Fluorescent probe for measuring aperture of ultrafiltration membrane as well as preparation method and application of fluorescent probe

Abstract

The invention relates to a fluorescent probe for measuring the aperture of an ultrafiltration membrane as well as a preparation method and application of the fluorescent probe. The fluorescent probe is a group of fluorescently-labeled proteins obtained by mixing a group of fluorescent dyes which have groups capable of reacting with amino groups in proteins and have non-interfering fluorescence signals with a group of proteins which have significant difference in equivalent diameter and can keep stable in a detection solution for reaction, and then performing purification and concentration. When the fluorescent probe is used as a reference substance to measure the aperture of the ultrafiltration membrane, the fluorescent probe is prepared into a mixed solution, then a filter membrane to be detected is used for filtering the mixed solution at one time, the fluorescence intensity is detected, and the rejection rate R of each fluorescent probe is further calculated. And obtaining apertures d50 and d90 according to a fitting curve of the equivalent diameter and the retention rate R, and drawing an aperture distribution diagram. The method can accurately measure the aperture of the ultrafiltration membrane of 2-20 nm, is convenient to operate, high in efficiency and low in test cost and complexity, and has important significance in research and application in the fields of materials, biology, environment and the like.

Description

Technical Field

[0001] The invention relates to a fluorescent probe for measuring the pore size of an ultrafiltration membrane and a preparation method and application thereof, belonging to the technical field of filter membrane characterization. Background Art

[0002] Ultrafiltration membrane has separation and filtration characteristics and is widely used in adsorption, separation, catalysis and other fields. The filtration pore size is an important property of ultrafiltration membrane, which has a significant impact on the material's permeability, permeation rate, filtration performance and other properties in practical applications. The characterization of the pore size data of the ultrafiltration membrane under actual working conditions is the technical guarantee for accurate control of the pore size. Therefore, it is very necessary to accurately test the pore size of the ultrafiltration membrane during the separation and filtration process.

[0003] At present, there are several methods for detecting the pore size of ultrafiltration membranes: mercury intrusion method, gas adsorption method, gas permeation method and molecular weight cut-off method. Mercury intrusion method and gas adsorption method have no great requirements on the material morphology, and can detect the data of all pores in porous materials, which are highly recognized internationally. The relative disadvantage is that the equipment value is high, and there is a certain difference between the data obtained by these two methods and the actual filtration capacity of the material. Relatively speaking, the results obtained by gas permeation method and molecular weight cut-off method are closer to practical applications. The gas permeation method requires the test object to be dried, and is more suitable for ultrafiltration membranes with gas separation performance. The interception method usually uses an ultraviolet spectrophotometer to detect the absorbance value of the sample. Only the interception rate of one substance can be measured at a time. The determination process is relatively cumbersome, and the demand for reagents is also large.

[0004] The fluorescent probe method is a newly developed method for measuring the pore size of filter membranes in recent years. It can directly measure the separation performance of the membrane and has the advantages of a wide range of test pore sizes, relatively accurate test results, simple operation, short test cycle, low test loss, and low test cost. It has been applied to the measurement of filter membrane pore size. For example, the applicant's previous patent CN114904397B discloses a method for measuring the pore size and pore size distribution of filter membranes. The patent selects a group of fluorescent particles with different particle sizes and different emission wavelengths as standards, uses the filter membrane to be tested to filter the prepared mixed suspension once, and then performs fluorescence detection on the obtained filtrate, and calculates the pore size of the filter membrane to be tested based on the retention rate R of the filter membrane to be tested for each fluorescent particle and its particle size.

[0005] Although the method for determining the pore size of filter membranes in this patent has outstanding characteristics such as high sensitivity, strong selectivity, simple operation, low cost, fast response speed, multi-channel simultaneous detection, real-time detection, visualization, and convenience, it can only be applied to filter membranes with a pore size range of 20 to 500 nm, and cannot detect ultrafiltration membranes with smaller pore sizes. Summary of the invention

[0006] In view of the above problems existing in the prior art, the purpose of the present invention is to provide a fluorescent probe for measuring the pore size of ultrafiltration membranes and a preparation method and application thereof. In this invention, the fluorescent probe can be used for pore size detection of ultrafiltration membranes with a pore size less than 20 nm.

