Fiber for protein concentration and liquid sample absorber which contains said fiber

Abstract

[Problem] In fields such as tumor marker test and pregnancy test kit, it is known to concentrate a target component (protein) by utilizing a superabsorbent polymer (SAP) as a method for improving test sensitivity. However, due to the powder form, SAP exhibits difficulties in handleability and formability and also causes problems such as falling off. Furthermore, the concentration effect is hardly at a sufficient level. The present invention has been made in view of such problems and on the basis of the discovery that a fiber containing an ammonium salt-type carboxyl group and a crosslinked structure serves as a material for protein concentration having excellent handleability and formability, in addition to having a high protein concentration effect. [Solution] A fiber for protein concentration is a fiber that contains an ammonium salt-type carboxyl group and a crosslinked structure, and is characterized by having a water absorption ratio of 4-300 times.

Description

Fiber for protein concentration and liquid sample absorbent containing said fiber

[0001] The present invention relates to a fiber for concentrating protein, a fiber structure for concentrating protein containing the same, and a liquid sample absorber containing the same, and can be applied to fields such as tumor marker test kits and pregnancy test reagents.

[0002] In the fields of tumor marker testing and pregnancy test kits, it is necessary to concentrate the target component (protein) in order to increase the sensitivity of the test.

[0003] To achieve this goal, it has been reported that superabsorbent polymers (SAPs) can be used (Non-Patent Document 1). SAPs consist of cross-linked polymers with a three-dimensional network structure, and when they are brought into contact with a protein solution, the solution moves into the SAP. If the protein size is larger than the size of the polymer network, it is blocked by the SAP surface, and the sieving function allows the solution to be concentrated.

[0004] Eiji Iritani, Yasuto Mukai, Chisato Ueki, Concentration and desalination of protein solutions using highly water-absorbent gel particles, Journal of the Society of Powder Technology, Vol. 38, No. 8, pp. 555-561, 2001

[0005] However, because SAP is in powder form, it is difficult to handle and mold. Furthermore, even when made into a product, fine powder tends to fall off, making it unsatisfactory for industrial use. Furthermore, the concentration effect is not necessarily at a sufficient level. To date, no material has been reported that overcomes these problems and has a sufficient protein concentration effect.

[0006] The present invention was made in consideration of the above-mentioned problems of the conventional technology. The present inventors discovered that fibers containing ammonium salt-type carboxyl groups and crosslinked structures can serve as a protein concentration material that has a high protein concentration effect and is excellent in handleability and moldability, and thus arrived at the present invention.

[0007] The present invention has the following configurations. (1) A fiber for protein concentration, which is a fiber containing an ammonium salt-type carboxyl group and a crosslinked structure and has a water absorption capacity of 4 to 300 times. (2) The fiber for protein concentration according to (1), which is a crosslinked polyacrylonitrile fiber. (3) The fiber for protein concentration according to (1) or (2), which has a water absorption capacity of 10 to 150 times. (4) A fiber structure containing the fiber for protein concentration according to any one of (1) to (3). (5) A liquid sample absorber containing the fiber structure according to (4). (6) A tumor marker test kit using the liquid sample absorber according to (5). (7) A pregnancy test agent using the liquid sample absorber according to (5).

[0008] By adopting the above-mentioned means, a protein concentration material having sufficient protein concentration effect and excellent handleability and moldability can be obtained. Such a fiber of the present invention can be suitably used for tumor marker tests or pregnancy test reagents, and can also be used in applications such as gel absorbents (concentrated gels) for the purpose of concentrating proteins.

[0009] Hereinafter, embodiments of the present invention will be described in detail.

[0010] In tumor marker tests and pregnancy test kits, it is necessary to concentrate the target components (proteins) contained in blood, urine, etc. to increase the sensitivity of the test. This requires a material that can concentrate the protein. While sufficient protein concentration is important, it also needs to be easy to handle and mold during industrial processing and product use. The great advantage of having a fibrous form is that it can be processed into various shapes such as nonwoven fabric, thread, and fabric, and there is no need to worry about powder generation during use.

[0011] There have been no reports of fibrous protein-enriched materials, and it was not possible to predict what kind of structure would be preferable. However, the inventors' investigations revealed that fibers containing ammonium salt-type carboxyl groups and cross-linked structures, and having the characteristic of swelling when immersed in water, would be excellent protein-enriched materials, leading to the present invention.

[0012] That is, the fiber for protein concentration of the present invention contains ammonium salt type carboxyl groups and a crosslinked structure, and has swelling properties in water.

