TW201636093A - A method for blood cell selection - Google Patents

Abstract

The invention provides a method for blood cell selection by using a zwitterionic-bias material which being a copolymer formed by zwitterionic structural units and charged structural units wherein the zwitterionic structural unit comprises at least one positive moiety and one negative moiety, a distance between the positive moiety and the negative moiety is a length of 1~5 carbon-carbon bonds, and the zwitterionic structural units and charged structural units are randomly arranged to have zwitterionic-bias.

Description

Method for screening blood cells

The present invention relates to a method of screening blood cells, and more particularly to a method of screening white blood cells using a dual ion charge bias type material.

Generally, white blood cells can be filtered from the blood by, for example, a filter containing a fibrous material, and the platelets and white blood cells are negatively charged, and the surface of the filter material is generally treated to be positively charged, except for the sponge structure (porous) using the filter material. The positively charged filter surface adsorbs negatively charged platelets and white blood cells. In addition, in order to avoid coagulation or activate platelets, an anticoagulant needs to be added to the blood sample, or the surface of the filter material needs to be further surface treated to prevent coagulation. U.S. Patent No. 5,407,581 discloses the treatment of a negatively charged filter material on the surface of blood, using a fiber material made of PET (polyethylene terephthalate) as a substrate, and then grafting a charged polymer on its surface to surface the filter material. With a little negative charge, because of the positively charged surface, it is easy to cause an increase in the concentration of bradykinin, which is easy to cause an allergic reaction during transfusion. However, as a material for blood cell screening, it is necessary to consider selectivity, cause no coagulation, allergic reaction, etc. (US Patent No. 5,407,581) No.), although the use of positively charged, negatively charged compounds reveals a positively charged surface, a negatively charged surface, or an electrically neutral surface, the overall surface charge performance, including three (positive, negative or Electrically neutral), however, there are only three types of blood reactions to materials. In addition, electrical neutrality also includes no charge or charge balance. The charge distribution also affects the blood's response to materials. Therefore, the development of materials for blood cell screening is still an important issue.

In view of the above background of the invention, in order to meet the requirements of the industry, an object of the present invention is to provide a method for screening white blood cells, the method comprising: providing a double ion charge deviation type material, the double ion charge deviation type material on platelets, red blood cells and The white blood cells have different selectivity; and the sample containing the white blood cells is contacted with the above-mentioned double ion charge deviation type material to achieve the purpose of selectively screening white blood cells.

One of the technical features of the present invention is a double ion charge deviation type material provided by the method for screening white blood cells, wherein the double ion charge deviation type material controls distance and distribution between charged groups having different charges. And the ratio, that is, the material having a specific range of double ion charge deviation, to achieve the effect of blood cell screening.

Furthermore, the second technical feature of the present invention is a double ion charge deviation type material provided by the method for screening white blood cells, which does not cause a blood coagulation reaction when screening and separating white blood cells from a blood sample, and does not cause The filter material is clogged.

In order to achieve the above object, according to an embodiment of the present invention, a method for screening white blood cells according to the present invention comprises: providing a double ion charge deviation type material, wherein the double ion charge deviation type material is a double ion structure unit and a charged structure. a copolymer composed of units, wherein the diionic building unit comprises at least a positively charged group and a negatively charged group, and the distance between the positively charged group and the negatively charged group is 1 to 5 carbon bonds, The charged structural unit and the double ion structural unit are randomly arranged to form a double ion charge deviation; and a sample containing white blood cells is brought into contact with the above-described double ion charge deviation type material.

In one embodiment, the diionic building block is derived from one or more of the following groups: diionic phosphobetaine; Zwitterionic phosphobetain (R ₁ =H or CH ₃ ;R ₂ =O or NH;m=1~5;n=1~5) diionic sulfobetaine; Zwitterionic sulfobetain (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5;n=1~5)diionic carboxybetaine;Zwitterionic carboxybetain (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1~5; n=1~5). Specifically, for example, 2-methacryloyloxyethyl phosphorylcholine (phophobetaine methacrylate (PBMA)) represented by the following formula Sulfuric betaine methacrylate (SBMA) Carboxybetaine methacrylate shown in the following formula (2-Carboxy- N, N -dimethyl- N - (2 '- (methacryloyloxy) ethyl) ethanamin-ium inner salt (carboxybetaine methacrylate, CBMA))

The above diionic building blocks are preferably derived from Sulfobetaine methacrylate.

