0234] [L] represents the concentration of free small molecules, where [L] = total small molecule concentration - (transmembrane small molecule concentration - the concentration of diffused small molecules).

[0235] K: The binding constant between proteins and small molecules.

[0236] N: The number of binding sites between proteins and small molecules.

[0237] Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

[0238] The binding constant K = 2.58 × 10⁻⁶ was obtained by analyzing the slope and intercept. 5 M -1 ( Figure 23 By comparing with the literature, the binding constant of trypsin to matrine obtained by multi-compartment electrophoresis is consistent with the value of 1.26 × 10⁻⁶ in the literature. 5 M -1 On the same order of magnitude, this demonstrates the accuracy of the results obtained by combining multi-chamber electrophoresis with constant determination.

[0239] The values ​​of the above elements are shown in Table 2 below.

[0240] Table 2

[0241]

[0242] The concentration of transmembrane small molecules minus the concentration of diffused small molecules is the same as the concentration of bound small molecules. It can also be calculated as follows: use HPLC-MS / MS to detect the supernatant of the experimental group and the control group to obtain the peak area Y1 of the experimental group and the peak area Y2 of the control group. Substitute the obtained peak areas into the standard curve equation of matrine Y=366X+29538, and calculate X1-X2 to obtain the concentration of bound small molecules.

[0243] To further verify the accuracy of multi-compartment electrophoresis in determining binding constants, the binding constants of trypsin and the drug molecule matrine were determined using fluorescence under the same conditions.

[0244] Solution preparation: Prepare an ammonium acetate buffer solution with pH = 8.00. Dissolve trypsin in the buffer solution to prepare a 5.0 μmol / L solution. -1 A solution of trypsin was prepared. A certain amount of matrine was dissolved in dimethyl sulfoxide solution to prepare a 1000 μmol / L solution. -1 Matrine solution.

[0245] Incubation: Add 2 mL of trypsin solution to the fluorescent sample cell, and add 5 μL to 10 μL of matrine solution to the sample cell each time, so that the drug concentration in the sample is 2.5 μmol / L. -1 5.0 μmol L -1 7.5 μmol L -1 10.0 μmol / L -1 15.0 μmol L -1 20.0 μmol L -1 25.0 μmol L -1 30.0 μmol L -1 35.0 μmol L -1 Each time matrine solution is added, it is mixed well, heated in a 37°C water bath for 5 minutes, mixed again, and then fluorescence detection is performed.

[0246] Fluorescence detection conditions: excitation wavelength 280 nm; scanning range 290–500 nm; excitation and emission band widths 5 nm; scanning speed 1000 nm / min. -1 Response time: 20ms.

[0247] Fluorescence spectra of trypsin at different drug concentrations ( Figure 24 It can be seen that as the concentration of the drug molecule matrine gradually increases, the fluorescence intensity of the protein gradually decreases.

[0248] Knowing the fluorescence intensity of trypsin at different drug concentrations, the Scatchard correction equation was used:

[0249]

[0250] in:

[0251] F: Fluorescence intensity of trypsin after drug addition;

[0252] F0: Trypsin fluorescence intensity;

[0253] [Dt]: Concentration of the added drug;

[0254] [Pt]: Protein concentration;

[0255] K: Associative constant;

[0256] N: Number of binding sites

[0257] Plot F0 / F against F0 / F0-F, and obtain the binding constant K and the number of binding sites N by using the slope and intercept.

[0258] Scatchard curve for determining the binding constant of trypsin and matrine by fluorescence method ( Figure 25 Y = 0.1856X - 1.3291, R 2 =0.9706, and the associative constant K is obtained from the slope and intercept as 1.86 × 10. 5 M -1 The binding constant measured by multi-chamber electrophoresis and the literature value K = 1.26 × 10⁻⁶ were compared with those obtained by multi-chamber electrophoresis. 5 M -1 This demonstrates the accuracy of multi-compartment electrophoresis in determining binding constants, and is on the same order of magnitude.

[0259] The protein in the following examples is human serum albumin, and the small molecule is indomethacin.