[0007] In order to achieve the above-mentioned invention object, the technical method adopted by the present invention is as follows:

[0008] One of the technical solutions of the present application provides a method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane, wherein the fluorescent probe is a fluorescently labeled protein, and the preparation method comprises the following steps:

[0009] S1.1 Select a group of fluorescent dyes with different emission wavelengths, add DMSO to prepare a group of fluorescent dye solutions a1, b1, c1, d1, e1;

[0010] S1.2 Select a group of proteins with different equivalent diameters, add NaHCO3 buffer solution, and prepare a group of protein solutions a2, b2, c2, d2, e2;

[0011] S1.3 Add fluorescent dye solutions a1, b1, c1, d1, e1 to the prepared protein solutions a2, b2, c2, d2, e2 respectively to obtain mixed solutions a3, b3, c3, d3, e3;

[0012] S1.4 Mix solutions a3, b3, c3, d3 and e3 by shaking until they are evenly mixed, and then react at room temperature in the dark;

[0013] S1.5 The mixed solutions a3, b3, c3, d3, and e3 after the reaction are placed in dialysis bags respectively, and both ends are sealed with sealing clips; the dialysis bags are placed in NaCl solution at room temperature and protected from light, NaCl solution at low temperature and protected from light, phosphate buffer solution at room temperature and protected from light, and phosphate buffer solution at low temperature and protected from light for dialysis in turn, so as to obtain solutions containing fluorescent probes a, b, c, d, and e for measuring the pore size of ultrafiltration membranes, wherein the equivalent diameters of the fluorescent probes a, b, c, d, and e are different from each other and the emission wavelengths are different from each other.

[0014] Furthermore, the fluorescent dye described in step S1.1 is required to have a group that can react with the amino group in the protein, and the fluorescent signals of the selected dyes in the same group should not interfere with each other, and the spectral crosstalk between the fluorescent probes in the mixed test should be less than 5%. The fluorescent dyes that can be used in the test include but are not limited to fluorescein, rhodamine, coumarin, Alexa series dyes, and Cy series dyes. The concentrations of the fluorescent dye solutions a1, b1, c1, d1, and e1 are all 5 to 20 mg / mL.

[0015] Furthermore, the protein equivalent diameter in step S1.2 is between 2 and 20 nm, and the equivalent diameters of the same group of proteins selected should have significant differences, and remain stable and different from each other in the detection solution, including but not limited to cytochrome C, ovalbumin, bovine serum albumin, bovine immunoglobulin, porcine immunoglobulin, and thyroglobulin; the concentrations of the protein solutions a2, b2, c2, d2, and e2 are all 1 to 5 mg / mL.

[0016] Furthermore, the role of adding the NaHCO3 buffer solution in step S1.2 is to stabilize the pH, protect the protein conformation, and provide a suitable environment during the experiment.

[0017] Furthermore, in step S1.3, the added amounts of the fluorescent dye solutions a1, b1, c1, d1, e1 and the protein solutions a2, b2, c2, d2, e2 satisfy that the ratio of the amount of fluorescent dye to the amount of protein is 5:1 to 50:1.

[0018] Furthermore, the uniform mixing in step S1.4 is manifested as uniform color of the entire solution; and the reaction time at room temperature in the dark is 2 to 4 hours.

[0019] Furthermore, the dialysis bag described in step S1.5 needs to be soaked in water for 30 to 60 minutes before use.

[0020] Furthermore, the concentration of the NaCl solution in step S1.5 is 0.1-0.2 mol / L, the concentration of the PBS solution is 0.01 mol / L, and the pH is 7.2-7.4; the dialysis time in the room temperature light-proof NaCl solution, the low temperature light-proof NaCl solution, the room temperature light-proof phosphate buffer solution, and the low temperature light-proof phosphate buffer solution is 4-6 h, 12-16 h, 4-6 h, and 12-16 h, respectively; the volume of dialysate used for each dialysis is 400-600 times the volume of solutions a3, b3, c3, d3, and e3; and the low temperature is preferably 4°C.

[0021] Furthermore, the purified fluorescent probes a, b, c, d, and e prepared in step S1.5 are concentrated by centrifugation and then stored in a refrigerator; the centrifugation conditions are 2000-4000 rpm, 20-30 min, and the freezing storage temperature is -18°C.

[0022] The second technical solution of the present application provides a fluorescent probe for measuring the pore size of an ultrafiltration membrane prepared by the above preparation method.