In one embodiment, the above charged structural unit comprises one or both of a positively charged structural unit and a negatively charged structural unit.

In one embodiment, the positively charged structural unit is derived from one or more of the following groups: primary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) secondary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5) tertiary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) quaternary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH; m=1~5). Specifically, for example, 2-aminoethyl methacrylate having the following formula Having the following formula of N- isopropyl acrylamide (N -Isopropylacrylamide) Dimethylaminoethyl methacrylate having the following formula [2-(Methacryloyloxy)ethyl]trimethylammonium; TMA)

In one embodiment, the negatively charged structural unit is derived from one or more of the following groups: sulfur-based monomers Carboxyl monomer Specifically, for example, 3-sulfopropyl methacrylate having the following formula 2-Carboxyethyl acrylate having the following formula

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 5% to 34%, and the double ion charge deviation type material has no activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 70 to 90 mol%, and the molar structure of the positively charged structural unit The ratio is 30~10 mol%.

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 35% to 75%, and the double ion charge deviation type material has an activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 30 to 60 mol%, and the molar ratio of the positively charged structural unit is It is made up of 70~40%.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 15% to 59%, and the double ion charge deviation type material has no activation effect on platelets. The above charge deviation range is when the sum of the double ion structural unit and the negatively charged structural unit is 100 mol%, and the double ion structure The molar ratio of the unit is 51-80 mol%, and the molar ratio of the negative-charged structural unit is 49-20 mol%.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 60% to 90%, and the double ion charge deviation type material has an activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the negatively charged structural unit, the molar ratio of the double ion structural unit is 30 to 50 mol%, and the molar structure of the negatively charged structural unit The ratio is 70~50 mol%.

In one embodiment, the double ion charge deviation type material has a positive charge deviation range of 5% to 35%. After a sample containing white blood cells passes through the double ion charge deviation type material, the white blood cell in the sample is subjected to the double ion charge deviation type. Material filtration. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 70 to 90 mol%, and the molar structure of the positively charged structural unit The ratio is 30~10 mol%.

In one embodiment, in the double ion structure unit, the distance between the positively charged group and the negatively charged group is preferably 2 to 4 carbon bonds.

Furthermore, according to another embodiment of the present invention, the method for screening white blood cells of the present invention comprises: providing a double ion charge deviation profile The double ion charge deviation type material is a copolymer composed of a positively charged structural unit and a negatively charged structural unit, wherein the positively charged structural unit and the negatively charged structural unit are separated by 1 to 5 carbon bonds. The length of the positively charged structural unit and the negatively charged structural unit are randomly arranged to form a double ion charge deviation; and a sample containing white blood cells is brought into contact with the above-described double ion charge deviation type material.

In one embodiment, the positively charged structural unit is a group derived from one or more of the following groups: a primary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5), secondary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH; R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5), tertiary amine monomer _{(R 1, = H or CH} 3; R 2 = O or NH; m = 1 ~ 5), four group monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH; m=1~5). 2-aminoethyl methacrylate having the following formula Having the following formula of N- isopropyl acrylamide (N -Isopropylacrylamide) Dimethylaminoethyl methacrylate having the following formula [2-(Methacryloyloxy)ethyl]trimethylammonium;TMA)

In one embodiment, the negatively charged structural unit is a group derived from one or more of the following groups: sulfur-based monomer Example: 3-sulfopropyl methacrylate (SA) Carboxyl monomer Example: 2-Carboxyethyl acrylate

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 15% to 65%, and the double ion charge deviation type material has an activation effect on platelets.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 50% to 80%, and the double ion charge deviation type material pair Platelets have no activation effect.

In one embodiment, the double ion charge deviation type material has a positive charge deviation range of 20% to 70%. After a sample containing white blood cells passes through the double ion charge deviation type material, the white blood cell in the sample is subjected to the double ion charge deviation type. Material filtration.

The dual ion charge-displacement material provided by the method of the present invention can be utilized as a hemoglobin screening application, and the distance, distribution and ratio between charged groups having different charges can be controlled to meet various blood cell separation requirements.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a bar graph showing the results of double ion charge deviation and platelet, red blood cell and leukocyte adsorption experiments of a double ion charge deviation type material (copolymer I) according to an exemplary embodiment of the present invention.

Fig. 2 is a bar graph showing the results of double ion charge deviation and platelet, red blood cell and leukocyte adsorption experiments of the double ion charge-biased material (copolymer II) according to Example 2 of the present invention.