[0260] Human serum albumin migration optimization experiment

[0261] To obtain the optimal migration rate of human serum albumin, this experiment optimized the migration rate of human serum albumin under the following electrophoresis conditions: 12V voltage, 10min electrophoresis time, and measurements were taken at pH = 6.80 and 8.00 for 75.3 μmol L⁻¹. -1 Human serum albumin mobility; measured at applied voltages of 12, 20, 40, and 60 V for 75.3 μmol / L at a buffer solution pH of 8.00 and an electrophoresis time of 10 min. -1 Human serum albumin mobility; measured at 75.3 μmol / L at 1, 3, 5, 10, and 15 min electrophoresis times under buffer solution pH = 8.00 and voltage 60 V. -1 Human serum albumin migration rate. All experiments were performed in triplicate. After the experiments, the solution sample from the negative receiving chamber was removed and analyzed by HPLC-UV at a wavelength of 280 nm, an injection volume of 5 μL, and a mobile phase of 100% buffer solution. Based on protein migration rate, the optimal migration conditions for human serum albumin were determined to be: buffer solution pH = 8.00, voltage 60 V, and electrophoresis time 10 min, under which the optimal migration rate of human serum albumin was 95%.

[0262] Example 8

[0263] Solution preparation: Prepare an ammonium acetate buffer solution with pH = 8.00 to dissolve human serum albumin, resulting in a concentration of 79.3 μmol / L. -1Human serum albumin solution was mixed slowly to avoid generating bubbles. Indomethacin was dissolved in dimethyl sulfoxide to prepare stock solutions of indomethacin at different concentrations: 558.99 μmol / L. -1 4192.40 μmol L -1 5589.90 μmol L -1 8384.80 μmol L -1 11179.80 μmol L -1 .

[0264] Preparation of experimental group sample solutions: Add 5 μL of indomethacin stock solution of different concentrations to 95 μL of protein solution to prepare 100 μL of experimental group sample solutions. At this time, the protein concentration in 100 μL of sample solution is approximately 75.3 μmol / L. -1 The concentrations of indomethacin were 27.95 μmol / L. -1 209.62 μmol L -1 279.50 μmol L -1 419.24 μmol L -1 558.99 μmol L -1 These are denoted as total protein concentration and total small molecule concentration, respectively.

[0265] Control solution: 100 μL of the control solution contained no protein, and the indomethacin concentration was the same as that of the experimental group, at 27.95 μmol / L. -1 209.62 μmol L -1 279.50 μmol L -1 419.24 μmol L -1 558.99 μmol / L -1 .

[0266] Incubation: The sample solutions of each experimental group were incubated in a water bath at 37°C for 30 min to obtain human serum albumin-indomethacin complex.

[0267] Multi-chamber electrophoresis separation: Install the separation chambers, add 100 μL of ammonium acetate buffer solution (pH=8.00) to the positive and negative receiving chambers respectively, and add 100 μL of the experimental group sample solution to the sample chamber respectively. The voltage is 60V and the electrophoresis time is 10 min.

[0268] Dissociation: Remove all the sample solution from the receiving chamber and place it in a centrifuge tube. Add 600 μL of methanol to the centrifuge tube and let it stand for at least 2.5 hours.

[0269] Centrifugation and concentration: Centrifuge the dissociated solution after standing at 12,000 rpm for 15 min, take the supernatant and concentrate it to less than 100 μL by nitrogen blowing, and then add methanol to make up to 100 μL.

[0270] Determination: The concentration of indomethacin in the supernatant was determined by HPLC-MS / MS and recorded as the transmembrane small molecule concentration.

[0271] Chromatographic conditions: Same as in Example 7.

[0272] Mass spectrometry conditions: Electrospray ionization (ESI); MRM detection mode; precursor ion m / z: 249.2; daughter ion m / z: 148.1; capillary voltage: 4 kV; collision voltage: 110 V; nebulizer gas (N2) flow rate: 13.0 L / min -1 Spray pressure: 30 psi; Dry gas temperature: 300℃; Positive ion mode.

