JPH0718883B2 - Water absorbent resin for concentrating biological fluid, method for producing the same and method for using the same - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to the field of biochemistry and medicine in the case of concentrating a biological fluid in order to analyze a substance of biological origin, for example, protein. The present invention relates to a water absorbing resin for concentrating a biological fluid, which is a polyacrylamide cross-linked product, which makes it possible to concentrate these components by absorbing substantially only water in the liquid without degrading the same, a method for producing the same and a method for using the same.

[Conventional technology]

2. Description of the Related Art Conventionally, it has been widely practiced to collect body fluid such as blood or lymph from an organism and analyze / identify protein components therein for the purpose of medical research / diagnosis. At this time, by separating and removing low-molecular components other than water and protein from the test sample in advance and increasing the concentration of protein component, the sensitivity of analysis can be improved.
Increasing precision is well known.

Conventionally, as described above, several methods have been used for increasing the concentration of protein components. For example, those obtained by bringing a test liquid into contact with an ultrafiltration membrane under pressure and allowing only water to pass therethrough, for example, those described in JP-A-60-145069 and JP-A-61-2284. There is something. Further, a water-absorbent resin composed of a cross-linked product of sodium polyacrylate has been conventionally known, and the water-absorbent resin is usually commercially available in a powder state.

[Problems to be Solved by the Invention]

However, some problems have been pointed out regarding the above-mentioned conventional techniques. For example, the concentration method using an ultrafiltration membrane requires special equipment for filtration, and its operation also requires skill.

In general, the biological fluid for analysis and identification targeted by the present invention is one in which several cc is used, and at the same time, the operation adopted from the fact that a large number of samples are subjected to the test in parallel is It is extremely important that it is simple and reliable. For example, it is easy for those skilled in the art to put the above-mentioned water-absorbing resin into a test liquid to absorb only water and concentrate the protein in the residual liquid.

However, since the water-absorbent resin does not have the ability to separate protein molecules from water molecules, it cannot be applied to this invention for the purpose of protein concentration. Further, when a water-absorbent resin in a powder state is used, it is necessary to separate the gel after water absorption from the test liquid, which makes the operation complicated.

[Means for Solving the Problems]

An object of the present invention is to solve the above problems,
While preserving the advantages of the above water-absorbent resin, by removing the drawbacks as much as possible, it is possible to greatly simplify the operation at the time of use, and further, various characteristics required to solve the problem. It is intended to provide a polyacrylamide crosslinked product for concentrating a biological fluid, that is, a water absorbing resin for concentrating a biological fluid, which has excellent concentrating performance by optimizing the above.

In the present invention, regarding the water-swellable gel containing acrylamide as a main component, the conditions for providing good protein separation performance, that is, the equilibrium water absorption capacity (Se) and the water absorption rate index (Sv)
By optimizing the above two conditions, a concentrating gel having satisfactory performance was reached.

Here, the water absorption capacity after leaving the water-absorbent resin for concentrating biological fluid in water for 24 hours is S24 and the water absorption capacity after leaving it for 3 hours is S24 and 3.
The absorption and absorption capacity after being left for a period of time is defined as S3, and Se = S24, S
If v = S3 / S24 is defined, the optimum range of the concentration gel for the purpose of the present invention can be represented by the following numerical range.

4 <Se <9, Sv> 0.80 Hereinafter, the meaning of each characteristic will be described.

If the water absorption rate of the concentrating gel is too low, it is natural that a large amount of concentrating gel is required per amount of the test liquid during use. From this phenomenon, Se> 4 is essential. On the other hand, a high equilibrium swelling rate means that the voids between the molecular chains at the time of swelling are wide, and corresponds to the increase in the molecular weight of the protein in the test solution that can be incorporated inside. As a result of examining the recovery efficiency of the protein molecule to be analyzed in the biological fluid, it was found that if Se <9, practically satisfactory efficiency is provided.

On the other hand, the size of the swelling speed of the concentration gel affects the time required for concentration and the stability of the concentration rate. Considering the convenience of actual consumers, it is desirable that the time required for concentration is around 3 hours. As described above, there is an upper limit to the range of equilibrium water absorption capacity as the degree of freedom to reach a satisfactory swelling ratio in this time zone. Therefore, the water absorption rate index Sv defined at the beginning is important as another degree of freedom. Needless to say, the water absorption rate index Sv is 3 after the start of water absorption.
Since this is a scale that approaches the equilibrium value at the time point, the larger this value, the smaller the increase in water absorption rate over time and the more stable the concentration rate. As a result of the examination, Sv> 0.
It turns out that 80 is a desirable indicator.

