Adsorbent and method for manufacturing the same

Abstract

To provide an absorbent capable of stably and selectively absorbing a protein such as viruses and a manufacturing method thereof.SOLUTION: Provided is an absorbent comprising a carboxymethylcellulose gel and a glycoside that modifies the surface of the carboxymethylcellulose gel, wherein the carboxymethylcellulose gel is a complex of the carboxymethylcellulose and an epoxy compound active at both ends that intermolecularly crosslinks the carboxymethylcellulose, the glycoside is a sugar derivative having an amino group at the end of an aglycone moiety, and a hydroxy group of the carboxymethylcellulose and the epoxy group of the epoxy compound active at both ends are addition bonded together, and the carboxymethyl group of carboxymethylcellulose gel and the amino group at the end of the aglycone part of the glycoside are bonded to form an amide bond.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an adsorbent that adsorbs proteins such as viruses and a method for producing the same. [Background Art]

[0002] In recent years, virus analysis technology has advanced, and the actual nature of viruses has been gradually elucidated. Examples of virus-carrying media include food, water, and air. Viruses have a wide variety of infection routes, and the concentration of viruses present in the media is dilute. Therefore, it has conventionally been extremely difficult to elucidate the infection routes of viruses that cause infectious disease damage. In order to elucidate the infection routes of viruses, a method for detecting viruses present in media with high sensitivity is required. In order to detect viruses with high sensitivity, means for adsorbing and concentrating viruses from media with high efficiency is required.

[0003] As a means for adsorbing viruses, virus adsorbents are known. As a virus adsorbent, for example, one obtained by pulverizing carbide of seed shells of pecan plants belonging to the Juglandaceae family and supporting or mixing the powder on a filter-shaped base material is known (see, for example, Patent Document 1). [Prior Art Literature] [Patent Literature]

[0004] [Patent Document 1] Japanese Unexamined Patent Publication No. 2007-190541 [Summary of the Invention] [Problem to be Solved by the Invention]

[0005] The virus adsorbent of Patent Document 1 is produced by carbonizing seed shells of pecan plants, so there is variation in quality, and it has not been possible to stably and selectively adsorb viruses.

[0006] The present invention has been made in view of the above circumstances, and aims to provide an adsorbent that can stably and selectively adsorb proteins such as viruses, and a method for producing the same. [Means for solving the problem]

[0007] The present invention has the following aspects. [1] An adsorbent comprising a carboxymethylcellulose gel and a glycoside that modifies the surface of the carboxymethylcellulose gel, The carboxymethylcellulose gel is a composite of carboxymethylcellulose and a terminally active epoxy compound that intermolecularly crosslinks the carboxymethylcellulose. The glycoside is a sugar derivative having an amino group at the aglycone end, The hydroxyl group of the carboxymethylcellulose and the epoxy groups of the terminally active epoxy compound are added together, An adsorbent in which the carboxymethyl group of the carboxymethylcellulose gel and the amino group at the aglycone end of the glycoside are bonded together by an amide bond. [2] The adsorbent according to [1], wherein the number of molecules of the terminally active epoxy compound bonded to each hydroxyl group of the carboxymethylcellulose is 0.10 or more and 1.00 or less. [3] The adsorbent according to [1] or [2], wherein the degree of substitution, which represents the proportion in which the hydroxyl groups of the carboxymethylcellulose are replaced by carboxymethyl groups, is 1.2 or less. [4] The adsorbent according to any one of [1] to [3], wherein the two-terminal active epoxy compound is at least one of polyethylene glycol diglycidyl ether and ethylene glycol diglycidyl ether. [5] The adsorbent according to any one of [1] to [4], wherein the weight-average molecular weight (Mw) of the carboxymethylcellulose is 83,000 or more. [6] A step of dissolving carboxymethylcellulose in an alkaline aqueous solution to prepare solution A containing the carboxymethylcellulose, The process involves stirring and mixing oil and surfactant to prepare solution B for the oil phase, The steps include adding solution A to solution B, microemulsifying solution A and solution B to form a water-in-oil emulsion, The steps include adding a terminally active epoxy compound to the water-in-oil emulsion, and intermolecularly crosslinking the carboxymethylcellulose with the terminally active epoxy compound to form a carboxymethylcellulose gel, The steps include: dissolving the carboxymethylcellulose gel in an organic solvent to prepare a solution C containing the carboxymethylcellulose gel; A method for producing an adsorbent, comprising the steps of: adding a glycoside having an amino group at the aglycone end to the solution C; forming an amide bond by linking the carboxymethyl group of the carboxymethylcellulose gel with the amino group at the aglycone end of the glycoside; and modifying the surface of the carboxymethylcellulose gel with the glycoside. [Effects of the Invention]

[0008] According to the present invention, it is possible to provide an adsorbent that can stably and selectively adsorb proteins such as viruses, and a method for producing the same. [Brief explanation of the drawing]