[0023] The third technical solution of the present application provides an application of a fluorescent probe for measuring the pore size of an ultrafiltration membrane, wherein the fluorescent probe is applied to the pore size detection of an ultrafiltration membrane with a pore size less than 20 nm, comprising the following steps:

[0024] S2.1 Select a group of fluorescent probes with different equivalent diameters and emission wavelengths as reference objects; the equivalent diameters of the fluorescent probes are all less than 20 nm;

[0025] S2.2 For each fluorescent probe used as a reference, draw a standard curve between its concentration and relative fluorescence intensity at its emission wavelength;

[0026] S2.3 uniformly disperse a group of fluorescent probes as reference materials in a PBS solution, and mix them to prepare a mixed solution in which the mass concentration of each fluorescent probe is C0;

[0027] S2.4 Filter the solution prepared in step S3 once using the filter membrane to be tested, and then perform fluorescence detection on the blank sample, the mixed solution and the obtained filtrate, and record the fluorescence intensities I0, I1, I2 of the fluorescent probes a, b, c, d, and e at their corresponding emission wavelengths, respectively. i ; The blank sample is PBS solution;

[0028] By fluorescence intensity I0, I, I i and formula The relative fluorescence intensity RFI of each fluorescent probe is calculated, and the relative fluorescence intensity RFI of each fluorescent probe is calculated; where RFI is the relative fluorescence intensity, I i is the fluorescence intensity of the corresponding emission peak of each fluorescent probe under the corresponding excitation light at different concentrations; I0 is the fluorescence intensity of the corresponding emission peak of the blank sample under the corresponding excitation light; I is the fluorescence intensity of the corresponding emission peak under the corresponding excitation light when the mass concentration of each fluorescent probe is C0μg / mL;

[0029] The mass concentration C of the filtrate of each fluorescent probe was calculated by the relative fluorescence intensity RFI calculated above and the standard curve drawn in S2.2. t and the test solution mass concentration C0;

[0030] S2.5 According to the mass concentration of each fluorescent probe in the filtrate and the test solution, the formula The retention rate R of the sample for each fluorescent probe is calculated;

[0031] S2.6 According to the equivalent diameter and retention rate R of a group of fluorescent labeled proteins obtained, select the S-curve in the Origin software and perform curve fitting to obtain the pore size d of the membrane to be tested. 50 and d 90 , and then according to d 50 ,d 90 The pore size distribution is plotted using the pore size distribution formula.

[0032] Furthermore, the fluorescent probe in step S2.1 is the fluorescent labeled protein prepared in the above-mentioned technical solution 1.

[0033] Furthermore, in some specific embodiments of the present application, step S2.1 selects the following group of fluorescent labeled proteins as reference substances:

[0034] FITC-labeled cytochrome C with an equivalent diameter of 3.4 nm and an emission wavelength of 490 nm;

[0035] HCCA labeled ovalbumin with an equivalent diameter of 5.46 nm and an emission wavelength of 386 nm;

[0036] Cy5-labeled bovine serum albumin with an equivalent diameter of 7.0 nm and an emission wavelength of 640 nm;

[0037] Cy7-labeled bovine immunoglobulin with an equivalent diameter of 11.0 nm and an emission wavelength of 740 nm;

[0038] TRITC-labeled thyroglobulin with an equivalent diameter of 17.0 nm and an emission wavelength of 550 nm.

[0039] Furthermore, the mixed solution in step S2.3 needs to be filtered using a microfiltration membrane before use to ensure that there are no other insoluble impurities in the solution.

[0040] Further, the C0 described in step S2.3 is preferably 0.1 μg / mL to 2 μg / mL;

[0041] Furthermore, the C0 described in step S2.3 is preferably 0.5 μg / mL to 1 μg / mL.

[0042] Furthermore, in some specific embodiments of the present application, the amount of the test solution used for the filtration operation in step S2.4 is 2 to 3 mL.

[0043] Furthermore, the one-time filtration described in step S2.4 is performed in a device for measuring the pore size of an ultrafiltration membrane.

[0044] Further, in some specific embodiments of the present application, in step S2.6, the Origin software is first used to prepare a nonlinear fitting curve between the equivalent diameter of the fluorescent probe and the interception rate R, and then the pore size d of the membrane to be tested is obtained from the curve. 50 and d 90 The aperture d 50 Means: 50% of the standard granular materials under this diameter cannot pass through the sample after filtration, and the passing rate is 50%. 90 It means that after filtering, 90% of the standard granular materials with a diameter of this value cannot pass through the sample, and the passing rate is 10%. According to the pore size distribution formula: The pore size distribution f(d) can be calculated and the pore size distribution diagram can be drawn, where: d is the pore size and π is the pi.