To reach the purpose of this invention, 50% acrylamide was used.
It is desirable to copolymerize 0.5 to 3.0 mol% of two or more water-soluble divinyl compounds with a nonionic acrylic monomer containing at least mol%. In this case, as the first cross-linking agent,
N, N-methylenebisacrylamide (abbreviation: BIS) is preferable, and as the second component, one kind of polyethylene glycol (meth) acrylate or a mixture of one or more kinds having ethylene glycol chains having different polymerization degrees ( Abbreviation, PEGD) is used.

In order to produce a product having a desired gel strength, which is a feature of the present invention, it is especially important to use two or more crosslinking agents in combination. As is well known, polyacrylamide gel produced using BIS alone as a cross-linking agent is extremely fragile, and during use, it is crushed by a slight external stimulus and remains in the concentrated liquid. It becomes necessary to add a separation operation to. In contrast, the use of cross-linking agents with the above combinations makes the product more elastic and significantly reduces the possibility of crushing.

Therefore, the gel for concentration of this product after absorbing water can be easily taken out from the liquid by an appropriate jig such as tweezers, and can be separated from the residual liquid.

In order to obtain such a tough gel for concentration, a water-soluble divinyl compound is added to a nonionic acrylic monomer in an amount of 0.5 to
When copolymerizing 3.0 mol%, the ratio of BIS and DEGDA is 1: 5.
It is desirable to be in the range of ˜4: 1, and particularly preferred range is 1: 3 to 1: 1.

Acrylamide is most favored as the nonionic acrylic monomer used in the present invention, but in the range of 50 mol% or less, hydrogen atoms between polymer chains can be obtained by copolymerizing with other nonionic acrylic monomers. The binding can be reduced and the water absorption rate can be improved.

As the nonionic acrylic monomer used for such a purpose, diacetone acrylamide, N-alkyl.
Acrylamide, N, N-dialkyl acrylamide,
Examples are N, N-mornellin acrylamide, hydroxyethyl (meth) acrylate, polyethylene glycol mono (meth) acrylate and the like.

These nonionic acrylic monomers are subjected to polymerization as a 15 to 30% by weight aqueous solution. Further, within the range of 10 mol% or less, an ionic acrylic monomer can be copolymerized. Examples of the ionic acrylic monomer applied for this purpose include acrylamide / alkyl sulfonate and (meth) acryloyloxyalkyl / trialkyl ammonium salt.

Polymerization of the aqueous monomer solution is convenient for imparting an appropriate specific surface area, so that it is placed in a mold such as a cylinder or a laminated plate and polymerized, and the obtained polyacrylamide gel is cut into a desired size and dried. And make it a product. If necessary, the polyacrylamide gel can be washed with water to remove a trace amount of soluble components remaining in the polyacrylamide gel before drying. The specific surface area Sa of the dry resin is preferably in the range below.

20 <Sa <100 cm ² / g As can be easily understood, the water absorption rate index Sv increases as the surface area per unit volume of gel increases. This means that the range of selection of the raw material monomer used is expanded. According to the experimental results, the effect was more than expected, and it became clear that the specific surface area Sa should be at least 20 cm ² / g.

On the other hand, the allowable upper limit of the specific surface area Sa is related to the ease of handling the polyacrylamide gel. For example, the commercially available gel introduced as the prior art is in a powder form, and its specific surface area Sa exceeds 100 cm ² / g. In such a state, filtration after concentration is indispensable, which brings about the practical inconvenience as described above. The polyacrylamide gel described in the present invention is far more elastic than the conventionally known so-called water-absorbing gel, but in actual use, in order to maintain a stable morphology, Sa <10
The upper limit was 0 cm ² / g.

In the actual test, the test liquid is taken in a test tube or beaker, the water-absorbing resin according to the present invention is put therein, and after leaving it for several hours, the swollen polyacrylamide gel is taken out using a jig such as tweezers and the residual liquid is left. To be tested.

Therefore, it is necessary that the shape of the water-absorbent resin be in consideration of the convenience of loading and unloading, and a columnar shape, a plate shape, or the like is desirable. From the above viewpoint, for example, when the shape is a cylindrical shape, the diameter is 1.5 mm or less, and when the shape is a plate, the thickness is 1.0 mm.
mm or less was suitable.