The foregoing and other objects, features, and advantages of the invention are set forth in the <RTIgt; In order to thoroughly understand the present invention, detailed steps and compositions thereof will be set forth in the following description. Obviously, the practice of the invention is not limited to the specific details that are apparent to those skilled in the art. On the other hand, well-known components or steps are not described in detail to avoid unnecessarily limiting the invention. The preferred embodiments of the present invention are described in detail below, but the present invention may be widely practiced in other embodiments, and the scope of the present invention is not limited by the scope of the following patents. .

In the application of blood treatment, the prior art (U.S. Patent No. 5,407,581) discloses a filter medium for charging a charged group as a blood, in particular, a surface of a filter material with a slight negative charge, which is suitable for use in blood treatment and does not occur. Allergic reaction. However, the inventors have found that the overall charge performance (positively charged, negatively charged or electrically neutral) of the surface of the filter material is not a major factor affecting the selectivity of the filter material to various blood cells, that is, various blood cells to the surface of the filter material (positively charged, There are only three types of negatively charged or electrically neutral surfaces. In addition, in the above literature, porous fibrous materials such as PET (polyethylene terephthalate) are used as the substrate of the filter material. The fiber material is subjected to surface modification treatment of a graft polymer or the like to obtain a surface of the filter material having a slight negative charge, so as to achieve an effect of not causing an allergic reaction.

However, the double ion charge bias provided by the method of the present invention In the case of a poor material, a fibrous material such as PET is not used as a substrate for a filter material for blood cell screening, and the above-described double ion charge-displacement type material itself has porosity, and the self-contained filter material for blood cell screening does not change the surface of the material. Processing such as quality treatment. However, the diionic charge-deviation type material described in the method for screening leukocytes provided by the present invention can be further graft-bonded to a polyester fiber material such as PP (polypropylene) fiber or PET, and can be used as a blood cell screening.

According to a first embodiment of the present invention, a method for screening white blood cells is disclosed, the method comprising: providing a dual ion charge deviation type material, wherein the double ion charge deviation type material is a copolymer composed of a double ion structure unit and a charged structure unit. And the diionic building unit comprises at least a positively charged group and a negatively charged group, and the distance between the positively charged group and the negatively charged group is 1 to 5 carbon bonds, and the charged structural unit is The diionic building blocks are randomly arranged to form a double ion charge deviation; and a sample containing white blood cells is brought into contact with the above-described double ion charge deviation type.

The diionic structural unit described in the above method for screening white blood cells has the positive ion-charged material in addition to the conventional anti-biomolecule adhesion effect, and has the addition of a positively and negatively charged group. Providing the property of double ion charge deviation, since the electrically neutral surface includes a surface having no charged groups at all and a surface having a charged group, and the surface with a charged group, the characteristics of which are accompanied by positively charged groups and The distance with a negatively charged group varies. Therefore, the above dual ion construction unit includes at least one The positively charged group and the negatively charged group have a length of 1 to 5 carbon bonds between the positively charged group and the negatively charged group, and the length of 2 to 4 carbon bonds is more ideal.

In one embodiment, the diionic building block is derived from one or more of the following groups: diionic phosphobetaine; Zwitterionic phosphobetain (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1~5; n=1~5) Example: 2-methacryloyloxyethyl phosphorylcholine (phophobetaine methacrylate; PBMA) Diionic sulfobetaine; Zwitterionic sulfobetain (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1~5; n=1~5) Example: sulfobetaine methacrylate [2-(Methacryloyloxy)ethyl]dimethyl(3) -sulfopropyl)-ammonium hydroxide(sulfobetaine methacrylate, SBMA) Diionic carboxybetaine; Zwitterionic carboxybetain _{(R 1, = H or CH} 3; R 2 = O or NH; m = 1 ~ 5; n = 1 ~ 5) Example: carboxybetaine methacrylate 2-Carboxy- N, N -dimethyl- N - (2 ' -(methacryloyloxy)ethyl)ethanamin-ium inner salt(carboxybetaine methacrylate,CBMA)

In one embodiment, the above charged structural unit comprises one or both of a positively charged structural unit and a negatively charged structural unit.