[0009] [Figure 1] This is a schematic perspective view showing an adsorbent according to one embodiment of the present invention. [Figure 2] This figure shows the relationship between the amount of WGA lectin adsorbed onto the adsorbent and the interaction time between the adsorbent and the mixture containing WGA lectin in Example 1. [Figure 3] This figure shows the relationship between the amount of WGA lectin adsorbed onto the adsorbent and the concentration of the adsorbent in the reaction solution in Example 2. [Figure 4] This figure shows the relationship between the amount of WGA lectin adsorbed onto the adsorbent and the type of glycoside immobilized on the adsorbent in Example 3. [Figure 5]It is a diagram showing the relationship between the adsorption amount of ECA lectin on the adsorbent and the types of glycosides immobilized on the adsorbent in Example 4. [Figure 6] It is a diagram showing the relationship between the adsorption amount of SSA lectin on the adsorbent and the types of glycosides immobilized on the adsorbent in Example 5. [Figure 7] It is a diagram showing the relationship between the adsorption amount of MAA lectin on the adsorbent and the types of glycosides immobilized on the adsorbent in Example 6. [Figure 8] It is a diagram showing the relationship between the adsorption amount of BSA on the adsorbent and the types of glycosides immobilized on the adsorbent in Example 7. [Figure 9] It is a fluorescence image captured by a fluorescence microscope (high magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 10] It is a superimposed image of a fluorescence image and a bright-field image captured by a fluorescence microscope (high magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 11] It is a bright-field image captured by a fluorescence microscope (high magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 12] It is a fluorescence image captured by a fluorescence microscope (low magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 13] It is a superimposed image of a fluorescence image and a bright-field image captured by a fluorescence microscope (low magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 14] It is a bright-field image captured by a fluorescence microscope (low magnification) showing the binding between the adsorbent and fluorescence-labeled WGA lectin in Example 8. [Figure 15] It is a diagram showing the relationship between the adsorption amount of WGA lectin on the adsorbent per 1 g of dried adsorbent and the concentration of WGA lectin in the reaction solution in Example 9. [Figure 16]In Example 9, it is a diagram showing data obtained by plotting the adsorption equilibrium concentration against the equilibrium adsorption amount and converting the data according to the Langmuir adsorption isotherm, with respect to the relationship between the adsorption amount of WGA lectin on the adsorbent and the concentration of WGA lectin in the reaction solution. DETAILED DESCRIPTION OF THE INVENTION

[0010] Embodiments of the adsorbent and the method for producing the adsorbent of the present invention will be described hereinafter. It should be noted that the present embodiment is specifically described for a better understanding of the spirit of the invention, and is not intended to limit the present invention unless otherwise specified.

[0011] [Adsorbent] Figure 1 is a perspective view schematically showing an adsorbent according to an embodiment of the present invention. As shown by formula (1) below, the adsorbent of the present embodiment is a compound comprising carboxymethyl cellulose gel (a) and a glycoside (b) modifying the surface of the carboxymethyl cellulose gel (a). In the adsorbent of the present embodiment, the carboxymethyl group of the carboxymethyl cellulose gel (a) and the amino group at the terminal aglycone portion of the glycoside (b) are bonded to form an amide bond.

[0012] [Chemical Formula]

[0013] As shown in Figure 1, the adsorbent 1 of the present embodiment comprises spherical carboxymethyl cellulose gel 2 and glycoside 3 distributed over the entire surface 2a of the carboxymethyl cellulose gel 2.

[0014] Carboxymethylcellulose gel (a) is a complex of carboxymethylcellulose (c) and a terminally active epoxy compound (d), as shown in formula (2) below. In carboxymethylcellulose gel (a), the hydroxyl group of carboxymethylcellulose (c) and the epoxy group of the terminally active epoxy compound (d) are added together, thereby creating intermolecular crosslinks of carboxymethylcellulose (c) with the terminally active epoxy compound (d).

[0015] [ka]

[0016] The number of molecules of the terminally active epoxy compound (d) bonded to each hydroxyl group of carboxymethylcellulose (c) is preferably 0.10 or more and 1.00 or less, and more preferably 0.25 or more and 0.60 or less. If the number of molecules is less than the lower limit, the adsorbent will partially dissolve when it comes into contact with water and will not be able to maintain its shape. If the number of molecules exceeds the upper limit, the adsorbent will become a solid powder and its surface area will decrease.

[0017] The adsorbent is spherical in shape. The average particle diameter of the adsorbent is preferably between 200 nm and 10,000 nm, and more preferably between 200 nm and 5,000 nm. If the average particle diameter of the adsorbent is below the lower limit, it is difficult to recover the adsorbent. If the average particle diameter of the adsorbent exceeds the upper limit, the surface area per particle mass decreases, and therefore the amount of lectin adsorbed decreases.

[0018] The average particle size of the adsorbent is calculated by selecting 100 particles arbitrarily from those observed with a scanning electron microscope and averaging the particle sizes of their primary particles.

[0019] In the adsorbent, the amount of glycoside used to modify the surface of the carboxymethylcellulose gel (a), that is, the amount of glycoside added when coating the surface of the carboxymethylcellulose gel (a) with the glycoside, is preferably 0.4 mol% to 500 mol%, more preferably 3 mol% to 200 mol%, and even more preferably 50 mol% to 150 mol%, when the number of moles of carboxymethyl groups contained in the carboxymethylcellulose gel (a) is taken as 100%. If the amount of glycoside added is less than the lower limit, the amount of target protein adsorbed onto the adsorbent will decrease significantly. If the amount of glycoside added exceeds the upper limit, the loss of glycosides will be large, meaning that the proportion of glycosides that are wasted without coating the adsorbent will increase, and the synthesis efficiency will decrease significantly.

[0020] "Carboxymethylcellulose gel" The carboxymethylcellulose gel (a) is spherical in shape. The average particle diameter of the carboxymethylcellulose gel (a) is preferably 200 nm to 10,000 nm, and more preferably 200 nm to 5,000 nm. If the average particle diameter of the carboxymethylcellulose gel (a) is below the lower limit, it is difficult to recover the particles of the carboxymethylcellulose gel (a). If the average particle diameter of the carboxymethylcellulose gel (a) exceeds the upper limit, the surface area per unit mass decreases, and therefore the amount of glycoside modification decreases.

[0021] The average particle size of the carboxymethylcellulose gel (a) can be measured by the same method as the method for measuring the average particle size of the adsorbent described above.