[0013] The amount of ammonium salt-type carboxyl groups contained in the fiber is 0.4 to 5.0 mmol / g, and preferably 1.0 to 3.0 mmol / g. If the amount of ammonium salt-type carboxyl groups is less than 0.4 mmol / g, the protein concentration effect and water absorbency may not be sufficient, while if it exceeds 5.0 mmol / g, the fiber properties may be poor, making handling during production and processing difficult. Furthermore, a crosslinked structure is necessary to prevent elution of the polymer that constitutes the fiber and to maintain the fiber properties.

[0014] The protein concentration fiber of the present invention has a water absorption capacity of 4 to 300 times its own weight, and preferably 10 to 150 times. If the water absorption capacity is less than 4 times, the protein concentration effect will be insufficient, and if the water absorption capacity exceeds 300 times, the fiber's physical properties will be poor, which may adversely affect processability and handleability, and may also completely absorb blood or urine, making the fiber unsuitable for use in a liquid sample absorber, as described below.

[0015] Although the fiber for protein concentration of the present invention is not particularly limited, crosslinked polyacrylonitrile fibers, which are relatively easy to produce, are suitable. Specific examples of such crosslinked polyacrylonitrile fibers include a water-absorbing layer having salt-type carboxyl groups on the surface obtained by crosslinking and hydrolyzing the surface of an acrylonitrile fiber, and a crosslinked polyacrylonitrile fiber having a core-sheath structure with an acrylonitrile fiber portion remaining in the center.

[0016] In particular, the above-mentioned crosslinked polyacrylonitrile-based fibers are more preferred because they have an acrylonitrile-based fiber portion in the center, which gives the fibers high physical strength and good handleability during processing, and also shows little change in the length direction of the fibers even when absorbing water, resulting in good dimensional stability of the product.

[0017] As described above, such crosslinked polyacrylonitrile fibers can be obtained by subjecting the surface of acrylonitrile fibers to crosslinking and hydrolysis. The crosslinking, i.e., the introduction of crosslinked structures, can be performed before, simultaneously with, or after hydrolysis. Furthermore, the introduction of crosslinked structures can utilize nitrile groups present in the acrylonitrile fibers or carboxyl groups generated by hydrolysis. However, methods utilizing carboxyl groups generated by hydrolysis tend to result in weak gel strength at the gel site formed on the fiber surface by hydrolysis, leading to problems such as the gel dropping off before the introduction of crosslinked structures or the leakage of some of the uncrosslinked carboxyl group-containing polymer. In contrast, the introduction of crosslinked structures using nitrile groups before or during hydrolysis increases gel strength after hydrolysis and reduces the amount of uncrosslinked carboxyl group-containing polymer, which is advantageous in terms of industrial handling and environmental impact. For these reasons, the introduction of crosslinked structures using nitrile groups before or during hydrolysis, or both, is preferred.

[0018] Below, we will explain an example of a method for producing crosslinked polyacrylonitrile fibers in which a crosslinked structure is introduced using nitrile groups. First, the acrylonitrile polymer constituting the raw acrylonitrile fibers is preferably a polymer containing 80% by weight or more, preferably 85% by weight or more, of acrylonitrile. Examples of copolymerizable monomers include vinyl halides and vinylidene halides such as vinyl chloride, vinyl bromide, and vinylidene chloride; ethylenically unsaturated carboxylic acids and their salts such as acrylic acid, methacrylic acid, maleic acid, and itaconic acid; (meth)acrylic acid esters such as methyl (meth)acrylate, ethyl (meth)acrylate, and butyl (meth)acrylate; vinyl esters such as vinyl acetate and vinyl propionate; ethylenically unsaturated sulfonic acids and their salts such as vinyl sulfonic acid, (meth)allylsulfonic acid, and p-styrenesulfonic acid; and vinyl compounds such as (meth)acrylamide, vinylidene cyanide, and methacrylonitrile. Examples of such acrylonitrile fibers include conventionally known acrylic fibers.

[0019] First, a method of performing a hydrolysis treatment after a crosslinking treatment using nitrile groups will be described. A commercially preferred method for introducing a crosslinked structure using nitrile groups into acrylonitrile-based fibers is to treat them in an aqueous solution with a crosslinking agent concentration of 0.1 to 10.0 wt % at a temperature of 50 to 120°C for 5 to 150 minutes. If the crosslinking agent concentration or treatment temperature falls below the lower limit, the amount of crosslinked structure introduced by covalent bonds will be insufficient. Conversely, if the crosslinking agent concentration or treatment temperature exceeds the upper limit, the amount of crosslinked structure introduced by covalent bonds will be too great. In either case, it will be difficult to obtain fibers that meet the absorbency range of the present invention.