[0045] Furthermore, a device for measuring the pore size of an ultrafiltration membrane is provided, the device comprising: a pressure reducing valve, a gas cylinder, a first regulating valve, a second regulating valve, a pressure gauge, a liquid storage tank, and a sample tank. The gas cylinder and the liquid storage tank are connected by a pipeline, and the pressure reducing valve, the first regulating valve, and the pressure gauge are arranged in sequence on the pipeline starting from one end of the gas cylinder; the liquid storage tank and the sample tank are connected by a pipeline, and the second regulating valve is arranged on the pipeline.

[0046] Furthermore, the method for using the device for measuring the pore size of an ultrafiltration membrane is:

[0047] S3.1 Open the gas cylinder and ensure that the gas supply is unobstructed;

[0048] S3.2 Operate the first regulating valve and accurately adjust the pressure in the pipeline to 0.1MPa by observing the pressure gauge; during this process, ensure that the second regulating valve remains closed;

[0049] S3.3 Pour the test liquid into the liquid reservoir, fix the membrane to be tested in the sample pool, and ensure that it is correctly positioned and well sealed. After the membrane to be tested is fixed, open the second regulating valve; at this time, the test liquid will pass through the membrane to be tested at a pressure of 0.1MPa; place a container at the lower end of the sample pool to collect the filtrate that passes through the membrane to be tested.

[0050] Compared with the prior art, the present invention has the following significant beneficial effects:

[0051] (1) The present invention aims to provide a fluorescent probe capable of accurately measuring the pore size of an ultrafiltration membrane in the range of 2 nm to 20 nm and a method for measuring the pore size of an ultrafiltration membrane using the fluorescent probe. Compared with the existing fluorescent probe method, the fluorescent probe prepared by the present invention has a significant detection range advantage and can cover the pore size detection range that is difficult to achieve with other technologies.

[0052] (2) In addition, compared with other pore size measurement technologies, the method of measuring the pore size of ultrafiltration membrane using the fluorescent probe in the present application is more convenient in operation, which not only improves the measurement efficiency, but also reduces the measurement cost and complexity. Through the method of measuring the pore size of ultrafiltration membrane using the fluorescent probe of the present invention, the pore size characteristics of the ultrafiltration membrane can be quickly and accurately evaluated, which has important practical significance for the research and application in the fields of materials science, bioengineering and environmental engineering. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 is a schematic diagram of the filtering device provided in Example 3;

[0054] The marks in the figure indicate:

[0055] 1 pressure reducing valve, 2 gas cylinder, 3 first regulating valve, 4 pressure gauge, 5 liquid storage tank, 6 second regulating valve, 7 sample tank;

[0056] Figure 2 It is a fluorescence emission spectrum in which the fluorescence signals of the five fluorescent marker proteins used in Example 2 do not interfere with each other;

[0057] Figure 3 is a standard curve between the concentration and relative fluorescence intensity of the five fluorescent marker proteins used in Example 4 at their emission wavelengths;

[0058] The marks in the figure indicate:

[0059] A is the standard curve of fluorescent labeled protein a, B is the standard curve of fluorescent labeled protein b, C is the standard curve of fluorescent labeled protein c, D is the standard curve of fluorescent labeled protein d, and E is the standard curve of fluorescent labeled protein e;

[0060] Figure 4 is a nonlinear fitting curve between the equivalent diameter of the fluorescent marker protein and the retention rate R described in Example 4;

[0061] Figure 5 This is the pore size distribution diagram obtained in Example 4. DETAILED DESCRIPTION

[0062] In order to enable those skilled in the art to better understand the technical solution of the present invention, the present invention is described in detail below in conjunction with specific embodiments. It should be noted that the following embodiments will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that for those of ordinary skill in the art, several variations and improvements can also be made without departing from the concept of the present invention. These all belong to the protection scope of the present invention.

[0063] All raw materials of the present invention have no particular limitation on their sources, and can be purchased from the market or prepared according to conventional methods known to those skilled in the art.

[0064] Example 1

[0065] This embodiment provides a method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane, comprising the following steps:

[0066] S1.1 Use an analytical balance to weigh 10 mg ± 1 mg of FITC (fluorescein isothiocyanate), HCCA (N-succinimidyl-7-hydroxycoumarin-3-carboxylate), Cy5 (sulfonic acid-Cy5-N-hydroxysuccinimide ester), Cy7 (sulfonic acid-Cy7-N-hydroxysuccinimide ester), and TRITC (tetramethylrhodamine isothiocyanate) fluorescent dyes, and add DMSO to prepare fluorescent dye solutions a1, b1, c1, d1, and e1 with a concentration of 10 mg / mL.

