CN109433017B - Microfiltration membrane and modification method - Google Patents
Microfiltration membrane and modification method Download PDFInfo
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- CN109433017B CN109433017B CN201811241166.7A CN201811241166A CN109433017B CN 109433017 B CN109433017 B CN 109433017B CN 201811241166 A CN201811241166 A CN 201811241166A CN 109433017 B CN109433017 B CN 109433017B
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- B01D67/0088—Physical treatment with compounds, e.g. swelling, coating or impregnation
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- B—PERFORMING OPERATIONS; TRANSPORTING
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- B01D61/00—Processes of separation using semi-permeable membranes, e.g. dialysis, osmosis or ultrafiltration; Apparatus, accessories or auxiliary operations specially adapted therefor
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- B01D61/147—Microfiltration
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- B—PERFORMING OPERATIONS; TRANSPORTING
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Abstract
The invention relates to a microfiltration membrane and a modification method, belongs to the technical field of membrane modification, and solves the problem that the microfiltration membrane in the prior art is difficult to release enriched cells. The microfiltration membrane is modified by using a temperature-sensitive water-soluble polymer solution. A microfiltration membrane modification method comprising: (1) preparing a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-6%; (2) and coating the temperature-sensitive water-soluble polymer solution on the microfiltration membrane for modification, and drying to obtain the modified microfiltration membrane. The invention enables the membrane filtration technology to be more widely applied in the medical field and the detection result to be more accurate.
Description
Technical Field
The invention relates to the technical field of membrane modification, in particular to a microfiltration membrane and a modification method.
Background
Circulating Tumor Cells (CTCs) and Circulating Tumor Microemboli (CTMs) present in peripheral blood are considered to be important causes and markers of tumor metastasis and recurrence.
Currently, membrane filtration technology (ISET) based on morphological enrichment is one of the major methods for detecting CTCs and CTMs. Compared with a specific enrichment detection technology (such as an EpCAM forward enrichment method) depending on tumor-related markers, the ISET technology is not influenced by the down-regulation of the expression of the tumor cell surface specific markers, has lower probability of detecting false negative results, and has the advantages of simple operation method, lower cost, high biological activity of the CTC and CTM obtained by enrichment and the like. In order to achieve an ideal and accurate detection target, the release rate of the microfiltration membrane to the enriched cells is an important index for determining the final detection result.
However, the interaction, adsorption and physical blocking of the existing microfiltration membrane and the enriched tumor cells can cause the great reduction of the release rate (the release rate is less than 10%). The releasing means such as forward and backward washing and soaking of the buffer solution can hardly achieve the releasing effect.
Disclosure of Invention
In view of the above analysis, the present invention aims to provide a microfiltration membrane and a modification method, so as to solve the problem that the existing microfiltration membrane is difficult to release the enriched cells.
The purpose of the invention is mainly realized by the following technical scheme:
in one aspect, the invention discloses a microfiltration membrane, wherein the microfiltration membrane is modified by coating a temperature-sensitive water-soluble polymer solution, and the temperature-sensitive water-soluble polymer can weaken the interaction between the enriched cells and the microfiltration membrane pores and can modify the pore size of the microfiltration membrane.
On the basis of the scheme, the invention is further improved as follows:
further, the volume percentage concentration of the temperature-sensitive water-soluble polymer solution is 0.1-6%.
Further, the temperature-sensitive water-soluble polymer comprises one or more of poly (isopropyl acrylamide), poly (N-isopropyl acrylamide), hydroxypropyl cellulose and polyvinyl alcohol.
Further, the material of the microfiltration membrane comprises parylene, polycarbonate or polyethylene terephthalate.
On the other hand, the invention also discloses a microfiltration membrane modification method, which comprises the following steps:
(1) preparing a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-6%;
(2) and coating the temperature-sensitive water-soluble polymer solution on a microfiltration membrane for modification, and drying to obtain the modified microfiltration membrane.
On the basis of the scheme, the invention is further improved as follows:
further, the volume percentage concentration of the temperature-sensitive water-soluble polymer solution is 1-6% or 0.1-1%.