[0022] "Carboxymethylcellulose" The weight-average molecular weight (Mw) of carboxymethylcellulose (c) is preferably 83,000 or more, and more preferably 100,000 to 120,000. If the weight-average molecular weight (Mw) of carboxymethylcellulose (c) is below the lower limit, it becomes difficult to form the carboxymethylcellulose gel (a) into spherical particles. If the weight-average molecular weight (Mw) of carboxymethylcellulose (c) exceeds the upper limit, the viscosity of carboxymethylcellulose (c) increases rapidly, making it difficult to uniformly mix it with the crosslinking agent during the synthesis of the carboxymethylcellulose gel (a).

[0023] The weight-average molecular weight (Mw) of carboxymethylcellulose (c) can be measured by gel filtration chromatography or rotational viscometer.

[0024] The degree of substitution, which represents the proportion of hydroxyl groups in carboxymethylcellulose (c) that are replaced by carboxymethyl groups, is expressed as "(number of hydroxyl groups replaced by carboxymethyl groups (total number of hydroxyl groups)) / (total number of hydroxyl groups before substitution with carboxymethyl groups)". The degree of substitution is preferably 1.2 or less, and more preferably 1.1 or less. If the degree of substitution exceeds the upper limit, the reaction with the crosslinking agent during the synthesis of carboxymethylcellulose gel (a) becomes difficult, and it becomes impossible to form carboxymethylcellulose gel (a) into spherical particles. The degree of substitution is preferably 0.5 or higher, and more preferably 0.7 or higher. If the degree of substitution is below the lower limit, carboxymethylcellulose (c) will not dissolve in water, and carboxymethylcellulose gel (a) cannot be obtained.

[0025] "Active epoxy compounds at both ends" The terminally active epoxy compound (d) is not particularly limited as long as it has epoxy groups at both ends that can bond to the hydroxyl group of carboxymethylcellulose (c). For example, at least one can be selected from monoepoxy compounds such as ethylene oxide, allyl glycidyl ether, 2-ethylhexyl glycidyl ether, methyl glycidyl ether, phenyl glycidyl ether, and epichlorohydrin (ECH); diepoxy compounds such as ethylene glycol diglycidyl ether (EGDE), polyethylene glycol diglycidyl ether, propylene glycol diglycidyl ether, polypropylene glycol diglycidyl ether, neopentyl glycol diglycidyl ether, and glycerin nobricidyl ether; triepoxy compounds such as glycerin triglycidyl ether and triglycidyl isocyanurate; and polyepoxy compounds such as glycerol polyglycidyl ether, trimethylolpropane polyglycidyl ether, pentaerythritol polyglycidyl ether, and sorbitol polyglycidyl ether. Among these, at least one of polyethylene glycol diglycidyl ether and ethylene glycol diglycidyl ether is preferred because it allows for the synthesis of carboxymethylcellulose gel (a) under mild reaction conditions.

[0026] "Glycoside" Glycoside (b) consists of a glycone portion (sugar portion) and an aglycone portion (non-sugar portion), with an amino group at the end of the aglycone portion. Glycoside (b) is not particularly limited as long as it is a glycoside having an amino group at the end of the aglycone portion, and includes compounds such as glycosylamines that do not have a spacer structure at the end of the aglycone portion. Examples of glycone portions include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylmannosamine, lactose, N-acetyllactosamine, cellobiose, chitobiose, sialyl lactose, sialyl-N-acetyllactosamine, lacto-N-neotetraose, Gb3, LewisX, etc. The sugar chain structure and type of the glycoside should be appropriately selected depending on the type of protein to be adsorbed.

[0027] The adsorbent of this embodiment comprises a carboxymethylcellulose gel and a glycoside that modifies the surface of the carboxymethylcellulose gel. Therefore, proteins such as viruses can be adsorbed onto the glycocone portion of the glycoside. Furthermore, because the adsorbent of this embodiment is spherical, proteins such as viruses can be uniformly adsorbed onto its surface. Moreover, by changing the type of glycocone portion of the glycoside, the adsorbent of this embodiment can selectively adsorb proteins by changing the type of proteins it can adsorb.

[0028] [Method for manufacturing adsorbents] A method for producing an adsorbent according to one embodiment of the present invention will be described. The method for producing the adsorbent of this embodiment is the method for producing the adsorbent of the embodiment described above. The method for producing the adsorbent of this embodiment comprises the steps of: preparing an aqueous solution of carboxymethylcellulose (solution A) (hereinafter referred to as "step 1"), preparing a solution for the oil phase (solution B) (hereinafter referred to as "step 2"), emulsifying solution A and solution B (hereinafter referred to as "step 3"), causing a crosslinking reaction to form a carboxymethylcellulose gel (hereinafter referred to as "step 4"), preparing a solution C containing the carboxymethylcellulose gel (hereinafter referred to as "step 5"), and modifying the surface of the carboxymethylcellulose gel with a glycoside (hereinafter referred to as "step 6").

[0029] "Process 1" In step 1, first, carboxymethylcellulose is added to an alkaline aqueous solution, and the aqueous solution and carboxymethylcellulose are stirred and mixed to dissolve the carboxymethylcellulose in the alkaline aqueous solution and prepare an aqueous carboxymethylcellulose solution (solution A).

[0030] The alkaline aqueous solution is not particularly limited, but examples include aqueous sodium hydroxide solution, aqueous potassium hydroxide solution, and aqueous barium hydroxide solution. Among these, aqueous sodium hydroxide solution is preferred due to its high versatility. The hydroxide ion (OH) concentration in the aqueous solution is preferably 0.2 M or more and 1.5 M or less, and more preferably 0.5 M or more and 1.0 M or less. If the hydroxide ion (OH) concentration is below the lower limit, the crosslinking reaction of carboxymethylcellulose by the epoxy compound will not proceed. If the hydroxide ion (OH) concentration exceeds the upper limit, hydrolysis of the carboxymethylcellulose molecules will proceed, and the yield of carboxymethylcellulose gel will decrease.