[0273] All experiments were performed in triplicate.

[0274] The control solution underwent the same treatment process as the experimental solution, namely, incubation, electrophoresis, dissociation, centrifugation and concentration. The concentration of indomethacin in the supernatant was detected by HPLC-MS / MS and recorded as the diffusion small molecule concentration.

[0275] Standard curve of the drug molecule indomethacin ( Figure 26 The standard curve equation is Y = 1333X + 34101, R. 2 =0.9998, proving that the drug molecule indomethacin is present at 13.98 μmol / L. -1 ~838.48 μmol L -1 The linear relationship is good within this range, so subsequent experiments will be conducted within this concentration range.

[0276] Similar to Example 7, based on the Scatchard model, the binding constant K of human serum albumin to indomethacin was obtained as 3.23 × 10⁻⁶ using the slope and intercept. 4 M -1 ( Figure 27 By comparing with the literature, the binding constant of human serum albumin to indomethacin obtained by multi-compartment electrophoresis was found to be 5.55 × 10⁻⁶ in the literature. 4 M -1 On the same order of magnitude, this demonstrates the accuracy of the results obtained by combining multi-chamber electrophoresis with constant determination.

[0277] The values ​​of the above elements are shown in Table 3 below.

[0278] Table 3

[0279]

[0280] The calculation method for the transmembrane small molecule concentration - diffused small molecule concentration, which is the same as that for the binding small molecule concentration, is the same as in Example 7.

[0281] To further verify the accuracy of multi-compartment electrophoresis in determining the binding constant, the binding constant of human serum albumin to the drug molecule indomethacin was determined using fluorescence under the same conditions.

[0282] Solution preparation: Same as in Example 7.

[0283] Incubation: Same as in Example 7.

[0284] Fluorescence detection conditions: Same as in Example 7.

[0285] Similar to Example 7, as the concentration of the drug molecule indomethacin gradually increases, the fluorescence intensity of the protein gradually decreases. Figure 28 ).

[0286] Similar to Example 7, the Scatchard curve of the binding constant of human serum albumin to indomethacin was determined by plotting F0 / F against F0 / F0-F using the Scatchard correction equation. Figure 29 Y = 0.0684X - 0.3513, R 2 =0.9326, and the associative constant K is obtained from the slope and intercept as 6.84 × 10. 4 M -1 The binding constant measured by multi-chamber electrophoresis and the literature value K = 5.55 × 10⁻⁶ were compared. 4 M -1 This demonstrates the accuracy of multi-compartment electrophoresis in determining binding constants, and is on the same order of magnitude.

[0287] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for determining the binding constant of proteins to small molecules, characterized in that, Includes the following steps: Provides proteins, small molecules, and buffer solutions; Using the buffer solution, prepare m portions of sample solutions containing the protein and the small molecule, where m ≥ 3; In each sample solution, the total protein concentration was the same, but the total small molecule concentration was different. Each of the sample solutions was processed as follows: The sample solution is incubated to form charged protein-small molecule complexes; The protein-small molecule complex was separated using multi-compartment electrophoresis. The separation was performed in a multi-compartment electrophoresis apparatus. The separation membrane of the apparatus was a double-passage membrane, comprising a double-passage base membrane and a hydrophobic material. The channels of the double-passage base membrane had a through-pore structure, and the hydrophobic material modified the upper and lower surfaces and the surface of the channels of the double-passage base membrane. The multi-compartment electrophoresis apparatus included a positive receiving chamber, a sample chamber, and a negative receiving chamber, separated from the positive and negative receiving chambers by separation membranes. Specifically, the separation included: adding the incubated sample solution to the sample chamber; adding the buffer solution to the positive and negative receiving chambers respectively; performing electrophoresis to allow the protein-small molecule complex to migrate through the separation membrane to the positive or negative receiving chamber; and removing all the solution containing the migrated protein-small molecule complex from the positive or negative receiving chamber. A dissociation agent is added to the separated protein-small molecule complex to dissociate it into protein and small molecules, resulting in a dissociation solution. The concentration of the small molecules in the dissociation solution is measured and recorded as the transmembrane small molecule concentration. Test the optimal migration rate of the protein; Based on the Scatchard model, the optimal migration rate of the protein, the total concentration of the protein, the total concentration of the small molecules in each sample solution, and the concentration of the transmembrane small molecules in each sample solution, the binding constant K between the protein and the small molecules is calculated. The equations for the Scatchard model are shown below: in, n = Concentration of transmembrane small molecules in each sample solution ÷ (Total protein concentration × Optimal protein migration rate), [L] represents the concentration of free small molecules in each sample solution, where [L] = total concentration of small molecules in each sample solution - concentration of transmembrane small molecules in each sample solution. K: The binding constant between proteins and small molecules. N: The number of binding sites between proteins and small molecules. Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