Further, in the step (2), a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 1-6% is coated on the microfiltration membrane in a spraying mode, and a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-1% is coated on the microfiltration membrane in a direct immersion or spraying mode.
Further, step (2) is followed by:
(3) filtering the sample by using the modified microfiltration membrane;
(4) the microfiltration membrane releases the cell population.
Further, in the step (4), the microfiltration membrane is washed by buffer solution, and cell groups with different sizes are collected.
Further, in the step (4), the microfiltration membrane after the sample is filtered in the step (3) is immersed in a buffer solution, heated in a water bath, and collected to obtain a cell population with a corresponding size.
The invention has the following beneficial effects:
(1) according to the invention, the temperature-sensitive water-soluble polymer capable of weakening the interaction between the enriched cells and the microfiltration membrane pores is coated on the surface of the microfiltration membrane, so that the release rate of the microfiltration membrane to the cells is improved, and the cell release rate is higher than 90%;
(2) according to the invention, the temperature-sensitive water-soluble polymer capable of modifying the aperture of the microfiltration membrane is coated on the surface of the microfiltration membrane, so that the grading and screening capabilities of the microfiltration membrane on cell populations with different sizes are enhanced;
(3) the aperture and the shape of the microfiltration membrane are accurately controlled by adjusting the concentration of the temperature-sensitive water-soluble polymer;
(4) the uniformity of the aperture of the microfiltration membrane is accurately controlled by adjusting the coating mode;
(5) accurately adjusting the aperture of the microfiltration membrane under the condition of buffer solution with specific dosage and temperature;
(6) the capture rate of the modified microfiltration membrane is not affected.
In the invention, the technical schemes can be combined with each other to realize more preferable combination schemes. Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof.
Drawings
The drawings are only for purposes of illustrating particular embodiments and are not to be construed as limiting the invention, wherein like reference numerals are used to designate like parts throughout.
FIG. 1 is a schematic representation of an unmodified microfiltration membrane;
FIG. 2 is an enlarged view of a portion of FIG. 1;
FIG. 3 is a diagram of the pore size morphology of a microfiltration membrane modified with a low concentration of a temperature sensitive water soluble polymer solution;
FIG. 4 is a graph of the pore size morphology of a microfiltration membrane modified with a high concentration of a temperature sensitive water soluble polymer solution;
FIG. 5 is a schematic view of a filter device used in an embodiment of the present invention.
Reference numerals:
1-an unmodified microfiltration membrane; 2-the upper part of the filtration unit; 3-lower part of the filtering device; 4-modified microfiltration membranes.
Detailed Description
The accompanying drawings, which are incorporated in and constitute a part of this application, illustrate preferred embodiments of the invention and together with the description, serve to explain the principles of the invention and not to limit the scope of the invention.
The invention discloses a modified microfiltration membrane 4, which is modified by using a temperature-sensitive water-soluble polymer, wherein the coated temperature-sensitive water-soluble polymer can weaken the interaction between enriched cells and microfiltration membrane pores and can modify the pore size of the microfiltration membrane.
In practice, a certain concentration of temperature-sensitive water-soluble polymer is coated on the unmodified microfiltration membrane 1 and dried, as shown in fig. 1.
Compared with the prior art, the microfiltration membrane provided by the embodiment has the beneficial effects that:
(1) according to the invention, the temperature-sensitive water-soluble polymer capable of weakening the interaction between the enriched cells and the microfiltration membrane pores is coated on the surface of the microfiltration membrane, so that the release rate of the microfiltration membrane to the cells is improved, and the cell release rate is higher than 90%;
(2) the temperature-sensitive water-soluble polymer capable of modifying the aperture of the microfiltration membrane is coated on the surface of the microfiltration membrane, so that the grading and screening capacity of the microfiltration membrane on cell populations with different sizes is enhanced;
(3) the capture rate of the modified microfiltration membrane is not affected.