[0020] The crosslinking agent is not particularly limited as long as it is a polyfunctional compound having two or more functional groups in one molecule that can chemically react with a nitrile group to form a covalent bond, and examples thereof include polyfunctional compounds having two or more functional groups such as amino groups, epoxy groups, etc. Specific examples include hydrazine hydrate, hydrazine sulfate, hydrazine hydrochloride, hydrazine nitrate, hydrazine bromate, diaminoethane, guanidine carbonate, 1,3-diaminopropane, and ethylene glycol diglycidyl ether.

[0021] The method for hydrolyzing the thus obtained fibers is preferably to prepare fibers to which an alkaline metal compound or an aqueous solution thereof has been attached so that the amount of the alkaline metal compound is within the range of 2.5 to 10.0 mmol / g, preferably 5.0 to 10.0 mmol / g, based on the dry weight of the fibers, and to heat the fibers at a temperature of 80° C. or higher for 5 to 180 minutes, preferably in a moist heat atmosphere at 100 to 150° C. for 10 to 120 minutes. In this hydrolysis treatment, a crosslinking agent may be added as necessary, or the method of simultaneous crosslinking and hydrolysis described below may be directly adopted.

[0022] The alkaline metal compound used herein refers to a substance whose 1.0 wt % aqueous solution has a pH of 7.5 or higher, and examples of such substances include hydroxides of alkali metals such as Na, K, Li, etc., and alkali metal salts of organic acids such as carbonic acid, acetic acid, formic acid, etc. Although water is industrially preferred as a solvent for preparing an aqueous solution of an alkaline metal compound, a mixed solvent of water and a water-miscible organic solvent such as alcohol, acetone, or dimethylformamide may also be used.

[0023] Next, a method for simultaneously carrying out crosslinking treatment using nitrile groups and hydrolysis treatment will be described. An aqueous solution containing a crosslinking agent and an alkaline metal compound is prepared by adhering fibers to the acrylonitrile fiber so that the amount of alkaline metal compound is in the range of 1.0 to 20.0 mmol / g, preferably 2.5 to 15.0 mmol / g, and the amount of crosslinking agent is in the range of 0.01 to 2.0 wt %, preferably 0.05 to 1.5 wt %, based on the dry weight of the fiber. The fibers are then heated at a temperature of 80°C or higher for 5 to 180 minutes, preferably in a moist, heated atmosphere at 100 to 150°C for 10 to 120 minutes. The crosslinking agent and alkali metal compound used can be the same as those described above.

[0024] The counter ions of the carboxyl groups of the fibers obtained in the above manner are ions (for example, sodium ions or potassium ions) derived from the alkali metal compound used in the hydrolysis. In order to replace these ions with ammonium ions, the fibers are first treated with an acid such as an aqueous solution of nitric acid or sulfuric acid to convert the counter ions of the carboxyl groups into hydrogen ions, which are then converted into the -COOH form, and then neutralized with ammonia gas. In this way, ammonium salt-type carboxyl groups (-COONH ４ The crosslinked polyacrylonitrile fiber of the present invention having the above structure can be obtained.

[0025] The crosslinked polyacrylonitrile fiber of the present invention described above uses ammonium salt as a counter ion of the carboxyl group because it has a significantly better protein concentration effect than other counter ions. The detailed reason for this has not been reported and is not clear, but it is presumed to be as follows.

[0026] In other words, by using the ammonium salt type, cross-linking occurs within the fiber structure via the hydrogen atoms of the ammonium ions, creating a fine mesh structure. It is thought that this structure prevents protein molecules with molecular weights in the tens of thousands from passing through the mesh and being absorbed into the fiber structure when the fiber absorbs water. This is thought to increase the protein concentration in the blood and urine that remain unabsorbed. Cross-linking via hydrogen is unique to ammonium ions, which contain hydrogen atoms, and does not occur with sodium or potassium ions. This is thought to be the reason why ammonium ions exhibit superior effects compared to other counter ions.

[0027] Furthermore, the cross-linking via the hydrogen atoms of ammonium ions is not as strong as the cross-linking via covalent bonds, and it is thought that the cross-linking can be reversibly dissolved. Therefore, it is thought that the lack of water absorption that occurs when a dense cross-linking structure via covalent bonds occurs will not occur, and that the protein can be efficiently concentrated by absorbing only water.