2. The method according to claim 1, characterized in that, The applied voltage for the multi-chamber electrophoretic separation method is 8-100V; The electrophoresis time for the multi-chamber electrophoresis separation method is less than 20 minutes.

3. The method according to claim 1 or 2, characterized in that, The applied voltage for the multi-chamber electrophoretic separation method is 8-60V; The electrophoresis time for the multi-chamber electrophoresis separation method is 2-20 min.

4. The method according to claim 1 or 2, characterized in that, The pH value of the buffer solution is 4-9.

5. The method according to claim 1 or 2, characterized in that, The concentration of the small molecules in the dissociated solution was determined using HPLC-MS / MS.

6. The method according to claim 1 or 2, characterized in that, The protein is trypsin, human serum albumin, bovine serum albumin, or α-glucosidase; The small molecule is a drug small molecule; The dissociation agent is one or more of methanol, ethanol, acetone, formaldehyde, and isopropanol.

7. The method according to claim 6, characterized in that, The small molecules of the drug are matrine, indomethacin, baicalin, or wogonin.

8. The method according to claim 1, characterized in that, The dual-pass membrane is a porous alumina dual-pass membrane; the hydrophobic material is one or more of polytetrafluoroethylene, polydimethylsiloxane, polymethyl methacrylate, polystyrene, polypropylene, and polyvinyl chloride. The contact angle of the dual-channel separation membrane is 50°-120°.

9. The method according to claim 1, characterized in that, The contact angle of the dual-channel separation membrane is 90°-120°.

10. The method according to claim 1, characterized in that, The dissociation includes: if the protein cannot precipitate in the dissociation solution, drying the taken solution to obtain a protein-small molecule complex solid; adding the dissociation agent to dissociate; centrifuging the dissociation solution after standing, and taking the supernatant; or, if the protein can precipitate in the dissociation solution, adding the dissociation agent to the taken solution to dissociate, centrifuging the dissociation solution after standing, and taking the supernatant. The detection includes: using HPLC-MS / MS to detect the concentration of the small molecules in the supernatant, which is denoted as the transmembrane small molecule concentration.

11. The method according to claim 1, characterized in that, Also includes: Prepare m control solutions containing the small molecule using the buffer solution, where m ≥ 3; The control solution is subjected to the same treatment process as the sample solution. The concentration of the small molecules in the resulting dissociated solution is recorded as the concentration of the diffused small molecules; Based on the Scatchard model, the optimal protein migration rate, the total protein concentration, the total concentration of small molecules in each sample solution, the concentration of transmembrane small molecules in each sample solution, and the concentration of diffusing small molecules in each control solution, the binding constant K between the protein and the small molecules is calculated. The equations for the Scatchard model are shown below: in, n = (concentration of transmembrane small molecules in each sample solution - concentration of diffusing small molecules in each control solution) ÷ (total protein concentration × optimal protein migration rate), [L] represents the concentration of free small molecules in each sample solution, where [L] = total concentration of small molecules in each sample solution - (concentration of transmembrane small molecules in each sample solution - concentration of diffusing small molecules in each control solution). K: The binding constant between proteins and small molecules. N: The number of binding sites between proteins and small molecules. Plot n against n / [L] and use the slope and intercept to obtain the binding constant K of the protein and the number of binding sites N.

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