JP7004978B2 - Cellulose nanofiber composite material and its manufacturing method - Google Patents

Description

The present invention relates to a cellulose nanofiber composite material to which an inorganic oxide is added and a method for producing the same, and more particularly to a cellulose nanofiber composite material suitable as a membrane for immunoassay such as an immunochromatographic carrier and a method for producing the same.

Cellulose has a structural unit called a microfibril having a diameter of 1 micron or less and a length of several microns. It has been proposed to produce a porous cellulose nonwoven fabric (Patent Document 1). Such cellulose non-woven fabrics are attracting attention as unique materials having high mechanical strength and low linear expansion rate, and are expected to be used as separators for power storage devices, functional filters, high-performance papers for daily life products, and the like. However, since it is difficult to adsorb proteins, it has a drawback that it is not suitable for applications such as a carrier for immunochromatography and an enzyme-immobilized membrane.

Therefore, Patent Document 2 proposes to improve the protein adsorption ability by maintaining a high porosity of the cellulose nonwoven fabric and appropriately inhibiting the affinity between the cellulose nonwoven fabric and water. Specifically, the fine cellulose fiber used as a raw material is crosslinked with a cross-linking agent such as an isocyanate compound, water-repellent treated with a water-repellent agent such as a silane coupling agent, and the fine cellulose fiber has a protein-adsorbing ability. As the functional group, a chemically binding functional group such as a sulfonic acid group, a carboxyl group, a phosphoric acid group, a quaternary ammonium group, a pyridium group, a primary amino group, a secondary amino group, a tertiary amino group, an azo group and a mercapto group. It is proposed to introduce a physically adsorptive functional group such as a group, a phenyl group and an alkyl group. Further, Patent Document 2 describes that less than 10% by weight of silica particles, inorganic particulate compounds such as alumina particles, and organic fine particles such as melamine resin may be added to the weight of the cellulose non-woven fabric. Has been done. However, Patent Document 2 does not disclose or suggest any TEMPO oxidation treatment of fine cellulose fibers, nor does it examine the development characteristics of labeling substances such as solvents and polystyrene latex particles in immunochromatography.

On the other hand, in Patent Document 3, mechanical properties are improved by adding plate-like nanoparticles to cellulose nanofibers obtained by TEMPO oxidation treatment and oxidizing at least a part of the hydroxyl groups on the surface to carboxyl groups to uniformly disperse them. The cellulose nanofiber composites that have been made are disclosed. However, Patent Document 3 does not describe the use of aluminum oxide, and deals with the improvement of protein adsorption ability and the development characteristics of labeling substances such as solvents and polystyrene latex particles in immunochromatography. do not have.

International Publication WO2006 / 004012 Japanese Unexamined Patent Publication No. 2012-167406 Japanese Unexamined Patent Publication No. 2013-010891

An object of the present invention is to provide a porous cellulose nanofiber composite material having excellent protein adsorption ability and capable of satisfactorily developing a labeling substance such as a solvent and polystyrene latex particles even when used as an immunochromatographic carrier, and a method for producing the same. It is in.

The present inventors have prepared a composite material obtained by adding a predetermined amount of an inorganic oxide such as aluminum oxide having a predetermined particle size to cellulose nanofibers in which at least a part of the hydroxyl groups on the surface is oxidized to a carboxyl group, and adsorbing the protein. We have found that it can be suitably used as a membrane for immunoassay such as an immunochromatographic carrier because it has excellent performance and has sufficient permeability and porosity to develop a labeling substance such as a solvent and polystyrene latex particles. Has been completed.

That is, according to one aspect of the present invention, at least a part of the hydroxyl group on the surface contains cellulose nanofibers oxidized to a carboxyl group and an inorganic oxide having a particle size of 0.1 to 300 μm, and the inorganic oxide is contained. Provided is a porous cellulose nanofiber composite material having a content of 800 to 6000 parts by mass with respect to 100 parts by mass of the cellulose nanofibers.