[0028] The shape of the fiber structure using the fiber for protein concentration of the present invention is not limited, and examples thereof include nonwoven fabric, paper, thread, woven fabric, knitted fabric, and the like.

[0029] In such a fiber structure, the fiber for protein concentration of the present invention may be used alone or in combination with other fibers, including, but not limited to, polyester, nylon, acrylic, polypropylene, polyethylene, vinylon, cotton, rayon, wool, and glass fiber.

[0030] The fiber structure of the present invention, when in the form of a nonwoven fabric, can be suitably used as a liquid sample absorber that absorbs and retains liquid samples such as blood and urine, among the components constituting tumor marker test kits and pregnancy test reagents. As described above, the protein concentration fiber of the present invention has the property of absorbing water and low-molecular-weight substances contained in blood and urine while not absorbing proteins, thereby enabling the protein concentration in the liquid sample to be increased. Therefore, even when the amount of protein in the liquid sample is small, the fiber structure of the present invention has the effect of concentrating the protein, making it possible to detect the presence of the target protein with high sensitivity.

[0031] The present invention will be described in detail below with reference to examples, but the scope of the present invention is not limited to these examples. Parts and percentages in the examples are by weight unless otherwise specified.

[0032] <Method for measuring water absorption capacity> Approximately 0.5 g of a sample was immersed in 300 ml of deionized water at 25°C for 30 minutes, and then centrifuged to dehydrate (160 G x 5 minutes, where G is the gravitational acceleration), and the weight of the adjusted sample (Y1 (g)) was measured. The sample was then dried in a vacuum dryer at 80°C until a constant weight was reached, and the weight of the resulting fiber (Y2 (g)) was measured and calculated using the following formula: water absorption capacity (times) = (Y1 - Y2) / Y2

[0033] <Method for Measuring the Amount of Ammonium Salt-Type Carboxyl Groups> Approximately 1 g of a fiber sample is immersed in 50 ml of 1 mol / l hydrochloric acid aqueous solution for 30 minutes. The fiber sample is then immersed in water at a bath ratio of 1:500. After 15 minutes, the bath pH is confirmed to be 4 or higher, and the sample is dried (if the bath pH is less than 4, the sample is washed again with water). Approximately 0.4 g of the fully dried fiber sample is then weighed (W1 [g]), 100 ml of water is added, and 15 ml of 0.1 mol / l sodium hydroxide aqueous solution, 0.4 g of sodium chloride, and phenolphthalein are added and stirred. After 15 minutes, the sample is titrated with 0.1 mol / l hydrochloric acid aqueous solution until the color of the phenolphthalein disappears, and the amount of hydrochloric acid aqueous solution consumed (V1 [ml]) is determined. The total amount of carboxyl groups is calculated from the obtained measurements using the following formula: (Formula) Total amount of carboxyl groups [mmol / g] = (0.1 × 15 - 0.1 × V1) / W1 Next, the amount of aqueous hydrochloric acid consumed is determined in the same manner as in the above method for measuring the total amount of carboxyl groups, except that the initial immersion in a 1 mol / L aqueous hydrochloric acid solution and the subsequent water washing are not performed, and the amount of acid-type carboxyl groups is calculated from the resulting measured value. The amount of acid-type carboxyl groups is subtracted from the above total amount of carboxyl groups to calculate the amount of ammonium salt-type carboxyl groups.

[0034] <Protein Concentration Factor> 10 mg of a flocculent evaluation sample was added to 400 μl of a protein-containing aqueous solution and allowed to stand for 10 minutes. The solution was then recovered, and the protein concentration in the solution was measured using the Bradford method. The proteins to be evaluated were BSA (bovine serum albumin) and gamma globulin, with the BSA concentration adjusted to approximately 300 ppm and the gamma globulin concentration adjusted to approximately 200 ppm. The protein concentration factor was calculated from the measured values ​​using the following formula: Protein concentration factor [fold] = (protein concentration in recovered solution) / (protein concentration in solution before sample addition). From the perspective of significantly improving protein detection, the protein concentration factor is preferably 1.50 times or more, more preferably 2.00 times or more, and even more preferably 2.50 times or more. There is no particular upper limit, but from the perspective of being suitable for use with the aforementioned liquid sample absorbent, it is preferably 5.00 times or less, and more preferably 4.00 times or less.