[0067] S1.2 Use an analytical balance to weigh about 1.0 mg ± 0.2 mg of cytochrome C, ovalbumin, bovine serum albumin, bovine immunoglobulin, and thyroid protein, respectively, add NaHCO3 buffer solution, and prepare protein solutions a2, b2, c2, d2, and e2 with a concentration of 2 mg / mL.

[0068] S1.3 Add a certain amount of fluorescent dye solution a1, b1, c1, d1, e1 to the prepared protein solutions a2, b2, c2, d2, e2 respectively to obtain mixed solutions a3, b3, c3, d3, e3. The ratio of the amount of fluorescent dye to protein is 5:1, and the matching is shown in Table 1.

[0069] Table 1 Fluorescent marker protein matching information table

[0070]

[0071] S1.4 Gently shake the mixed solutions a3, b3, c3, d3, and e3 to make them evenly mixed (the overall color of the solution is uniform), and react at room temperature in the dark for 2 h.

[0072] The S1.5 dialysis bag needs to be soaked in water for 30 minutes before use. The mixed solutions a3, b3, c3, d3, and e3 are placed in the dialysis bag respectively, and the two ends are sealed with sealing clips. The dialysis bag is placed in 0.5L, 0.15mol / L NaCl solution, dialyzed at room temperature in the dark for 4 hours, then replaced with fresh NaCl solution, and dialyzed at 4℃ in the dark for 12 hours. After that, the dialysis bag is placed in 0.5L PBS solution (mass concentration 0.01mol / L, pH 7.2-7.4), dialyzed at room temperature in the dark for 4 hours, then replaced with fresh PBS solution and dialyzed at 4℃ in the dark for 12 hours, and the purified solution containing fluorescent probes a, b, c, d, and e can be obtained. The obtained solution containing probes a, b, c, d, and e was concentrated by centrifugation at 2500 rpm for 25 min and then stored in a refrigerator to obtain probes a, b, c, d, and e. The proteins and fluorescent dyes corresponding to probes a, b, c, d, and e are shown in Table 1, respectively.

[0073] Example 2

[0074] This embodiment provides a method for determining the spectral crosstalk of a fluorescent probe, comprising the following steps:

[0075] (1) The fluorescently labeled proteins (i.e., fluorescent probes) obtained in Example 1 are sequentially added to the PBS solution to prepare a mixed solution in which the mass concentration of each fluorescent probe is 1 μg / mL, and a solution abcde is obtained. abcde refers to the probes corresponding to the serial numbers a, b, c, d, and e in Table 1. The solutions bcde (referring to the probes corresponding to the serial numbers b, c, d, and e in Table 1, the same below), acde, abde, abce, and abcd are prepared in the same way, that is, each solution lacks a fluorescently labeled protein. The fluorescent probes corresponding to the numbers are consistent with those in Table 1. Solutions abcde, bcde, acde, abde, abce, and abcd are used as test solutions.

[0076] (2) Prepare a PBS solution without any fluorescent probe as a blank sample.

[0077] (3) Test the fluorescence signal of the test solution under the same test parameters to confirm that when testing the fluorescence intensity of the target fluorescent probe, the spectral crosstalk generated by other fluorescent probes is less than 5%. For example, when the target fluorescent probe is a, select 490nm excitation light to test the fluorescence signal intensity I of abcde, bcde and blank solution at 520nm. abcde-520 , I bcde-520 and I 0-520 According to the formula Calculate, if the spectral crosstalk is greater than 5%, the fluorescent probe needs to be tested separately. In the formula, SO is the spectral crosstalk, I bcde-520 is the fluorescence signal intensity of bcde solution at 520 nm, I 0-520 is the fluorescence signal intensity of the blank solution at 520 nm, I abcde-520 is the fluorescence signal intensity of abcde solution at 520nm.

[0078] Figure 2 As shown, the excitation wavelength and emission wavelength of the selected five fluorescent marker proteins are different, and the fluorescent signals do not interfere with each other, so they can be used as benchmarks.