Polymerization of the monomer is performed by a polymerization initiator and radicals generated by irradiation with ultraviolet rays. As a polymerization initiator, a peroxide such as ammonium persulfate (abbreviation: APS) and reduction of hydrogen sulfite (abbreviation: SL), N, N, N ′, N′-tetramethylethylenediamine (abbreviation: TEMED), etc. The redox type polymerization initiator that is used in combination with the agent is prized,
It is not particularly limited. The above-mentioned peroxide and reducing agent are 0.001 to 1% by weight, preferably
0.002-0.5% by weight is used. The polymerization temperature is not particularly limited as long as it is a temperature at which the initiator functions, but is usually preferably in the range of 5 to 50 ° C.

[Action]

The polyacrylamide cross-linked product for concentrating a biological fluid according to the present invention is a water-absorbent resin having a property of absorbing only water in a short time while eliminating proteins.

The above properties can be achieved by imparting appropriate crosslink density and specific surface area to a water-absorbent resin composed of a crosslinked nonionic acrylic polymer mainly composed of acrylamide.

That is, in this polyacrylamide crosslinked product for concentrating biological fluid, when the equilibrium water absorption capacity is set to 9 or less, protein molecules cannot penetrate into the crosslinked resin and only water is absorbed. As a result of having the hydrophilicity of the polymer functional group mainly composed of acrylamide and the high specific surface area of 20 cm ² / g or more, this polyacrylamide gel can complete water absorption in a short time and give a constant concentration rate.

Further, by using N, N-methylenebisacrylamide and polyethylene glycol (meth) acrylate in combination as a cross-linking agent, a tough gel for concentration can be obtained in a water-absorbed state, and can be easily separated from the residual liquid. it can.

The shape of the water-absorbent gel is columnar, plate-like, etc., and can be put into a test liquid in a test tube, a beaker or the like to easily perform a protein concentration operation.

〔Example〕

Next, examples according to the present invention will be described, but the present invention is limited to the following examples unless it exceeds the scope of the technical idea constituted by the matters described in the claims. Of course, it is not a thing.

Example-1 (Sample preparation method) A glass tall beaker having a capacity of 200 ml was charged with the amount of the monomer shown in Table-1 and 120 ml of distilled water, nitrogen aeration was performed, and the internal temperature was adjusted to 5 ° C, followed by polymerization. NN ′ as an initiator
Azobisamidinopropane hydrochloride (V-50 manufactured by Wako Pure Chemical Industries)
0.2% aqueous solution 1.5 ml, ammonium persulfate 0.1% aqueous solution 1.5
ml and 1.5 ml of a 0.1% aqueous solution of sodium hydrogen sulfite were sequentially added and mixed. After uniformly mixing the polymerization initiator, length 12
0 mm, 2.5 mm inner diameter, 0.02 mm wall thickness polypropylene cylinder 10
A bundle of 0 pieces was bundled and inserted into the glass beaker so that the direction of the axis was the vertical direction, and the polymerization was continued.

2 hours after the initiation of polymerization, place each glass beaker at 55 ° C for 6 hours.
The temperature was kept warm to complete the polymerization.

After the completion of the polymerization, the bundle of polypropylene cylinders was separated and cut into pieces each having a length of 4.5 mm, which were then air-dried at 70 ° C., and the columnar polymer was taken out in a dried state and subjected to a test.

The monomer composition is shown in Table 1. The abbreviations used in Table-1 have the following meanings.

AAM: Acrylic amide, BIS: Methylenebisacrylic amide, and DEGDM: Diethylene glycol dimethacrylate [Test method] (1) Measurement of water absorption: Put 50 ml of distilled water in a beaker with a capacity of 200 ml, and put 3 samples in each beaker. After 3 hours, 24 hours, and 48 hours, take out one piece each with tweezers, absorb the water on No. 2 filter paper, and then measure the weight with a balance that can measure up to the last 4 digits, and after 3 hours Of water absorption (that is, water absorption ratio S3)
The water absorption rate index Sv was calculated from the water absorption after 24 hours (that is, the water absorption capacity S24).

Note that Sv = S3 / S24.

(2) Measurement of BSA (bovine serum albumin) recovery rate; BSA was used as an index for protein concentration. Prepare 5 ml of BSA 0.05% solution in the Wasselman test tube and use 15 to 20 samples (weight, about 0.06 g) in total in each test tube so that the residual liquid of the BSA solution becomes about 0.5 ml. Concentrated.