In one embodiment, the positively charged structural unit is derived from one or more of the following groups: a primary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) Example: 2-aminoethyl methacrylate Secondary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH; R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5) Example: N -Isopropylacrylamide Tertiary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) Example: dimethylaminoethyl methacrylate Quaternary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1~5) Example: [2-(Methacryloyloxy)ethyl]trimethylammonium (TMA)

In one embodiment, the negatively charged structural unit is derived from one or more of the following groups: sulfur-based monomers Example: 3-sulfopropyl methacrylate (SA) Carboxyl monomer Example: 2-Carboxyethyl acrylate

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 5% to 34%, and the double ion charge deviation type material has no activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 70 to 90 mol%, and the molar structure of the positively charged structural unit The ratio is 30~10 mol%.

The above charge deviation range is defined as a case where the double ion charge deviation type material is a positively charged ionic monomer (for example, TMA) and a negatively charged ionic monomer (for example, SA), and is defined as a peak according to an X-ray photoelectron spectroscopy (XPS). Position 399eV is the signal contributed by the quaternary nitrogen group (N ⁺ ) on the positively charged ionic monomer (TMA), which is contributed by the peak position 168eV as the sulfur element (SO ₃ ^- ) on the negatively charged ionic monomer (SA) The signals are divided into two peaks and the integrated area under the peak is calculated. The ratio of the area used is the content of the positive and negative charge monomers in the double ion charge deviation type blood cell screening material.

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 35% to 75%, and the double ion charge deviation type material has an activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 30 to 60 mol%, and the molar structure of the positively charged structural unit The ratio is 70~40 mol%.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 15% to 59%, and the double ion charge deviation type material has no activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the negatively charged structural unit, the molar ratio of the double ion structural unit is 51 to 80 mol%, and the molar structure of the negatively charged structural unit is The ratio is 49~20 mol%.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 60% to 90%, and the double ion charge deviation type material has an activation effect on platelets. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the negatively charged structural unit, the molar ratio of the double ion structural unit is 30 to 50 mol%, and the molar structure of the negatively charged structural unit The ratio is 70~50 mol%.

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 5% to 35%. After a sample containing white blood cells passes through the double ion charge deviation type material, the white blood cells in the sample are electrically charged by the double ion. Charge-type material filtration. When the above charge deviation range is 100 mol% for the sum of the double ion structural unit and the positively charged structural unit, the molar ratio of the double ion structural unit is 70 to 90 mol%, and the molar structure of the positively charged structural unit The ratio is 30~10 mol%.

Furthermore, according to a second embodiment of the present invention, a method for screening white blood cells comprises: providing a dual ion charge deviation type material, wherein the double ion charge deviation type material is a positively charged structural unit and a negatively charged structural unit. a copolymer, wherein the distance between the positively-charged structural unit and the negatively-charged structural unit is 1 to 5 carbon bonds, and the positively-charged structural unit and the negatively-charged structural unit are randomly arranged to form a double ion charge deviation And contacting a sample containing white blood cells with the above-described double ion charge deviation type material.

This embodiment differs from the first embodiment in that the dual ion charge-displacement type material described in the method of the present embodiment uses only the positively-charged structural unit and the negatively-charged structural unit to constitute the copolymer of the present invention. The distance between the positive electric structural unit and the negatively charged structural unit is 1 to 5 carbon bonds, for example, a specific group is selected, and the positively charged structural unit and the negatively charged structural unit are randomly arranged to form a double ion charge deviation.

In one embodiment, the positively charged structural unit is a group derived from one or more of the following groups: a primary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) Example: 2-aminoethyl methacrylate Secondary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH; R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5) Example: N -Isopropylacrylamide Tertiary amine monomer (R ₁ ,=H or CH ₃ ;R ₂ =O or NH;m=1~5) Example: dimethylaminoethyl methacrylate Quaternary amine monomer (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1~5) Example: [2-(Methacryloyloxy)ethyl]trimethylammonium (TMA)

In one embodiment, the negatively charged structural unit is a group derived from one or more of the following groups: sulfur-based monomer Example: 3-sulfopropyl methacrylate Carboxyl monomer, Example: 2-Carboxyethyl acrylate

In one embodiment, the dual ion charge deviation type material has a positive charge deviation range of 15% to 65%, and the double ion charge deviation type material has an activation effect on platelets.

In one embodiment, the double ion charge deviation type material has a negative charge deviation range of 50% to 80%, and the double ion charge deviation type material has no activation effect on platelets.