Further, according to another aspect of the present invention, an inorganic oxide having a particle size of 0.1 to 300 μm is 800 to 6000 with respect to 100 parts by mass of cellulose nanofibers in which at least a part of the hydroxyl groups on the surface is oxidized to a carboxyl group. Provided is a method for producing a porous cellulose nanofiber composite, which comprises forming a slurry containing at least a part by mass into a film and drying it.

In the porous cellulose nanofiber composite material of the present invention, cellulose nanofibers in which at least a part of the hydroxyl groups on the surface are oxidized to carboxyl groups and inorganic oxides such as aluminum oxide having a particle size of 0.1 to 300 μm are mixed in a predetermined ratio. Since it contains and has a porous structure in which both cellulose nanofibers and inorganic oxides are uniformly dispersed without agglomeration, it has excellent protein adsorption ability and is suitable as a membrane for immunoassay, as well as a solvent and polystyrene latex particles and the like. Since it has sufficient permeability and porosity to develop the labeling substance of, it is suitable as an immunochromatographic carrier and can be used as an alternative to the conventional nitrocellulose membrane in the immunoassay.

Explanatory drawing which shows the fluorescence intensity measuring apparatus used in an Example. The graph which shows the development performance of the polystyrene particle containing a fluorescent dye of the sample thin film obtained in an Example. The graph which shows the relationship between the alumina addition concentration and the particle mobility of the sample thin film obtained in an Example. The photograph which shows the protein adsorption ability of the sample thin film obtained in an Example. a is a plan view showing an immunochromatographic test strip prepared in Examples, and b is a vertical cross-sectional view of the immunochromatographic test strip shown in a.

1. 1. Porous Cellular Nanofiber Composite Material The porous cellulose nanofiber composite material of the present invention is mainly composed of cellulose nanofibers in which at least a part of the hydroxyl groups on the surface are oxidized to carboxyl groups and an inorganic oxide such as aluminum oxide. ..

1-1. Cellulose Nanofibers As the cellulose nanofibers, pulps obtained by defibrating pulp to microfibrils or similar units by a known method can be used. The cellulose nanofibers are not particularly limited as long as they have a maximum fiber diameter of 1 micron or less, but usually, the maximum fiber diameter is 1000 nm or less and the number average fiber diameter is 2 nm or more and 150 nm or less, and the maximum fiber diameter is preferable. Is 500 nm or less and the number average fiber diameter is 2 nm or more and 100 nm or less, and more preferably, the maximum fiber diameter is 30 nm or less and the number average fiber diameter is 2 nm or more and 10 nm or less. The maximum fiber diameter and the number average fiber diameter can be determined based on an image obtained by observing with an electron microscope at a magnification of 5000 times, 10000 times, 50,000 times, etc. according to the method described in JP2013-10891. can.

The method for obtaining the oxidized cellulose nanofibers is not particularly limited as long as it can oxidize at least 10% of the hydroxyl groups on the surface of the cellulose nanofibers to a carboxyl group. Masanori Otsuka, Tsuguyuki Saito, Toshiharu Emae, Akira Isogai: Characteristic analysis of TEMPO-catalyzed oxidized pulp sheet, Journal of Functional Paper Research, No. 48, p24 (2009). As a typical oxidation method, there is a method in which an N-oxyl compound is used as a catalyst component in an aqueous solvent such as water, and an oxidizing agent is allowed to act on cellulose before defibration or cellulose nanofibers after defibration. By this oxidation treatment, at least a part of the primary hydroxyl groups exposed on the surface of the cellulose nanofibers is oxidized to a carboxyl group. The concentration of cellulose and cellulose nanofibers in the reaction solution is not particularly limited as long as the reaction proceeds, but it is usually preferable that the concentration is 5% or less by mass of the entire reaction solution. The cellulose nanofibers used in the present invention preferably have 15% or more of the hydroxyl groups on the surface oxidized to carboxyl groups, and more preferably 20% or more of the hydroxyl groups on the surface are oxidized to carboxyl groups. It is particularly preferable that 30% or more of the hydroxyl groups on the surface are oxidized to the carboxyl group.