[0035] Examples 1 and 4 A mixed aqueous solution of 35% sodium hydroxide aqueous solution and 0.1% hydrazine aqueous solution was applied to the surface of acrylic fibers in an amount equal to the weight of the acrylic fibers, and hydrolysis was carried out for 15 minutes at 108° C. Thereafter, the fibers were added to an aqueous nitric acid solution to adjust the pH to 2.5, and then neutralized with ammonia, to obtain fibers for protein concentration of the present invention according to Examples 1 and 4.

[0036] Example 2: Acrylic fibers were immersed in a 1.0% aqueous hydrazine solution and then held at 85°C for 40 minutes to introduce a crosslinked structure via covalent bonds, yielding crosslinked acrylic fibers. Subsequently, a mixed aqueous solution of 35% aqueous sodium hydroxide and 0.1% aqueous hydrazine was applied to the fiber surface in an amount equal in weight to the crosslinked acrylic fibers, and hydrolysis was carried out at 113°C for 18 minutes. The fibers were then added to an aqueous nitric acid solution to adjust the pH to 2.5, and then neutralized with ammonia, yielding the fiber for protein concentration according to Example 2 of the present invention.

[0037] Example 3: Acrylic fibers were immersed in a 2.0% aqueous hydrazine solution and then held at 85°C for 120 minutes to introduce a crosslinked structure via covalent bonds, yielding crosslinked acrylic fibers. Subsequently, a mixed aqueous solution of 35% aqueous sodium hydroxide and 0.1% aqueous hydrazine was applied to the fiber surface in an amount equal to the weight of the crosslinked acrylic fibers, and hydrolysis was carried out at 113°C for 18 minutes. The fibers were then added to an aqueous nitric acid solution to adjust the pH to 2.5, and then neutralized with ammonia, yielding a fiber for protein concentration according to Example 3 of the present invention.

[0038] Comparative Example 1 Fibers according to Comparative Example 1 were prepared in the same manner as in Example 1, except that the ammonia neutralization was not carried out.

[0039] Comparative Examples 2 and 7 Fibers according to Comparative Examples 2 and 7 were produced in the same manner as in Example 1, except that the treatment with the nitric acid aqueous solution and the ammonia neutralization were not carried out.

[0040] Comparative Example 3 Fibers according to Comparative Example 3 were produced in the same manner as in Example 2, except that the treatment with the nitric acid aqueous solution and the ammonia neutralization were not carried out.

[0041] Comparative Example 4 Fibers according to Comparative Example 4 were produced in the same manner as in Example 3, except that the treatment with the nitric acid aqueous solution and the ammonia neutralization were not carried out.

[0042] Comparative Example 5 A fiber according to Comparative Example 5 was produced in the same manner as in Example 1, except that instead of ammonia neutralization, treatment was carried out in an aqueous solution containing calcium nitrate in an amount equal to the weight of the acrylic fiber at 30° C. for 30 minutes.

[0043] Comparative Example 6 A fiber according to Comparative Example 6 was produced in the same manner as in Example 1, except that instead of ammonia neutralization, treatment was carried out in an aqueous solution containing magnesium nitrate in an amount equal to the weight of the acrylic fiber at 30° C. for 30 minutes.

[0044] Table 1 shows the evaluation results of the protein concentration effect of the fibers obtained in each of the Examples and Comparative Examples.

[0045]

[0046] A good protein concentration effect was observed in Examples 1 to 4, which used fibers for protein concentration characterized by containing ammonium salt-type carboxyl groups and crosslinked structures, but a sufficient protein concentration effect was not observed in fibers (Comparative Examples 1 to 7) that used ions other than ammonium salt-type ions as counter ions. In particular, comparisons between Example 1 and Comparative Examples 2, 5, and 6, Example 2 and Comparative Example 3, Example 3 and Comparative Example 4, and Example 4 and Comparative Example 7 show that even if the amount of carboxyl groups is roughly the same, when the counter ion is an ammonium ion, the protein concentration ratio is significantly improved compared to when the counter ion is an ammonium ion.

Claims

1. A fiber for protein concentration, which contains ammonium salt-type carboxyl groups and a crosslinked structure and has a water absorption capacity of 4 to 300 times.

2. The fiber for protein concentration according to claim 1, which is a cross-linked polyacrylonitrile fiber.

3. The fiber for protein concentration according to claim 1, characterized in that the water absorption rate is 10 to 150 times.

4. A fiber structure comprising the fiber for protein concentration according to any one of claims 1 to 3.

5. A liquid sample absorbent comprising the fiber structure according to claim 4.

6. A tumor marker test kit using the liquid sample absorbent according to claim 5.

7. A pregnancy test agent characterized by using the liquid sample absorbent material according to claim 5.

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