Considering that the modified microfiltration membrane is used for filtering cells, the selected temperature-sensitive water-soluble polymer should not have toxicity to the cells, the temperature-sensitive water-soluble polymer in the embodiment is one or more of poly (isopropyl acrylamide) (PIPAAM), poly (N-isopropyl acrylamide) (PNIPAM), hydroxypropyl cellulose (HPC) and polyvinyl alcohol (PVA), and the polymer has good continuous film forming property, is non-toxic and has a lower (32-38 ℃) minimum critical solution temperature (LCST).
Illustratively, the material of the microfiltration membrane of the present embodiment may be parylene, Polycarbonate (PC) or polyethylene terephthalate (TETP).
The invention also discloses a microfiltration membrane modification method, which comprises the following steps:
(1) preparing a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-6%;
(2) and coating the temperature-sensitive water-soluble polymer solution on a microfiltration membrane for modification, and drying to obtain the modified microfiltration membrane.
Compared with the prior art, the microfiltration membrane modification method provided by the embodiment has the beneficial effects that: the pore diameter and the shape of the microfiltration membrane are accurately controlled by adjusting the concentration of the temperature-sensitive water-soluble polymer, so that the separation of cell groups with different sizes is realized, and the grading and screening capacity of the microfiltration membrane on the cell groups with different sizes is improved.
In experiments, the temperature-sensitive water-soluble polymer solution is found to be incapable of improving the cell release rate of the microfiltration membrane when the volume percentage concentration is less than 0.1%, the solution is changed into gel when the concentration is more than 6%, the gel cannot be coated on the surface of the microfiltration membrane, and the pore diameter of the microfiltration membrane is seriously shrunk when the concentration is too high, so that the microfiltration membrane loses the filtering function. Therefore, the volume percentage concentration of the temperature-sensitive water-soluble polymer solution prepared by the embodiment is 0.1-6%, and the temperature-sensitive water-soluble polymer solution with the concentration can improve the cell release rate of the microfiltration membrane and can realize the control of the pore size of the microfiltration membrane.
Specifically, the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 1-6% has an obvious shrinkage effect on the pore diameter of the microfiltration membrane, and the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-1% has no influence on the pore diameter appearance of the microfiltration membrane.
It is worth noting that, because the concentration is high, if the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 1% -6% is coated on the microfiltration membrane in a direct immersion mode, the aperture of the microfiltration membrane can be uneven, therefore, the spraying mode is selected when the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 1% -6% is coated in the embodiment, the aperture of the microfiltration membrane can be even due to the coating mode, cells can conveniently pass through the microfiltration membrane, and the aperture uniformity of the microfiltration membrane is further improved.
Illustratively, the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-1% can be coated on the micro-filtration membrane by a direct immersion method or a spraying method.
Considering that the sizes of the cell populations to be enriched are different and the ways of releasing the cell populations by the microfiltration membrane should be different, in this embodiment, for the cell populations enriched to be larger than a certain size, the microfiltration membrane after the sample filtration is immersed in a buffer solution, heated in a water bath, and collected to obtain the cell populations with corresponding sizes; for enriching a cell population with a certain size in a certain interval range, buffer solutions with different volumes and temperatures are selected to flush the microfiltration membrane so as to achieve the purpose of adjusting the aperture of the microfiltration membrane, so that the cell population with the corresponding size is flushed from the microfiltration membrane, enters filtrate, and is collected to obtain the cell populations with different sizes.
Depending on the size of the cell population to be enriched, two cases can be distinguished: first, only cell populations larger than a certain size are enriched in the system to be separated (for example, a cell population sample with two sizes: cell population A has an average size of R1, cell population B has an average size of R2, and R1< R2); secondly, in the system to be separated, it is necessary to separate and enrich a cell population with a certain size range (taking a cell population sample with three sizes as an example: the average size of the cell population A is R1, the average size of the cell population B is R2, the average size of the cell population C is R3, the average size of the cell population D is R4, and R1< R2< R3< R4).