In one embodiment, the double ion charge deviation type material has a positive charge deviation range of 20% to 70%. After a sample containing white blood cells passes through the double ion charge deviation type material, the white blood cell in the sample is subjected to the double ion charge deviation type. Material filtration. More preferably, when the sum of the positively-charged structural unit and the negatively-charged structural unit is 100% by mole, the molar ratio of the positively-charged structural unit is 80% by mole, and the molar ratio of the negatively-charged structural unit is 20%. ear%.

Example 1:

A dual ion charge-deviation material for the synthesis of blood cells, which is a copolymer of a positively charged structural unit (TMA) and a negatively charged structural unit (SA): SAmTMAn (where m and n represent SA and TMA, respectively) The molar ratio, such as SA8TMA2, indicates that the molar ratio of SA and TMA is 8:2, and the same representation is used below.

TMA ([2-(Methacryloyloxy)ethyl]trimethylammonium chloride solution) is mixed with SA (3-Sulfopropyl methacrylate potassium salt) according to different ratios and stirred uniformly, and then the raw material of the monomer unit (that is, the mixture of TMA and SA) is 90 wt%, a crosslinking agent NMBA ( N, N- Methylenebisacryl amide, 96%, manufactured by ACROS Co.) (8 wt%) was added and stirred until uniform, and the initiator APS (1 wt%) was added at room temperature ( At 25 ° C), the monomer and the crosslinking agent are subjected to radical polymerization, and finally the catalyst TEMED (N, N, N', N'-Teramethylethylenediamine, 99%) (1% by weight) is added to accelerate the polymerization reaction, and the mixed solution is taken out. Into the module for preparing the copolymer I, the reaction was completed to form the copolymer I. Two hours after the reaction time, the copolymer II was taken out and immersed in deionized water and stored in a refrigerator at 4 ° C, and washed three times with deionized water every 24 hours to ensure that the copolymer I was stored in a clean environment. The double ion charge deviation and the diiodomethane contact angle of each copolymer I are shown in Table 1. The diiodomethane contact angle was measured by diiodomethane as a test liquid, and dropped onto the surface of the copolymer I to be tested to observe the contact angle with the surface of the copolymer I. When the contact angle is larger, it indicates that the surface of the copolymer I is more hydrophilic; on the contrary, when the contact angle becomes smaller, it means that the surface of the copolymer I is more hydrophobic. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a bar graph showing the results of double ion charge deviation and platelet, red blood cell and leukocyte adsorption experiments of a material for diionic charge-deviating blood cell screening (copolymer I) according to an exemplary embodiment of the present invention.

Example 2:

A dual ion charge-deviation material for the synthesis of blood cells, which is a copolymer composed of a double ion structural unit (SBMA) and a charged structural unit (positively charged structural unit (TMA) or negatively charged structural unit (SA)). Substance II: SpTMAq (where p and q represent the molar ratio of S and TMA, respectively, for example, S8TMA2 indicates that the molar ratio of SBMA and TMA is 8:2, the same representation is used below) or SpSAr (where p and r respectively) Indicates the molar ratio of S and SA. For example, S8SA2 indicates that the molar ratio of SBMA and SA is 8:2, and the same expression is used hereinafter.

SBMA(2-(Methacryloyloxy)ethyl]dimethyl(3-sulfopropyl)-ammonium hydroxide) is mixed with TMA ([2-(Methacryloyloxy)ethyl]trimethylammonium chloride solution) or SA (3-Sulfopropyl methacrylate potassium salt) according to different ratios. Stir well, then the raw material of the monomer unit (that is, the mixture of SBMA and TMA or SA) is 90% by weight, and the crosslinking agent NMBA (8wt%) is added and mixed until the mixture is stirred until After homogenization, the initiator ammonium persulfate APS (1wt%) was added, and the monomer and the cross-linking agent were subjected to radical polymerization at room temperature (25 ° C), and finally the catalyst TEMED (1 wt%) was added to accelerate the polymerization. The mixed solution was taken out and placed in a module for preparing the copolymer II, and the reaction was completed to form a copolymer II. Two hours after the reaction time, the copolymer II was taken out and immersed in deionized water and stored in a refrigerator at 4 ° C, and washed three times with deionized water every 24 hours to ensure that the copolymer II was stored in a clean environment. The double ion charge deviation and the diiodomethane contact angle of each copolymer II are shown in Table 2. Fig. 2 is a bar graph showing the results of the double ion charge deviation and the results of platelet, red blood cell and leukocyte adsorption experiments of the material for the double ion charge-deviating blood cell screening (copolymer II) according to Example 2 of the present invention.