Examples of the N-oxyl compound include TEMPO (2,2,6,6-tetramethyl-1-piperidin-N-oxyl) and TEMPO derivatives having various functional groups at the C4 position, for example, 4-acetamide TEMPO. 4-Carboxy TEMPO, 4-phosphonooxy TEMPO and the like can be used. Of these, TEMPO and 4-acetamide TEMPO are preferable from the viewpoint of reaction rate. The amount of the N-oxyl compound used may be a catalytic amount sufficient to promote the oxidation reaction, preferably in a concentration range of 0.05 to 4 mmol / L, and more preferably in a concentration range of 0.05 to 2 mmol / L. ..

Depending on the type of oxidizing agent, the N-oxyl compound may be combined with bromide or iodide as a catalyst component. Examples of such bromides and iodides include ammonium salts such as ammonium bromide and ammonium iodide, alkali metals bromide such as lithium bromide, potassium bromide and sodium bromide, lithium iodide, potassium iodide and iodide. Use alkali metal iodide such as sodium, alkaline earth metal bromide such as calcium bromide, magnesium bromide and strontium bromide, and alkaline earth metal iodide such as calcium iodide, magnesium iodide and strontium iodide. Can be done. When the hypochlorite alkali metal salt is used as an oxidant, it is preferable to use a catalyst component in which an N-oxyl compound is combined with a bromide or an iodide, and the chlorite alkali metal salt is used as an oxidant. In this case, it is preferable to use the N-oxyl compound alone as a catalyst component.

Oxidizing agents include hypohaloic acid or a salt thereof, hypohaloic acid or a salt thereof, perhalogenic acid or a salt thereof, halogen, halogen oxide, nitrogen oxide, hydrogen peroxide, peracetic acid, persulfate, and persulfate. Peracids such as benzoic acid can be mentioned, and here, chlorine, bromine, iodine and the like can be mentioned as halogens. Of these oxidizing agents, hypohaloic acid alkali metal salts and haloanoic acid alkali metal salts are preferable, and hypochlorite alkali metal salts and chlorite alkali metal salts are more preferable. The amount of the oxidizing agent used may be an amount sufficient to allow the oxidation reaction to proceed, and is preferably in the range of 1 to 50 mmol / L.

1-2. Inorganic oxides Examples of the inorganic oxides include metal oxides such as aluminum oxide and inorganic oxides such as silicon oxide, but aluminum oxide is preferable. As the inorganic oxide, one having a particle size of 0.1 to 300 μm is used. When the particle size is smaller than 0.1 μm, the cellulose nanofibers fill the voids in the porous cellulose nanofiber composite material, which narrows the pore size of the voids, such as a solvent in immunochromatography and polystyrene latex particles. It causes the development of the labeling substance to be hindered. Further, when the particle size exceeds 300 μm, the voids of the porous cellulose nanofiber composite material become larger as the particle size of the inorganic oxide used as the carrier becomes larger, and the porous cellulose nanofiber composite material is bonded to the developed label and the composite material. The frequency of interaction with the protein decreases and the detection sensitivity decreases. The particle size of the inorganic oxide is preferably 0.5 to 250 μm, more preferably 1.0 to 150 μm. The particle size of the inorganic oxide can be measured as an average particle size using a particle size distribution meter based on the dynamic light scattering method.