For the first case, the method comprises the following steps:
(1) preparing temperature sensitive water soluble polymer solution. Preparing a high-concentration polymer aqueous solution in an environment higher than the lowest critical solution temperature of the temperature-sensitive water-soluble polymer, and diluting the high-concentration polymer aqueous solution to 0.1-1% in volume percentage according to different application scenes for later use (the low-concentration temperature-sensitive water-soluble polymer solution has almost no influence on the aperture of the filter membrane);
(2) and (5) modifying the microfiltration membrane. The pore size of the prepared or purchased microfiltration membrane ranges from R1 to R2. And coating the temperature-sensitive water-soluble polymer solution with the customized concentration on the microfiltration membrane by using a direct immersion or spraying mode. The treated microfiltration membrane is placed at normal temperature or 50 ℃ for full drying for standby, and the influence on the pore diameter morphology is almost unchanged due to the low concentration of the selected polymer solution, as shown in figure 3;
(3) the sample was filtered using a modified microfiltration membrane. The microfiltration membrane is fixed to the filtration device and, as shown in fig. 5, alcohol is injected from the opening of the upper part 2 of the filtration device to wet the microfiltration membrane and the microfiltration membrane is rinsed with phosphate buffered saline. Passing the sample through the microfiltration membrane until all the liquid passes through, at which time the cell population A is discharged from the lower part 3 of the filtration device through the pores of the microfiltration membrane, as shown in FIG. 5, and the cell population B is retained on the filtration membrane;
(4) the microfiltration membrane releases the cell population to be enriched. Transferring the microfiltration membrane into a container filled with phosphate buffer salt solution, performing water bath heating according to the critical dissolving temperature of the corresponding modification material, finishing the release stage after 1-5min, taking out the microfiltration membrane, and releasing the cell group B into the container.
For the second case, the method comprises the following steps:
(1) preparing temperature sensitive water soluble polymer solution. Preparing a high-concentration polymer aqueous solution in an environment higher than the lowest critical solution temperature of the temperature-sensitive water-soluble polymer, and diluting the high-concentration polymer aqueous solution to 1-6% by volume percent according to different application scenes for later use (the temperature-sensitive water-soluble polymer solution with the concentration of 1-6% has a relatively obvious shrinkage effect on the aperture of the filter membrane);
(2) and (5) modifying the microfiltration membrane. The pore size of the prepared or purchased microfiltration membrane is larger than R4. The temperature-sensitive water-soluble polymer solution with customized concentration is coated on the microfiltration membrane by a spraying mode so as to ensure the uniformity of the aperture. Placing the modified microfiltration membrane at normal temperature or 50 ℃ for fully drying for later use, wherein the pore diameter of the microfiltration membrane can be influenced to different degrees due to the high concentration of the selected polymer solution, and the pore diameter of the treated microfiltration membrane is reduced to be between R1 and R2 as shown in figure 4;
(3) the sample was filtered using a modified microfiltration membrane. The membrane was fixed to the filtration device and as shown in figure 5, alcohol was injected from the opening in the upper part 2 of the filtration device to wet the membrane and the membrane was rinsed with phosphate buffered saline. Passing the sample through the microfiltration membrane until all of the liquid has passed, whereupon the cell population a is discharged from the lower part 3 of the filtration device through the pores of the microfiltration membrane, the cell population B, C, D being retained on the filtration membrane;
(4) the microfiltration membrane releases the cell population to be enriched.
1) And (3) flushing the microfiltration membrane for a certain time by using a buffer solution at a specific temperature to expand the diameter of the microfiltration membrane to be between R2 and R3, fixing the aperture of the microfiltration membrane by using a normal-temperature buffer solution, and collecting the filtrate at the lower part 3 of the filtering device. At this point, cell population B is effectively isolated and collected;
2) and (3) flushing the microfiltration membrane for a certain time by using a buffer solution at a specific temperature to expand the diameter of the microfiltration membrane to be between R3 and R4, fixing the aperture of the microfiltration membrane by using a normal-temperature buffer solution, and collecting the filtrate at the lower part 3 of the filtering device. At this point, cell population C is effectively isolated and collected;
3) if the collection of the cell population D is not needed, the operation is ended; if the cell population D is to be collected, the above operation is repeated, the diameter of the microfiltration membrane is expanded to be larger than R4, the pore diameter of the microfiltration membrane is fixed by using a normal temperature buffer solution, and the filtrate at the lower part 3 of the filtration apparatus is collected. At this time, cell population D was efficiently isolated and collected.