The concentration of BSA in the residual liquid of about 0.5 ml was quantified by liquid chromatography, and the recovery rate was calculated by comparison with the theoretical concentration rate by the concentration ratio. Liquid chromatograph conditions are columns;
Clear pack GFS-3.8 x 300 mm, lysis solution; 20 mM phosphate buffer (pH 7.0), 0.2 M NaCl, detector; UV215 nm2.0 AUSF, injection volume; 100 μ (microliter), chart speed; 1
It is 0 cm / hr.

The results of each evaluation test are shown in Table-2.

Example-2 Acrylamide 30 was added to a device similar to that used in Example-1.
g, BIS 0.3 g, DEGDM 0.9 g, and 120 ml of distilled water were charged, except that a bundle of glass tubes having various inner diameters of 120 mm in length was inserted, a dried polymer sample was prepared in the same manner as in Example-1. Obtained.

Table 3 shows the relationship between the dry matter column diameter of the cylindrical gel and the specific surface area Sa.
Shown in.

Further, the aqueous monomer solution having the above composition was polymerized in the gap between the glass plates sandwiching the spacer, and then shredded into 2 cm squares by a cutter, dried while preventing deformation, and subjected to the test. Table 4 shows the thickness and specific surface area Sa of the dried plate.

For the samples described in Table-3 and Table-4, the results of the evaluation test performed in the same manner as in Example-1 are shown in Table-5.

Example-3 BIS0.3 with two acrylic monomers in the amounts listed in Table-6
By the same procedure as in Example-1, except that an aqueous monomer solution consisting of g, 0.9 g of DEGDM, and 120 ml of distilled water was used.
A sample of the dried polymer was obtained and subjected to the test.

The evaluation test results for each sample are shown in Table-7. In addition,
The abbreviations used in Table 6 have the following meanings.

AAM; acrylic amide, DAAAM; diacetone acrylic amide, DEGMM; diethylene glycol monomethacrylate, HEA; hydroxyethyl acrylate, and Na-AAC;
Sodium Acrylate Example-4 Two crosslinkers in the amounts listed in Table-8 and acrylamide
A sample of the dried polymer was obtained by the same procedure as in Example 1 except that an aqueous monomer solution consisting of 30 g and 120 ml of distilled water was used and subjected to the test.

The results of the evaluation test for each test are shown in Table-9.

The abbreviations used in Table-8 have the following meanings.

DEGDA; diethylene glycol diacrylate, and TEGD
A: Triethylene glycol diacrylate

Claims (3)

[Claims] 1. A cross-linked water-absorbing polymer in the form of small particles is introduced into a test biological fluid in a container to absorb water and low-molecular-weight components in the test solution, and then the cross-linked product and the residual liquid. When the residual liquid is separated into an analytical sample, the composition of the crosslinked product is a water-soluble divinyl compound consisting of methylenebisacrylamide and polyethylene glycol (meth) acrylate,
And a nonionic monovinyl monomer copolymer containing acrylamide in an amount of 50 mol% or more, wherein the total amount of the water-soluble divinyl compound is 0.5 to 3 mol% with respect to acrylamide, and the crosslinked product absorbs water for 24 hours. Later water absorption ratio is 4
The water-absorbent resin for concentrating biological fluid is characterized in that the cross-linked product has a specific surface area of 20 to 100 cm ² / g. 2. A water-soluble divinyl compound comprising methylenebisacrylamide and polyethylene glycol (meth) acrylate, and an aqueous solution of a nonionic monovinyl monomer containing acrylamide in an amount of 50 mol% or more are copolymerized. When polymerizing the monomer aqueous solution adjusted so that the total amount of the compound is 0.5 to 3 mol% with respect to acrylamide, use a container in which the specific surface area of the resulting water-absorbent resin is 20 to 100 cm ² / g. A method for producing a water-absorbent resin for concentrating a biological fluid, which comprises drying a hydrogel obtained by polymerization in the container. 3. The water-absorbent resin for concentrating a biological fluid according to claim 1 is put into a biological fluid for a test in a container so that the water-absorbent resin selectively absorbs only water and low molecular weight components. A method for concentrating a biological fluid, which comprises concentrating a protein component in the residual liquid by separating it from the residual liquid later.