[0079] Example 3

[0080] This embodiment provides a device for detecting the pore size and pore size distribution of an ultrafiltration membrane. Figure 1The structure of the device includes a pressure reducing valve 1, a gas cylinder 2, a first regulating valve 3, a second regulating valve 6, a pressure gauge 4, a liquid storage tank 5, and a sample tank 7. The gas cylinder 2 is connected to the liquid storage tank 5 through a pipeline, and the pressure reducing valve 1, the first regulating valve 3, and the pressure gauge 4 are arranged in sequence on the pipeline starting from one end of the gas cylinder 2. The liquid storage tank 5 is connected to the sample tank 7 through a pipeline, and the second regulating valve 6 is arranged on the pipeline. Among them, the function of the pressure reducing valve 1 is to reduce the high-pressure gas in the gas cylinder to the low-pressure gas required by the working equipment and ensure its stable output. Among them, the function of the gas cylinder 2 is to provide pressure for the filtering operation. Among them, the function of the first regulating valve 3 is to adjust the pressure in the pipeline to a filtering pressure of 0.05 to 0.2 MPa. Among them, the function of the pressure gauge 4 is to display the actual pressure value in the pipeline. Among them, the function of the liquid storage tank 5 is to store the test liquid. Among them, the function of the second regulating valve 6 is to control the pressure entering the sample tank. Among them, the function of the sample tank 7 is to fix the filter membrane to be tested.

[0081] The operating steps of the device are:

[0082] (1) Open gas cylinder 2 and ensure that the gas supply is unobstructed.

[0083] (2) Operate the first regulating valve 3 and accurately adjust the pressure in the pipeline to 0.1 MPa by observing the pressure gauge 4. During this process, ensure that the second regulating valve 6 remains closed.

[0084] (3) Pour the test liquid into the liquid reservoir 5, fix the membrane to be tested in the sample pool, and ensure that it is correctly positioned and well sealed. After the membrane to be tested is fixed, open the second regulating valve 6; at this time, the test liquid will pass through the membrane to be tested at a pressure of 0.1 MPa; place a container at the lower end of the sample pool 7 to collect the filtrate that passes through the membrane to be tested.

[0085] Example 4 A method for detecting the pore size and pore size distribution of ultrafiltration membrane

[0086] The method comprises the following steps:

[0087] S2.1 Select a group of fluorescent labeled proteins (Table 1) with different equivalent diameters and non-interfering fluorescence signals as reference substances; Figure 2 As shown, the excitation wavelengths and emission wavelengths of the five selected fluorescent marker proteins are different, and the fluorescent signals do not interfere with each other, so they can be used as benchmarks;

[0088] S2.2 For the five fluorescent-labeled proteins (a, b, c, d, and e in Table 1) used as reference materials, standard curves were drawn for concentrations at their emission wavelengths (the concentrations were 0.1, 0.2, 0.4, 0.6, 0.8, and 1.0 μg / mL) and relative fluorescence intensity. Figure 3As shown, A is the standard curve of cytochrome C labeled with fluorescein isothiocyanate, B is the standard curve of ovalbumin labeled with N-succinimidyl-7-hydroxycoumarin-3-carboxylate, C is the standard curve of bovine serum albumin labeled with sulfonate-Cy5-N-hydroxysuccinimide ester, D is the standard curve of bovine immunoglobulin labeled with sulfonate-Cy7-N-hydroxysuccinimide ester, and E is the standard curve of thyroglobulin labeled with tetramethylrhodamine isothiocyanate;

[0089] S2.3 The five fluorescently labeled proteins used as the reference materials were uniformly dispersed in a PBS buffer solution to prepare a mixed suspension with a concentration of 1 ug / mL, and the solution was filtered using a microfiltration membrane to ensure that there were no other insoluble impurities in the solution;

[0090] S2.4 Take 3 mL of the mixed suspension and filter it once using a 25 mm diameter filter membrane (a commercially available 50 KDa ultrafiltration membrane, the filtration device is referenced Figure 1 ), then take 1 mL of the filtrate and perform fluorescence detection at an emission wavelength of 520 nm corresponding to the fluorescent marker protein a, fluorescence detection at an emission wavelength of 448 nm corresponding to the fluorescent marker protein b, fluorescence detection at an emission wavelength of 670 nm corresponding to the fluorescent marker protein c, fluorescence detection at an emission wavelength of 780 nm corresponding to the fluorescent marker protein d, and fluorescence detection at an emission wavelength of 580 nm corresponding to the fluorescent marker protein e; then according to Figure 2 The five standard curves shown are used to calculate the mass concentrations of the five fluorescent labeled proteins in the filtrate and the original solution. t and C0. According to the following formula:

[0091]

[0092] The retention rates R of the tested filter membranes for the five fluorescent labeled proteins were calculated respectively, as shown in Table 2:

[0093] Table 2 Retention rate R of the tested filter membrane for 5 fluorescent labeled proteins

[0094]

[0095] S2.5 Based on the equivalent diameters and retention rates R of the 5 fluorescent labeled proteins obtained in the above table, the S-shaped curve in the Origin software was used to make a nonlinear fitting curve between the equivalent diameters and retention rates R of the fluorescent labeled proteins (see Figure 4 Then the fitting curve can be obtained:

[0096] When the interception rate is 50%, d 50 =5.05nm. When the interception rate is 90%, d 90=7nm. According to the pore size distribution formula:

[0097]

[0098] The pore size distribution f(d) can be calculated and the pore size distribution diagram can be drawn (see Figure 5 As shown), where: d is the aperture and π is the circumference of a circle.

[0099] The above description of the embodiments is to facilitate the understanding and use of the invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments, and improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be within the scope of protection of the present invention.

Claims

1. A method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane, characterized in that: The fluorescent probe is a fluorescent labeled protein, and the preparation method comprises the following steps: S1.1 Select a group of fluorescent dyes with different emission wavelengths, add DMSO to prepare a group of fluorescent dye solutions a1, b1, c1, d1, e1; S1.2 Select a group of proteins with different equivalent diameters, add NaHCO3 buffer solution, and prepare a group of protein solutions a2, b2, c2, d2, e2; S1.3 Add fluorescent dye solutions a1, b1, c1, d1, e1 to the prepared protein solutions a2, b2, c2, d2, e2 respectively to obtain mixed solutions a3, b3, c3, d3, e3; S1.4 Mix solutions a3, b3, c3, d3 and e3 by shaking until they are evenly mixed, and then react at room temperature in the dark; S1.5 The mixed solutions a3, b3, c3, d3, and e3 after the reaction are placed in dialysis bags respectively and the two ends are sealed; the dialysis bags are placed in NaCl solution at room temperature and away from light, NaCl solution at low temperature and away from light, phosphate buffer solution at room temperature and away from light, and PBS solution at low temperature and away from light for dialysis in turn, so as to obtain solutions containing fluorescent probes a, b, c, d, and e for measuring the pore size of ultrafiltration membranes, wherein the equivalent diameters of the fluorescent probes a, b, c, d, and e are different from each other and the emission wavelengths are different from each other.

2. The method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 1, characterized in that: The fluorescent dye in step S1.1 has a group that can react with the amino group in the protein, and the fluorescent signals of the selected dyes in the same group do not interfere with each other, including but not limited to fluorescein, rhodamine, coumarin, Alexa series dyes, and Cy series dyes; the concentrations of the fluorescent dye solutions a1, b1, c1, d1, and e1 are all 5 to 20 mg / mL.

3. The method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 1, characterized in that: The equivalent diameter of the protein in step S1.2 is between 2 and 20 nm, and the equivalent diameters of the same group of proteins have significant differences, and remain stable in the detection solution, including but not limited to cytochrome C, ovalbumin, bovine serum albumin, bovine immunoglobulin, porcine immunoglobulin or thyroglobulin; the concentrations of the protein solutions a2, b2, c2, d2, and e2 are all 1 to 5 mg / mL.

4. The method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 1, characterized in that: In step S1.3, the added amounts of the fluorescent dye solutions a1, b1, c1, d1, e1 and the protein solutions a2, b2, c2, d2, e2 satisfy the following conditions: the ratio of the amount of fluorescent dye to the amount of protein is 5:1 to 50:1; The reaction time in step S1.4 at room temperature and away from light is 2 to 4 hours.

5. A fluorescent probe for measuring the pore size of an ultrafiltration membrane prepared by the preparation method according to any one of claims 1 to 4.