The temperature of the buffer solution in the above steps should be greater than or equal to the lowest critical solution temperature of the temperature-sensitive water-soluble polymer, and in order to avoid cell death in a high temperature environment, the temperature of the buffer solution should be lower than 38 ℃.
Example 1
There are two cell populations in the cell system, the A cell population having an average size of 6 μm (e.g., red blood cells), the B cell population having an average size of 15 μm (e.g., A549 cells), and the subject to be enriched is the B cell population. The material of the microfiltration membrane is parylene, and the aperture is 8 mu m.
Preparing poly N-isopropyl acrylamide (PNIPAM) into a solution with the volume percentage concentration of 0.1%; immersing the microfiltration membrane in the solution for 1min, taking out and naturally drying. And placing the treated microfiltration membrane into a filtering device. Injecting the cell mixed solution into the upper part 5 of the filtering device, transferring the microfiltration membrane into a container filled with phosphate buffer salt solution after the liquid completely passes through, heating in a water bath at 34 ℃, ending the release stage after 1min, taking out the filtration membrane, releasing the cell group B into the container, counting the number of the cell group B, and further calculating to obtain the release rate of the cell group B of 92%.
Example 2
There are two cell populations in the cell system, the A cell population having an average size of 6 μm (e.g., red blood cells), the B cell population having an average size of 15 μm (e.g., A549 cells), and the subject to be enriched is the B cell population. The material of the microfiltration membrane is parylene, and the aperture is 8 mu m.
Preparing poly N-isopropyl acrylamide (PNIPAM) into a solution with the volume percentage concentration of 1%; immersing the microfiltration membrane in the solution for 1min, taking out and naturally drying. And placing the treated microfiltration membrane into a filtering device. Injecting the cell mixed solution into the upper part 2 of the filtering device, transferring the microfiltration membrane into a container filled with phosphate buffer salt solution after the liquid completely passes through, heating in a water bath at 34 ℃, ending the release stage after 1min, taking out the filtration membrane, releasing the cell group B into the container, counting the number of the cell group B, and further calculating to obtain the release rate of the cell group B of 97%.
Example 3
There are three cell populations in the cell system, the A cell population having an average size of 2 μm (e.g., platelets), the B cell population having an average size of 6 μm (e.g., red blood cells), and the C cell population having an average size of 15 μm (e.g., red blood cells). The material of the microfiltration membrane is polyethylene terephthalate, and the aperture is 8 mu m.
Preparing hydroxypropyl cellulose into a solution with the volume percentage concentration of 2%; the solution is evenly coated on the micro-filtration membrane by adopting a spraying method and is naturally dried (the aperture is modified to be about 3-4 mu m according to an empirical value). And (4) placing the treated microfiltration membrane into a filtering device. Injecting the cell mixed solution into the upper part 5 of the filtering device, and collecting filtrate (A cell group) after the liquid completely passes through; washing the microfiltration membrane in the device with phosphate buffer solution at about 34 deg.C for 5min (according to empirical value, the pore diameter is modified to about 7-10 μm), further washing the microfiltration membrane with normal temperature buffer solution, and collecting filtrate (B cell population); transferring the microfiltration membrane to a container containing phosphate buffered saline, heating in a water bath at 34 deg.C, ending the release stage after 5min, and taking out the filtration membrane, wherein the cell population C is released into the container. The numbers of the a cell population, the B cell population and the C cell population were counted, and the release rate of the a cell population, the release rate of the B cell population and the release rate of the C cell population were calculated to be 90%, 91% and 93%, respectively.
Example 4
There are four cell populations in the cell system, the A cell population having an average size of less than 1 μm (e.g., bacteria), the B cell population having an average size of 2 μm (e.g., platelets), the C cell population having an average size of 15 μm (e.g., red blood cells), and the D cell population having an average size of 25 μm (e.g., primary cells). The micro-filtration membrane is made of polycarbonate and has a pore size of 30 mu m.