[0031] In the preparation of solution A, carboxymethylcellulose is completely dissolved in the aqueous solution. The content of carboxymethylcellulose relative to 100% by mass of the total amount of the aqueous solution is preferably 5% by mass or more and 20% by mass or less, and more preferably 10% by mass or more and 15% by mass or less. If the content of carboxymethylcellulose is below the lower limit, the crosslinking reaction does not proceed easily, and spherical particles of carboxymethylcellulose gel cannot be obtained. If the content of carboxymethylcellulose exceeds the upper limit, the viscosity increases rapidly, and carboxymethylcellulose gel with fine particle size cannot be obtained.

[0032] "Process 2" In step 2, the oil and surfactant are stirred and mixed to prepare solution B for the oil phase.

[0033] The oil is not particularly limited, but examples include vegetable oils, castor oil, grease, liquid paraffin, and silicone oil. Among these, silicone oil is preferred from the viewpoint of chemical stability in alkaline aqueous solutions.

[0034] The surfactant is not particularly limited, but examples include polyoxyethylene alkyl ethers, fatty acid sorbitan esters, alkyl polyglycosides, fatty acid diethanolamides, alkyl monoglycerol ethers, and polyether-modified silicones. Among these, polyether-modified silicones are preferred from the viewpoint of chemical stability.

[0035] The amount of surfactant added is adjusted according to the amounts of solution A and solution B used in the following step 3. Specifically, the amount of surfactant contained in solution B used in step 3 is preferably 0.02 parts by mass or more and 0.1 parts by mass or less, and more preferably 0.05 parts by mass or more and 0.08 parts by mass or less, per 100 parts by mass of aqueous carboxymethylcellulose solution (solution A). If the amount of surfactant added is below the lower limit, the particle size of the carboxymethylcellulose gel increases. If the amount of surfactant added exceeds the upper limit, the particle size of the carboxymethylcellulose gel decreases drastically.

[0036] "Process 3" In step 3, first, the carboxymethylcellulose aqueous solution (solution A) prepared in step 1 is mixed with the terminally active epoxy compound and stirred.

[0037] The amount of the terminally active epoxy compound added to solution A is preferably 2 parts by mass or more and 8 parts by mass or less, and more preferably 3 parts by mass or more and 5 parts by mass or less, per 100 parts by mass of aqueous solution of carboxymethylcellulose (solution A). If the amount of terminally active epoxy compound added is less than the lower limit, the crosslinking reaction will be insufficient. If the amount of terminally active epoxy compound added exceeds the upper limit, the crosslinking density will increase and it will become a solid powder, so a sufficient surface area cannot be obtained.

[0038] Next, solution A is added to solution B and microemulsified using a homogenizer or the like (to form a water-in-oil (W / O) emulsion containing carboxymethylcellulose).

[0039] The amount of oil added to solution B is preferably 100 parts by mass or more, and more preferably 250 parts by mass or more, per 100 parts by mass of aqueous carboxymethylcellulose solution (solution A). If the amount of oil added is less than the lower limit, it is difficult to form nanoparticles of carboxymethylcellulose gel.

[0040] In step 3, homogenization is preferred for microemulsification. A solution containing carboxymethylcellulose and a crosslinking agent (solution A) is added to solution B (for example, a silicone oil containing a surfactant), and the mixture is homogenized to prepare an emulsion. By micronizing the water-in-oil (W / O) droplets, the average particle size of the resulting carboxymethylcellulose gel can be made smaller.

[0041] The vibration frequency in the homogenizer treatment is preferably 10,000 rpm to 25,000 rpm, and more preferably 15,000 rpm to 22,000 rpm. If the vibration frequency is below the lower limit, the particle size of the resulting emulsion will be large, and a sufficient surface area will not be obtained. If the vibration frequency exceeds the upper limit, the particles of the resulting emulsion will be too fine.

[0042] "Process 4" In step 4, the water-in-oil emulsion formed in step 3 is allowed to stand, and the carboxymethylcellulose molecules are intermolecularly crosslinked by the terminally active epoxy compounds. This forms a carboxymethylcellulose gel, which is a composite of carboxymethylcellulose and the terminally active epoxy compounds. In step 4, the carboxymethyl group and the unsubstituted hydroxyl group in carboxymethylcellulose are added to the epoxy group of the terminally active epoxy compound.

[0043] The standing time and conditions are not particularly limited, as long as the carboxymethylcellulose undergoes intermolecular crosslinking, but for example, standing at room temperature for 24 hours is a possible example.

[0044] In step 4, after the reaction in which the hydroxyl group of carboxymethylcellulose and the epoxy groups of the terminally active epoxy compound are added together is completed, the solution containing the complex of carboxymethylcellulose and the terminally active epoxy compound is sequentially washed with hexane and alcohol. Specifically, hexane is added to the solution containing the complex to extract the oil (e.g., silicone oil) derived from solution B into the hexane, and then the particles are recovered by centrifugation. Next, alcohol is added to remove sodium hydroxide, unreacted terminally active epoxy compound, etc., and fine particles of the complex are produced. The alcohol is not particularly limited, but examples include ethanol, denatured ethanol, methanol, and isopropanol.

[0045] Furthermore, the fine particles of the composite are added to water to swell and gel the composite particles with water. Then, the gelled composite is centrifuged to obtain a carboxymethylcellulose gel.

[0046] "Process 5" In step 5, the carboxymethylcellulose gel formed in step 4 is dissolved in an organic solvent to prepare solution C containing the carboxymethylcellulose gel.