For the platelet adsorption test, the water gel (copolymer I or II) to be tested was placed in a 24-well culture dish, and 1 mL of PBS was added to each well plate, and then placed in an oven at 37 ° C for one hour. PBS was removed, the reaction in order to eliminate the anticoagulant and facilitates platelet activation, first of all the need to add 1mM Mg ²⁺ and 2.5mM Ca ²⁺ containing platelet-rich human plasma solution (Platelet Rich Plasma, PRP). 1 mL of PRP was added to each well, and the reaction was carried out at 37 ° C for 2 hours, and then washed 5 times with PBS to wash the platelets which were not attached to the surface of the water gel. Next, the platelets attached to the surface of the water gel were fixed, and 1 mL of a 2.5 vol% glutaraldehyde solution was added to the 24-well culture dish, and the solution was placed in a refrigerator at 4 ° C for 24 hours, and then the glutaraldehyde solution was further added. After taking out, it was washed repeatedly with PBS, and the water gel was dried by a freeze dryer. After the dry operation, the water glue is fixed on the stage by using a carbon tape, and a conductive metal is plated on the surface by a plasma technique, and then the scanning electron microscope (SEM) is used to observe that the platelets are attached to the surface of the water gel. Type.

In the red blood cell adsorption experiment, the water gel (copolymer I or II) to be tested was placed in a 24-well culture dish, and 1 mL of PBS was added to each well plate, and then placed in an oven at 37 ° C for one hour. The PBS was taken out and 1 mL of red blood cell thick liquid was added to the water gel to be tested, and placed in an oven at 37 ° C for 2 hours. After the red blood cell thick liquid was aspirated and washed with PBS to remove unadsorbed blood cells, 1 mL of a 2.5 vol% glutaraldehyde solution was added and left to stand in a refrigerator at 4 ° C for 24 hours. Finally, a conjugated-focus laser scanning electron microscope (CLSM) was used to observe the image of red blood cells attached to the surface of the water gel.

In the white blood cell adsorption experiment, the water gel (copolymer I or II) to be tested was placed in a 24-well culture dish, and 1 mL of PBS was added to each well plate and placed in an oven at 37 ° C for one hour. The PBS was taken out and 1 mL of a white blood cell thick solution was added to the water gel to be tested, and placed in an oven at 37 ° C for 2 hours. After the white blood cell thick liquid was aspirated and washed with PBS to remove unadsorbed blood cells, 1 mL of a 2.5 vol% glutaraldehyde solution was added, and the mixture was allowed to stand in a refrigerator at 4 ° C for 24 hours. Finally, a conjugated-focus laser scanning electron microscope (CLSM) was used to observe the condition in which white blood cells were attached to the surface of the water gel.

From the results of the above-mentioned platelet, red blood cell and white blood cell adsorption experiments, it can be known that when synthesizing the double ion charge-displacement type material of the present invention, the desired blood cell screening characteristics can be obtained by controlling the double ion charge deviation. Table 3 shows Different double ion charge deviation range, the material has different blood cell screening characteristics, which uses TMS and SA to induce PP fiber film with different SA-TMA bias range by using plasma-induced surface grafting technology (pore 30um, fiber diameter) 2um, thickness 0.5mm), a modified 2.5cm diameter film was placed in the holder, and 5cc human red blood cell thick solution was filtered by pressure. The filtration effect was measured by a blood analyzer (LH780-COULTER® LH 780 Hematology Analyzer, Beckman Coulter Co.) tested the filtered blood cell content. In Table 3, when the red blood cell thick liquid was filtered by the double ion charge-displacement blood cell screening material SA2TMA8, the white blood cell removal rate was 99.98%, and the platelet recovery rate was 3.825%, which was extremely useful for red blood cell recovery of the red blood cell thick liquid.

In addition, when the double ion charge-displacement type material S7TMA3 (the sample with the highest white blood cell adsorption rate in Fig. 2) is used, when the platelet thick solution is filtered, the platelet recovery rate is 99.1%, and the white blood cell removal rate is 99.15%. Liquid platelet recovery is extremely useful.

The above-described examples are merely illustrative, and various modifications and changes can be made without departing from the spirit and scope of the invention.

In summary, the double ion charge deviation type material according to the present invention can be utilized as a material for blood cell screening, and the material itself has porosity by controlling the distance, distribution and ratio between charged groups having different charges. Self-contained is a filter material for blood cell screening, and has not been subjected to surface modification treatment of materials, etc., and meets various blood cell separation requirements, but can also be combined with fiber materials such as PP (polypropylene) fiber or PET, and used as a blood cell. The use of screening, especially in the field of screening white blood cells.