The purity of the inorganic oxide is not particularly limited, but is preferably 95% or more, more preferably 99% or more, from the viewpoint of suppressing variation in the protein adsorption ability in the sheet due to the mixture of impurities.
The crystal type of aluminum oxide preferably used as an inorganic oxide is not particularly limited, but a three-dimensional crystal type having a large specific surface area is preferable from the viewpoint of protein adsorption ability. The blending amount of the inorganic oxide is 800 to 6000 parts by mass with respect to 100 parts by mass of the cellulose nanofibers. When this compounding amount is less than 800 parts by mass, the proportion of the oxidized cellulose nanofibers becomes high, and it becomes difficult to form a porous structure. On the other hand, if it exceeds 6000 parts by mass, the proportion of the oxidized cellulose nanofibers decreases, so that the properties of the inorganic oxide become stronger, which causes the protein adsorption ability to decrease. The blending amount of the inorganic oxide is preferably 2000 to 4500 parts by mass, and more preferably 2700 to 3300 parts by mass with respect to 100 parts by mass of the cellulose nanofibers.

2. 2. Method for producing a porous cellulose nanofiber composite material In the porous cellulose nanofiber composite material of the present invention, the cellulose nanofibers having been oxidized as described above are subjected to the above-mentioned inorganic oxide having a predetermined particle size in the cellulose nanofiber 100. It can be produced by forming a slurry obtained by mixing 800 to 6000 parts by mass with respect to parts by mass into a film shape and drying it.

As the solvent of the slurry, an organic solvent can be used in addition to water. Of these, since the oxidation treatment of the cellulose nanofibers is performed in an aqueous solvent, it is preferable to use water as the solvent. As the organic solvent, either an aqueous organic solvent or a non-aqueous organic solvent can be used, but an organic solvent having a low boiling point, for example, an alcohol solvent such as ethanol is preferable because it is easy to dry.

The method for producing a composite material using the above slurry is not particularly limited as long as a desired film-like composite material can be obtained. For example, the slurry is sufficiently stirred in a disperser to uniformly disperse and mix the contents, then the slurry is applied on an appropriate support, dried, and then the support is removed to make the slurry porous. A cellulose nanofiber composite material having a structure can be obtained. Further, after drying, the cellulose nanofiber composite material may be supported on the support without removing the support. As the support, for example, a screen, a woven cloth, a slide glass, a membrane filter, a filter paper, a plastic film such as polyethylene, or the like can be used. When used as a carrier for immunochromatography, an impermeable material such as a plastic film is preferably used as the support. The cellulose nanofiber concentration of the slurry is not particularly limited as long as a desired porous structure can be obtained, but is preferably 0.05% by mass to 4% by mass, more preferably 0.1% by mass to 2% by mass. The drying can be performed by using known means, and examples thereof include natural drying, vacuum drying, heat drying, suction drying and the like.

3. 3. Physical characteristics of the porous cellulose nanofiber composite material The porosity of the porous cellulose nanofiber composite material of the present invention is preferably 40% to 99%, more preferably 50% to 85%. Such porosity can be achieved by appropriately adjusting the concentration of the slurry and the dispersion solvent. From this point of view, the concentration of the slurry is preferably 1.0% by volume to 60% by volume, more preferably 15% by volume to 50% by volume. When the porosity is in this range, when used as an immunochromatographic carrier for lateral flow, not only the developing solvent can be easily developed by capillarity, but also the conjugate to which the granular labeling substance is bound is good. Can be moved to. In addition, the porosity can be appropriately adjusted depending on the drying time of the slurry.
When the composite material of the present invention is used as a carrier for immunochromatography, the thickness is preferably 40 to 300 μm, more preferably 100 to 200 μm. As the labeling substance, known substances can be used, for example, in addition to metal colloids such as gold colloid and platinum colloid, synthetic latex particles such as polystyrene latex colored with each pigment such as red and blue, and synthetic latex particles such as polystyrene latex. Latex particles such as natural rubber latex can be mentioned. The nitrocellulose membrane conventionally used as a carrier for immunochromatography is inferior in the development performance of a conjugate using polystyrene particles as a labeling substance, but the carrier for later flow chromatography made of the composite material of the present invention is used. Is also excellent in the deployment performance of conjugates using polystyrene particles.