[0047] The organic solvent disperses the carboxymethylcellulose gel and does not inhibit the amide bond formation reaction with the glycoside. The organic solvent is not particularly limited, but examples include dimethylformamide and dimethyl sulfoxide (DMSO). Among these, dimethyl sulfoxide is preferred from the viewpoint of efficiently carrying out the reaction in step 6.

[0048] The carboxymethylcellulose gel content in solution C is preferably 0.01% to 10% by mass, and more preferably 0.05% to 2% by mass, based on 100% by mass of the total amount of solution C. If the carboxymethylcellulose gel content is below the lower limit, the reaction efficiency of the glycoside decreases. If the carboxymethylcellulose gel content exceeds the upper limit, the viscosity increases, and the reaction efficiency decreases.

[0049] "Process 6" In step 6, a glycoside having an amino group at the aglycone end is added to solution C prepared in step 5, and an amide bond is formed by bonding the carboxymethyl group of the carboxymethylcellulose gel with the amino group at the aglycone end of the glycoside. This modifies the carboxymethylcellulose gel with the glycoside, thereby obtaining the adsorbent of this embodiment.

[0050] The amount of glycoside added to solution C is preferably 0.6 parts by mass or more and 1000 parts by mass or less per 100 parts by mass of carboxymethyl groups of the carboxymethylcellulose gel contained in solution C, more preferably 3 parts by mass or more and 200 parts by mass or less, and even more preferably 50 parts by mass or more and 100 parts by mass or less. If the amount of glycoside added is less than the lower limit, the adsorption rate of the adsorbent decreases significantly. If the amount of glycoside added exceeds the upper limit, the viscosity of the reaction solution increases and the reaction efficiency decreases.

[0051] It is preferable to add a condensing agent to solution C in order to form an amide bond by linking the carboxymethyl group of the carboxymethylcellulose gel with the amino group at the end of the aglycone portion of the glycoside. Examples of condensing agents, though not particularly limited, include 1-[bis(dimethylamino)methylene]-1H-benzotriazolium 3-oxide hexafluorophosphate (HBTU) and benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphate salt (BOP).

[0052] The amount of condensing agent added to solution C is preferably 2.5 parts by mass or more and 4000 parts by mass or less per 100 parts by mass of carboxymethyl groups of the carboxymethylcellulose gel contained in solution C, more preferably 12.5 parts by mass or more and 800 parts by mass or less, and even more preferably 200 parts by mass or more and 400 parts by mass or less. If the amount of condensing agent added is less than the lower limit, the binding rate of the glycoside decreases significantly. If the amount of condensing agent added exceeds the upper limit, the viscosity of the reaction solution increases and the reaction efficiency decreases.

[0053] It is preferable to add a reaction inhibitor to solution C in order to promote the reaction that forms an amide bond by linking the carboxymethyl group of the carboxymethylcellulose gel with the amino group at the end of the aglycone portion of the glycoside, and to suppress side reactions. Examples of reaction inhibitors, though not particularly limited, include 1-hydroxybenzotriazole (HOBt).

[0054] The amount of reaction inhibitor added to solution C is preferably 0.7 parts by mass or more and 1100 parts by mass or less per 100 parts by mass of carboxymethyl groups of the carboxymethylcellulose gel contained in solution C, more preferably 3.4 parts by mass or more and 220 parts by mass or less, and even more preferably 55 parts by mass or more and 110 parts by mass or less. If the amount of reaction inhibitor added is less than the lower limit, the structural uniformity of the adsorbent decreases. If the amount of reaction inhibitor added exceeds the upper limit, the viscosity of the reaction solution increases and the reaction efficiency decreases.

[0055] According to the method for manufacturing the adsorbent of this embodiment, the adsorbent of the above-described embodiment can be manufactured. [Examples]

[0056] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0057] [Synthesis Example 1] "Synthesis of carboxymethylcellulose gel" 30 mL of 0.5 M sodium hydroxide solution was poured into a 100 mL beaker. Carboxymethylcellulose (trade name: Cekol 700, manufactured by CP Kelco) was added to this sodium hydroxide aqueous solution, and the mixture was stirred and mixed to dissolve the carboxymethylcellulose in the sodium hydroxide aqueous solution, thereby preparing an aqueous solution of carboxymethylcellulose. The carboxymethylcellulose content was set to 10% by mass per 100% by mass of the total solution volume. Next, while continuing to stir the aqueous carboxymethylcellulose solution, 1.2 mL of ethylene glycol diglycidyl ether was added and mixed uniformly to prepare solution I. Next, an oil phase was prepared by mixing 15 mL of silicone oil (product name: KF-96L-0.65cs, manufactured by Shin-Etsu Chemical Co., Ltd.) with 0.3 mL of a solution silicone-based surfactant (product name: KF-6048, manufactured by Shin-Etsu Chemical Co., Ltd.). 5 mL of solution I was added to this oil phase and emulsified using a homogenizer (T25 Digital Ultra Turrax, manufactured by IKA) at a frequency of 20,000 rpm for 3 minutes. The crosslinking reaction was allowed to proceed at room temperature for 24 hours to produce a carboxymethylcellulose gel consisting of carboxymethylcellulose and ethylene glycol diglycidyl ether. Next, the solution containing the carboxymethylcellulose gel was transferred to a 50 mL centrifuge tube, 10 mL of hexane was added and shaken to extract the silicone oil and surfactant into the hexane phase, and the carboxymethylcellulose gel was purified by centrifugation (8000 rpm, 10 minutes). Washing with hexane was performed twice. Next, 10 mL of ethanol was added, and the washing was performed twice in the same manner. This removed sodium hydroxide and ethylene glycol diglycidyl ether. This yielded a carboxymethylcellulose gel. Next, 2 mL of dimethyl sulfoxide (DMSO) was added to 20 mg of carboxymethylcellulose gel, and the solvent was replaced with DMSO by centrifugation (8000 rpm, 10 minutes). This procedure was repeated three times to prepare a dimethyl sulfoxide dispersion of carboxymethyl DMSO cellulose gel.