The present invention has been described in detail with reference to the preferred embodiments of the present invention, and the scope of the invention is not limited thereto, and it is understood that various modifications and changes can be made without departing from the spirit and scope of the invention. In addition, any of the objects or advantages or features of the present invention are not required to be achieved by any embodiment or application of the invention. In addition, the abstract sections and headings are only used to assist in the search of patent documents and are not intended to limit the scope of the invention.

Claims (29)

A method for screening white blood cells, the method comprising: providing a double ion charge deviation type material, wherein the double ion charge deviation type material is a copolymer composed of a double ion structure unit and a charged structure unit, wherein the double ion structure unit includes at least a positively charged group and a negatively charged group, the distance between the positively charged group and the negatively charged group being 1 to 5 carbon bonds, and the charged structural unit and the double ion structural unit are randomly arranged A double ion charge deviation is formed; and a sample containing white blood cells is brought into contact with the above-described double ion charge deviation type material. The method for screening white blood cells according to the first aspect of the patent application, wherein the diionic building unit is derived from a group of one or more of the following groups: Zwitterionic phosphobetain having the following general formula: (R ₁ =H or CH ₃ ; R ₂ =O or NH;m=1~5; n=1~5), Zwitterionic sulfobetain having the following general formula: (R ₁ =H or CH ₃ ; R ₂ =O or NH;m=1~5; n=1~5) and Zwitterionic carboxybetain having the following general formula: _{(R 1, = H or CH} 3; R 2 = O or NH; m = 1 ~ 5; n = 1 ~ 5). A method of screening white blood cells according to the first aspect of the patent application, wherein the charged structural unit comprises one or both of a positively charged structural unit and a negatively charged structural unit. A method of screening white blood cells according to item 3 of the patent application, wherein the positively charged structural unit is derived from a group of one or more of the following groups: a monoamine monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH;m=1~5), a secondary amine monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH; R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5), a tertiary amino group monomer having the following general formula _{(R 1, = H or CH} 3; R 2 = O or NH; m = 1 ~ 5) having the following general formula and a group of four monomers (R ₁ =H or CH ₃ ; R ₂ =O or NH; m=1~5). A method for screening white blood cells according to item 3 of the patent application, wherein the negatively charged structural unit is derived from a group of one or more of the following groups: a sulfur-based monomer having the following general formula _{(R 1 = H or CH 3} ; R 2 = O or NH; m = 1 ~ 5) and carboxylic monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH; m=1~5). The method for screening white blood cells according to item 3 of the patent application scope, wherein the double ion charge deviation type material has a positive charge deviation range of 5% to 34%, and the double ion charge deviation type material has no activation effect on platelets. According to the method for screening white blood cells according to Item 6 of the patent application, wherein the sum of the double ion structural unit and the positively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 70 to 90 mol%, The molar ratio of the positively charged structural unit is 30 to 10 mol%. The method for screening white blood cells according to item 3 of the patent application scope, wherein the double ion charge deviation type material has a positive charge deviation range of 35% to 75%, and the double ion charge deviation type material has an activation effect on platelets. According to the method for screening white blood cells according to Item 8 of the patent application, wherein the sum of the double ion structural unit and the positively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 30 to 60 mol%, The molar ratio of the positively charged structural unit is 70 to 40 mol%. According to the method for screening white blood cells according to Item 3 of the patent application, wherein the double ion charge deviation type material has a negative charge deviation range of 15% to 59%, and the double ion charge deviation type material has no activation effect on platelets. fruit. The method for screening white blood cells according to claim 10, wherein when the sum of the double ion structural unit and the negatively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 51 to 80 mol%. The molar ratio of the negatively charged structural unit is 49-20 mol%. According to the method for screening white blood cells according to Item 3 of the patent application, wherein the material for double ion charge deviation type blood cell screening has a negative charge deviation range of 60% to 90%, the double ion charge deviation type material has an activation effect on platelets. The method for screening white blood cells according to claim 12, wherein when the sum of the double ion structural unit and the negatively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 30 to 50 mol%, The molar ratio of the negatively charged structural unit is 70 to 50 mol%. According to the method of screening white blood cells according to