Preparing polyvinyl alcohol (PVA) into a solution with the volume percentage concentration of 6%; the solution is uniformly coated on the microfiltration membrane by adopting a spraying method and naturally dried (the aperture is modified to be closed according to an empirical value). Placing the treated microfiltration membrane into a filtering device, soaking the microfiltration membrane in the device by using phosphate buffer solution with the temperature of about 34 ℃, and pouring out the buffer solution when the microfiltration membrane begins to leak; injecting the cell mixed solution into the upper part 5 of the filtering device, and collecting filtrate (A cell group) after the liquid completely passes through; washing the microfiltration membrane in the device with phosphate buffered saline solution at about 34 deg.C for 5min (according to empirical value, the pore diameter is modified to about 7-10 μm), further washing the microfiltration membrane with normal temperature buffer solution, and collecting filtrate (B cell population); washing the microfiltration membrane in the device with phosphate buffer solution at about 34 deg.C for 5min (according to empirical value, the pore diameter is modified to about 16-20 μm), further washing the microfiltration membrane with normal temperature buffer solution, and collecting filtrate (C cell population); transferring the filter membrane into a container containing phosphate buffer saline solution, heating in a water bath at 34 ℃, ending the release stage after 3min, taking out the filter membrane, and releasing the cell group D into the container. The numbers of the a cell population, the B cell population, the C cell population and the D cell population were obtained by statistics, and the release rate of the a cell population, the release rate of the B cell population, the release rate of the C cell population, and the release rate of the D cell population were calculated to be 92%, 90%, and 94%, respectively.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.
Claims (6)
1. A microfiltration membrane modification method is characterized by comprising the following steps:
(1) preparing a temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-6%;
(2) coating the temperature-sensitive water-soluble polymer solution on a microfiltration membrane for modification, and drying to obtain a modified microfiltration membrane;
the temperature-sensitive water-soluble polymer can weaken the interaction between the enriched cells and the microfiltration membrane pores and can modify the pore diameters of the microfiltration membrane;
the capture rate of the modified microfiltration membrane is not affected;
the temperature-sensitive water-soluble polymer comprises one or more of poly (isopropyl acrylamide), poly (N-isopropyl acrylamide), hydroxypropyl cellulose and polyvinyl alcohol;
the material of the microfiltration membrane comprises parylene, polycarbonate or polyethylene terephthalate.
2. The microfiltration membrane modification method according to claim 1, wherein the volume percentage concentration of the temperature-sensitive water-soluble polymer solution is 1-6% or 0.1-1%.
3. The microfiltration membrane modification method according to claim 1, wherein in the step (2), the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 1-6% is coated on the microfiltration membrane by spraying, and the temperature-sensitive water-soluble polymer solution with the volume percentage concentration of 0.1-1% is coated on the microfiltration membrane by directly immersing or spraying.
4. The microfiltration membrane modification method according to claim 1, wherein the step (2) is further followed by:
(3) filtering the sample by using the modified microfiltration membrane;
(4) the microfiltration membrane releases the cell population.
5. The method for modifying a microfiltration membrane according to claim 4, wherein in the step (4), the microfiltration membrane is washed with a buffer solution, and the cell population having different sizes is collected.
6. The method for modifying a microfiltration membrane according to claim 4, wherein in the step (4), the microfiltration membrane after the sample is filtered in the step (3) is immersed in a buffer solution, heated in a water bath, and collected to obtain a cell population of a corresponding size.
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| CN112608820B (en) * | 2020-12-15 | 2022-04-15 | 北京大学 | Method and device for separating and enriching high-cell-activity rare cells and application |
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| 温敏性抗污染纳滤膜的制备及性能研究;胥建美，徐国荣，李露，吴水波，邵天宝，赵河立;《净水技术》;20170925(第S2期);第31页第2-5段；第32页第1栏最后一段及附图3，第33页第3段 * |
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| CN109433017A (en) | 2019-03-08 |
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