[0058] [Synthesis Example 2] "Synthesis of adsorbents" 5 mg of carboxymethylcellulose gel obtained in Synthesis Example 1 was mixed with 500 μL of dimethyl sulfoxide, and the mixture was stirred with a magnetic stirrer to disperse the carboxymethylcellulose gel in the dimethyl sulfoxide, thereby preparing dispersion B. Next, while continuing to stir dispersion B, 27.9 mg of benzotriazolyl-N-hydroxytrisdimethylaminophosphonium hexafluorophosphate salt dissolved in 500 μL of dimethyl sulfoxide and 2.3 mg of 1-hydroxybenzotriazole were added to dispersion B, and the mixture was stirred for 15 minutes. Next, while continuing to stir dispersion B, 7.4 mg of LacNAc-glycoside (R. Masaka, et al., Bioorg. Med. Chem., 18, 621 (2010)) dissolved in 250 μL of dimethyl sulfoxide was added to dispersion B, and the mixture was stirred for 48 hours to bind the carboxymethylcellulose gel to the glycoside (LacNAc-glycoside) represented by the following formula (3), forming an adsorbent. Next, the adsorbent was centrifuged at 12,000 rpm for 3 minutes using a centrifuge (step α-1). Next, the supernatant liquid in the container containing the adsorbent was removed (step α-2). Next, 1 mL of dimethyl sulfoxide was injected into the container containing the adsorbent (step α-3). Processes α-1 to α-3 were repeated. Next, the adsorbent was centrifuged at 12,000 rpm for 3 minutes using a centrifuge (step β-1). Next, the supernatant liquid in the container containing the adsorbent was removed (step β-2). Next, 1 mL of distilled water was poured into the container containing the adsorbent (step β-3). Processes β-1 to β-3 were repeated. Through the above process, 3.9 mg of an adsorbent consisting of carboxymethylcellulose gel and LacNAc-glycoside was obtained.

[0059] [ka]

[0060] [Synthesis Example 3] "Synthesis of adsorbents" The adsorbent was obtained in the same manner as in Synthesis Example 2, except that the glycoside represented by the following formula (4) (Lac-glycoside) (R. Masaka, et al., Bioorg. Med. Chem., 18, 621 (2010)) was used instead of LacNAc-glycoside.

[0061] [ka]

[0062] [Synthesis Example 4] "Synthesis of adsorbents" The adsorbent was obtained in the same manner as in Synthesis Example 2, except that the glycoside represented by the following formula (5) (GlcNAc-glycoside) (R. Masaka, et al., Bioorg. Med. Chem., 18, 621 (2010)) was used instead of LacNAc-glycoside.

[0063] [ka]

[0064] [Synthesis Example 5] "Synthesis of adsorbents" The adsorbent was obtained in the same manner as in Synthesis Example 2, except that the glycoside represented by the following formula (6) (Neu5Acα2,6LacNAc-glycoside) (M. Ogata, et al., ACS Omega, 5, 21940 (2020)) was used instead of LacNAc-glycoside.

[0065] [ka]

[0066] [Synthesis Example 6] "Synthesis of adsorbents" The adsorbent was obtained in the same manner as in Synthesis Example 2, except that the glycoside represented by the following formula (7) (Neu5Acα2,3LacNAc-glycoside (M. Ogata, et al., ACS Omega, 5, 21940 (2020))) was used instead of LacNAc-glycoside.

[0067] [ka]

[0068] [Example 1] The adsorbent synthesized in Synthesis Example 2 was dispersed in 20 μL of distilled water to prepare dispersion C1. The concentration of the adsorbent in dispersion C1 was 1.25 mg / mL. Wheat germ lectin (WGA lectin) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D1. The concentration of WGA lectin in solution D1 was 50 μM. Solution D1 was added to dispersion C1, mixed, and allowed to stand (interact) at 4°C for 5 seconds, 30 seconds, 60 seconds, 300 seconds, 600 seconds, 1800 seconds, or 3600 seconds. After the interaction was complete, the adsorbent with WGA lectin adsorbed on its surface was precipitated using a centrifuge. The supernatant containing the WGA lectin that was not adsorbed was filtered, and the absorbance (280 nm) was measured using a micro spectrophotometer (Thermo Fisher Scientific, Nano Drop Lite). The amount of WGA lectin adsorbed per unit mass of adsorbent was calculated using a pre-prepared calibration curve to determine the relationship between absorbance and WGA concentration. The results are shown in Figure 2. From the results in Figure 2, it was found that the amount of WGA lectin adsorbed onto the adsorbent reaches saturation when the mixture of dispersion C1 and solution D1 is allowed to stand for more than 5 minutes.

[0069] [Example 2] The adsorbent synthesized in Synthesis Example 2 was dispersed in 20 μL of distilled water to prepare dispersion C2. The concentrations of the adsorbent in dispersion C2 were set to 0.31 mg / mL (adsorbent concentration in reaction solution: 6.26 μg / 40 μL), 0.63 mg / mL (adsorbent concentration in reaction solution: 12.5 μg / 40 μL), 1.25 mg / mL (adsorbent concentration in reaction solution: 25 μg / 40 μL), 2.5 mg / mL (adsorbent concentration in reaction solution: 50 μg / 40 μL), and 5 mg / mL (adsorbent concentration in reaction solution: 100 μg / 40 μL). Solution D1 was added to dispersion C2, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with WGA lectin adsorbed on its surface was precipitated using a centrifuge, and the supernatant containing WGA lectin that was not adsorbed on the adsorbent was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of WGA lectin adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 3. From the results in Figure 3, it was found that the amount of WGA lectin adsorbed onto the adsorbent in solution D1 reached its maximum value when the concentration of the adsorbent in the reaction solution was between 6.26 μg / 40 μL and 25 μg / 40 μL.