6. An application of a fluorescent probe for measuring the pore size of an ultrafiltration membrane as claimed in claim 5, characterized in that: The fluorescent probe is applied to the pore size detection of an ultrafiltration membrane with a pore size less than 20 nm, and comprises the following steps: S2.1 Select a group of fluorescent probes with different equivalent diameters and emission wavelengths as reference objects; the equivalent diameters of the fluorescent probes are all less than 20 nm; S2.2 For each fluorescent probe used as a reference, draw a standard curve between its concentration and relative fluorescence intensity at its emission wavelength; S2.3 uniformly disperse a group of fluorescent probes as reference materials in a PBS solution, and mix them to prepare a mixed solution in which the mass concentration of each fluorescent probe is C0; S2.4 Filter the mixed solution prepared in step S2.3 once using the filter membrane to be tested to obtain a filtrate, and then perform fluorescence detection on the blank sample, the mixed solution and the obtained filtrate, and record the fluorescence intensities I0, I1, I2 of the fluorescent probes a, b, c, d, and e at their corresponding emission wavelengths, respectively. i ; The blank sample is PBS solution; By fluorescence intensity I0, I, I i and formula The relative fluorescence intensity RFI of each fluorescent probe is calculated; where RFI is the relative fluorescence intensity, I i is the fluorescence intensity of the corresponding emission peak of each fluorescent probe under the corresponding excitation light at different concentrations; I0 is the fluorescence intensity of the corresponding emission peak of the blank sample under the corresponding excitation light; I is the fluorescence intensity of the corresponding emission peak under the corresponding excitation light when the mass concentration of each fluorescent probe is C0μg / mL; The mass concentration C of the filtrate of each fluorescent probe was calculated by the relative fluorescence intensity RFI calculated above and the standard curve drawn in S2.

2. t and the test solution mass concentration C0; S2.5 According to the mass concentration of each fluorescent probe in the filtrate and the test solution, the formula The retention rate R of the sample for each fluorescent probe is calculated; S2.6 According to the equivalent diameter and retention rate R of a group of fluorescent labeled proteins obtained, select the S-curve in the Origin software and perform curve fitting to obtain the pore size d of the membrane to be tested. 50 and d 90 , and then according to d 50 d 90 The pore size distribution is plotted using the pore size distribution formula.

7. The use of a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 6, characterized in that: The fluorescent probe in step S2.1 is obtained by the method for preparing a fluorescent probe for measuring the pore size of an ultrafiltration membrane as described in any one of claims 1 to 5; The C0 in step S2.3 is 0.1 μg / mL to 2 μg / mL; The one-time filtration described in step S2.4 is performed in a device for measuring the pore size of an ultrafiltration membrane.

8. The use of a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 7, characterized in that: Step S2.1 selects the following group of fluorescently labeled proteins as reference substances: FITC-labeled cytochrome C with an equivalent diameter of 3.4 nm and an emission wavelength of 490 nm; HCCA labeled ovalbumin with an equivalent diameter of 5.46 nm and an emission wavelength of 386 nm; Cy5-labeled bovine serum albumin with an equivalent diameter of 7.0 nm and an emission wavelength of 640 nm; Cy7-labeled bovine immunoglobulin with an equivalent diameter of 11.0 nm and an emission wavelength of 740 nm; TRITC-labeled thyroglobulin with an equivalent diameter of 17.0 nm and an emission wavelength of 550 nm.

9. The use of a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 6, characterized in that: The pore size distribution formula in step S2.6 is:

10. The use of a fluorescent probe for measuring the pore size of an ultrafiltration membrane according to claim 7, characterized in that: The device for measuring the pore size of an ultrafiltration membrane comprises the following structure: A pressure reducing valve (1), a gas cylinder (2), a first regulating valve (3), a second regulating valve (6), a pressure gauge (4), a liquid storage tank (5), and a sample tank (7); the gas cylinder (2) and the liquid storage tank (5) are connected via a pipeline, and the pressure reducing valve (1), the first regulating valve (3), and the pressure gauge (4) are arranged in sequence on the pipeline starting from one end of the gas cylinder (2); the liquid storage tank (5) and the sample tank (7) are connected via a pipeline, and the second regulating valve (6) is arranged on the pipeline; The method for using the device for measuring the pore size of an ultrafiltration membrane is as follows: S3.1 Open the gas cylinder (2) and ensure that the gas supply is unobstructed; S3.2 Operate the first regulating valve (3) and accurately adjust the pressure in the pipeline to 0.1 MPa by observing the pressure gauge (4); during this process, ensure that the second regulating valve (6) remains closed; S3.3 Pour the test liquid into the liquid storage tank (5), fix the membrane to be tested in the sample tank, and ensure that it is correctly positioned and well sealed; after the membrane to be tested is fixed, open the second regulating valve (6); at this time, the test liquid will pass through the membrane to be tested at a pressure of 0.1MPa; place a container at the lower end of the sample tank (7) to collect the filtrate that passes through the membrane to be tested.

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