Item 3 of the patent application, wherein the double ion charge deviation type material has a positive charge deviation range of 5% to 35%, and a sample containing white blood cells passes through the double ion charge deviation type material, and the sample is in the sample. The white blood cells are filtered by the double ion charge deviation type material. a method for screening white blood cells according to item 14 of the patent application, wherein When the sum of the double ion structural unit and the positively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 70 to 90 mol%, and the molar ratio of the positively charged structural unit is 30 to 10 mol. ear%. The method for screening white blood cells according to claim 1, wherein the distance between the positively charged group and the negatively charged group is 2 to 4 carbon bonds in the double ion structure unit. A method for screening white blood cells, the method comprising: providing a double ion charge deviation type material, wherein the double ion charge deviation type material is a copolymer composed of a positively charged structural unit and a negatively charged structural unit, wherein the positively charged structure The distance between the unit and the negatively charged structural unit is 1 to 5 carbon bonds, and the positively charged structural unit and the negatively charged structural unit are randomly arranged to form a double ion charge deviation; and contact with a sample containing white blood cells The above double ion charge deviation type material. A method for screening white blood cells according to claim 17 wherein the positively charged structural unit is derived from a group of one or more of the following groups: a monoamine monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH;m=1~5), a secondary amine monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH; R ₃ =CH ₃ or CH(CH ₃ ) ₂ ;m=1~5), a tertiary amino group monomer having the following general formula (R ₁ ,=H or CH ₃ ; R ₂ =O or NH;m=1 to 5) and a quaternary amine monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH; m=1~5). A method for screening white blood cells according to claim 17, wherein the negatively charged structural unit is derived from a group of one or more of the following groups: a sulfur-based monomer having the following general formula (R ₁ =H or CH ₃ ; R ₂ =O or NH;m=1~5) and having the following general carboxy monomer (R ₁ =H or CH ₃ ; R ₂ =O or NH; m=1~5). The method for screening white blood cells according to claim 17 of the patent application, wherein the double ion charge deviation type material has a positive charge deviation range of 15% to 65%, and the double ion charge deviation type material has an activation effect on platelets. The method for screening white blood cells according to claim 17 of the patent application, wherein the double ion charge deviation type material has a negative charge deviation range of 50% to 80%, and the double ion charge deviation type material has no activation effect on platelets. The method for screening white blood cells according to claim 17 of the patent application scope, wherein the double ion charge deviation type material has a positive charge deviation range of 20% to 70%, and a sample containing white blood cells passes through the double ion charge deviation type material in the sample. The white blood cells are filtered by the double ion charge deviation type material. The method for screening white blood cells according to claim 1 or 17, wherein the above-mentioned diionic charge-displacement type material is grafted to polypropylene fibers or polyester fibers and used as a blood cell screen. A method of screening white blood cells according to the first aspect of the patent application, wherein the diionic building unit is derived from Sulfobetaine methacrylate. According to the method for screening white blood cells according to claim 17, wherein the sum of the positively-charged structural unit and the negatively-charged structural unit is 100 mol%, the molar ratio of the positively-charged structural unit is 80 mol%, The molar ratio of the negatively charged structural unit is 20 mol%. According to the method for screening white blood cells according to the first aspect of the patent application, wherein the sum of the double ion structural unit and the positively charged structural unit is 100 mol%, the molar ratio of the double ion structural unit is 70 mol%, with positive The molar ratio of the charge building unit was 30 mol%. A method for screening white blood cells according to the first aspect of the patent application, wherein the diionic building unit is derived from 2-methacryloyloxyethyl phosphorylcholine (phophobetaine methacrylate; PBMA)) [2-(Methacryloyloxyethyl)ethyl]dimethyl(3-sulfopropyl)-ammonium hydroxide (sulfobetaine methacrylate, SBMA) Or 2-Carboxy- N , N- dimethyl- N- (2 ' -(methacryloyloxy)ethyl)ethanamin-ium inner salt(carboxybetaine methacrylate (CBMA)) A method for screening white blood cells according to item 3 of the patent application, wherein the positively charged structural unit is derived from 2-aminoethyl methacrylate having the following formula: Having the following formula of N- isopropyl acrylamide (N -Isopropylacrylamide) Dimethylaminoethyl methacrylate having the following formula [2-(Methacryloyloxy)ethyl]trimethylammonium;TMA) A method for screening white blood cells according to the third aspect of the patent application, wherein the negatively charged structural unit is derived from 3-sulfopropyl methacrylate having the following formula: 2-Carboxyethyl acrylate having the following formula