[0070] [Example 3] The adsorbent synthesized in Synthesis Example 3 was dispersed in 20 μL of distilled water to prepare dispersion C3. The concentration of the adsorbent in dispersion C3 was 1.25 mg / mL. The adsorbent synthesized in Synthesis Example 4 was dispersed in 20 μL of distilled water to prepare dispersion C4. The concentration of the adsorbent in dispersion C4 was 1.25 mg / mL. Solution D1 was added to each of the above dispersions C1, C3, and C4, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with WGA lectin adsorbed on its surface was precipitated using a centrifuge, and the supernatant containing WGA lectin that was not adsorbed on the adsorbent was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of WGA lectin adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 4. From the results in Figure 4, it was found that the adsorbents immobilized with LacNAc-glycoside and GlcNAc-glycoside had superior ability to adsorb WGA lectin. On the other hand, the adsorbent immobilized with Lac-glycoside had inferior ability to adsorb WGA lectin.

[0071] [Example 4] ECA lectin (Erythrina variegata) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D2. The concentration of ECA lectin in solution D2 was 50 μM. Solution D2 was added to each of the above dispersions C1, C3, and C4, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with ECA lectin adsorbed on its surface was precipitated using a centrifuge, and the supernatant containing ECA lectin that was not adsorbed on the adsorbent was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of ECA lectin adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 5. From the results in Figure 5, it was found that LacNAc-glycoside and the adsorbent immobilized with Lac-glycoside had superior ability to adsorb ECA lectins. On the other hand, the adsorbent immobilized with GlcNAc-glycoside was found to have inferior ability to adsorb ECA lectins.

[0072] [Example 5] The adsorbent synthesized in Synthesis Example 5 was dispersed in 20 μL of distilled water to prepare dispersion C5. The concentration of the adsorbent in dispersion C5 was 0.625 mg / mL. The adsorbent synthesized in Synthesis Example 6 was dispersed in 20 μL of distilled water to prepare dispersion C6. The concentration of the adsorbent in dispersion C6 was 0.625 mg / mL. Japanese elderberry bark lectin (SSA lectin) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D3. The concentration of SSA lectin in solution D3 was 12.5 μM. Solution D3 was added to each of the above dispersions C1, C3, C5, and C6, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with SSA lectin adsorbed on its surface was precipitated using a centrifuge, and the supernatant containing SSA lectin that was not adsorbed on the adsorbent was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of SSA lectin adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 6. From the results in Figure 6, it was found that the adsorbent immobilized with Neu5Acα2,6LacNAc-glycoside had superior ability to adsorb SSA lectin. On the other hand, the other adsorbents were found to have inferior ability to adsorb SSA lectin. Furthermore, as reported in Biomacromolecules, 10, 1894-1903 (2009), SSA lectin and human influenza virus have identical binding affinity, and it is known that if SSA lectin can be adsorbed, human influenza virus can also be adsorbed. In other words, the results in Figure 6 show that the adsorbent immobilized with Neu5Acα2,6LacNAc-glycoside can adsorb human influenza virus, and this adsorbent can be used as both an adsorbent and a detection agent for human influenza virus.

[0073] [Example 6] Canine lectin (MAA lectin) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D4. The concentration of MAA lectin in solution D4 was 12.5 μM. Solution D4 was added to each of the above dispersions C5 and C6, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with MAA lectin adsorbed on its surface was precipitated using a centrifuge, and the supernatant containing MAA lectin that was not adsorbed was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of MAA lectin adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 7. From the results in Figure 7, it was found that the adsorbent immobilized with Neu5Acα2,3LacNAc-glycoside had superior ability to adsorb MAA lectin. On the other hand, it was found that the adsorbent immobilized with Neu5Acα2,6LacNAc-glycoside had inferior ability to adsorb MAA lectin. The results from Examples 3, 4, 5, and 6 show that changing the type of glycoside used in the adsorbent changes the type of lectin protein that the adsorbent can adsorb.

[0074] [Example 7] Bovine serum albumin (BSA) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D5. The concentration of BSA in solution D5 was 50 μM. Solution D5 was added to each of the above dispersions C1, C3, C4, C5, and C6, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with BSA adsorbed on its surface was precipitated using a centrifuge, and the supernatant liquid containing BSA that was not adsorbed was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. From the obtained absorbance, the amount of BSA adsorbed per unit mass of adsorbent was calculated in the same manner as in Example 1. The results are shown in Figure 8. From the results in Figure 8, it was found that all of the adsorbents were inferior in their ability to adsorb BSA. The results from Examples 3, 4, 5, 6, and 7 showed that the adsorbent selectively adsorbs specific proteins depending on the type of glycoside used.

[0075] [Example 8] The adsorbent synthesized in Synthesis Example 2 was dispersed in 20 μL of distilled water to prepare dispersion C7. The concentration of the adsorbent in dispersion C7 was 5 mg / mL. WGA lectin modified with fluorescein isothiocyanate (FITC, a fluorescent dye) (FITC-WGA lectin) was dissolved in 20 μL of phosphate-buffered saline to prepare solution D6. The concentration of FITC-WGA lectin in solution D6 was 1 mg / mL. Solution D6 was added to dispersion C7 in a brown bottle, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent body on which FITC-WGA had been adsorbed was washed with distilled water, and then the adsorbent body was recovered. The obtained adsorbent was examined using a fluorescence microscope to observe the fluorescence originating from the FITC of the adsorbent at an excitation wavelength of 490 nm and an emission wavelength of 520 nm. The results are shown in Figures 9 to 14. Figures 9 and 12 are fluorescence images. Figures 10 and 13 are superimposed fluorescence and bright-field images. Figures 11 and 14 are bright-field images. The results in Figures 9 to 14 show that the fluorescence is emitted in a spherical shape, consistent with the bright-field images. Therefore, it was determined that the adsorbent is spherical. Furthermore, it was found that the protein is uniformly adsorbed on the surface of the adsorbent.

[0076] [Example 9] A dispersion C2 (20 μL) with an adsorbent concentration of 0.31 mg / mL was prepared in the same manner as in Example 2. Solution D7 was prepared by dissolving WGA lectin in 20 μL of phosphate-buffered saline. The concentrations of WGA lectin in solution D7 were 5.9 mM (concentration of WGA lectin in reaction solution: 3.0 mM / 40 μL), 8.8 mM (concentration of WGA lectin in reaction solution: 4.4 mM / 40 μL), 13.2 mM (concentration of WGA lectin in reaction solution: 6.6 mM / 40 μL), and 19.8 mM (concentration of WGA lectin in reaction solution: 9.9 mM / The reaction solutions were set at 40 μL, 29.6 mM (concentration of WGA lectin in the reaction solution: 14.8 mM / 40 μL), 44.4 mM (concentration of WGA lectin in the reaction solution: 22.2 mM / 40 μL), 66.7 mM (concentration of WGA lectin in the reaction solution: 33.4 mM / 40 μL), and 100 mM (concentration of WGA lectin in the reaction solution: 50.0 mM / 40 μL). Solution D7 was added to dispersion C2, mixed, and allowed to stand (interact) at 4°C for 30 minutes. After the interaction was complete, the adsorbent with WGA adsorbed on its surface was precipitated using a centrifuge, and the supernatant liquid containing WGA that was not adsorbed was filtered and collected. The absorbance of the obtained filtrate was measured at a wavelength of 280 nm. The absorbance of the filtrate was measured in the same manner as in Example 1. The relationship between the adsorption equilibrium concentration and the amount of adsorbed material at equilibrium was determined from the obtained absorbance data. The results are shown in Figure 15. From the results in Figure 15, it was found that the amount of WGA lectin adsorbed per gram of dry adsorbent reaches saturation when the concentration of WGA lectin in the reaction solution is 22 mM / 40 μL or higher. Furthermore, the maximum adsorption amount and adsorption model were determined using the adsorption isotherm equation. The results are shown in Figure 16. The results in Figure 16 show data plotted between the adsorption equilibrium concentration and the amount of adsorbed material at equilibrium, converted to conform to the Langmuir adsorption isotherm equation. The square of the correlation coefficient R is very close to 1, indicating single-phase adsorption and specific adsorption to the adsorbent surface. It was also found that the theoretical maximum adsorption amount per 1 g of dry adsorbent is 2.30 g. [Explanation of Symbols]

[0077] 1 Adsorbent 2. Carboxymethylcellulose gel 3. Glycosides

Claims

1. An adsorbent comprising a carboxymethylcellulose gel and a glycoside that modifies the surface of the carboxymethylcellulose gel, The carboxymethylcellulose gel is a composite of carboxymethylcellulose and a terminally active epoxy compound that intermolecularly crosslinks the carboxymethylcellulose. The glycoside is a sugar derivative having an amino group at the aglycone end, The hydroxyl group of the carboxymethylcellulose and the epoxy groups of the terminally active epoxy compound are added together, An adsorbent in which the carboxymethyl group of the carboxymethylcellulose gel and the amino group at the end of the aglycone portion of the glycoside are bonded together by an amide bond, thereby adsorbing lectins.

2. The adsorbent according to claim 1, wherein the number of molecules of the terminally active epoxy compound bonded to each hydroxyl group of the carboxymethylcellulose is 0.10 or more and 1.00 or less.

3. The adsorbent according to claim 1 or 2, wherein the degree of substitution, which represents the proportion in which the hydroxyl groups of the carboxymethylcellulose are replaced by carboxymethyl groups, is 1.2 or less.

4. The adsorbent according to claim 1 or 2, wherein the two terminally active epoxy compounds are at least one of polyethylene glycol diglycidyl ether and ethylene glycol diglycidyl ether.

5. The adsorbent according to claim 1 or 2, wherein the weight-average molecular weight (Mw) of the carboxymethylcellulose is 83,000 or more.

6. A method for producing an adsorbent that adsorbs lectins, A step of preparing solution A containing carboxymethylcellulose by dissolving carboxymethylcellulose in an alkaline aqueous solution, The process involves stirring and mixing oil and surfactant to prepare solution B for the oil phase, The process involves adding solution A to solution B, microemulsifying solution A and solution B to form a water-in-oil emulsion, The steps include adding a terminally active epoxy compound to the water-in-oil emulsion, and intermolecularly crosslinking the carboxymethylcellulose with the terminally active epoxy compound to form a carboxymethylcellulose gel, A step of dispersing the carboxymethylcellulose gel in an organic solvent to prepare a dispersion C containing the carboxymethylcellulose gel, A method for producing an adsorbent, comprising the steps of: adding a glycoside having an amino group at the end of the aglycone portion to the dispersion C; forming an amide bond by linking the carboxymethyl group of the carboxymethylcellulose gel with the amino group at the end of the aglycone portion of the glycoside; and modifying the surface of the carboxymethylcellulose gel with the glycoside.

Images