The porous cellulose nanofiber composite material of the present invention has excellent protein adsorption properties, and proteins can be fixed in the same manner as a normal nitrocellulose membrane. Examples of the protein to be immobilized include various antibodies and antigens. Protein fixation can be performed in the same manner as a conventional nitrocellulose membrane. Therefore, the porous cellulose nanofiber composite material of the present invention can be used as an alternative to the nitrocellulose membrane in any immunoassay, and is a lateral flow type immunochromatographic carrier, a flow-through type immunochromatographic carrier, and a western blot. , Can be used as a membrane for immunostaining, dot blot, etc.

When the composite material of the present invention is used as a carrier for lateral flow type immunochromatography, the average pore size thereof is not particularly limited as long as it can develop a labeling substance such as a solvent or polystyrene latex particles. It is preferably 1 μm to 20 μm, more preferably 5 μm to 10 μm. If the average pore size is less than 1 μm, the labeling substance or the like cannot develop in the porous cellulose nanofiber composite material, causing clogging and a non-specific reaction. On the other hand, if the average pore size is 20 μm or more, the reaction efficiency between the labeling substance or the like and the capture antibody immobilized on the carrier may decrease, and the detection sensitivity may decrease. The average pore size can be determined by direct observation of the pores with an electron microscope, and can also be measured by using a pore distribution measuring device by a mercury intrusion method or a gas adsorption method.

(1) Preparation of TEMPO oxidized cellulose
Adjustment of TEMPO Oxidized Cellulose has been reported so far (Masanori Otsuka, Tsuguyuki Saito, Toshiharu Emae, Akira Isogai: Characteristic Analysis of TEMPO Catalyst Oxidized Pulp Sheet, Journal of Functional Paper Research Society, No. 48, p24 (2009) )) Was followed.
1 g of shredded pulp (pulp derived from coniferous tree provided by Taiko Paper Co., Ltd.) and 100 mL of distilled water were stirred with a mixer. Transfer the prepared sample to a beaker with an internal volume of 200 mL, TEMPO reagent (2,2,6,6-tetramethylpiperidine 1-oxyl) (product of Nacalai Tesque Co., Ltd.) 0.0125 g and sodium bromide 0. 125 g was added and dissolved. A 0.5 M aqueous sodium hydroxide solution was added to the obtained solution to adjust the pH to 10. Next, 10 mL of 0.1 M aqueous sodium hypochlorite solution was added while stirring the obtained solution, and the pH was maintained at 10 using 0.5 M hydrochloric acid and 0.5 M aqueous sodium hydroxide solution during the reaction. The reaction was terminated when all the added sodium hypochlorite was consumed and the pH did not fluctuate. Sodium hypochlorite, which may remain in the reaction solution, was inactivated by adding 5 mL of ethanol. The obtained TEMPO oxide cellulose was suction-filtered using a glass filter, and then washed with distilled water and suction-filtered repeatedly until the pH of the filtrate became around 7. The obtained filtration residue was used as a TEMPO oxidized cellulose sample. The TEMPO oxidized cellulose sample was dispersed in distilled water, and the 1% by mass sample was stirred with a mixer for 10 minutes to perform a defibration treatment.

(2) Preparation of aluminum oxide-added cellulose nanofiber composite material In a triangular flask with an internal volume of 100 mL, 30 mL of the 0.1% by mass defibrated TEMPO oxide cellulose sample solution prepared in (1) above and aluminum oxide (Wako Pure Chemical Industries, Ltd.) The company's product (particle size 75 μm) was added, and degassing was performed by ultrasonic treatment for 10 minutes with an ultrasonic cleaner. Aluminum oxide was added so that the concentration in the sample solution was 20, 23, 25, 27, 30 or 33 mg / mL. The sample solution was applied to the petri dish on which the slide glass was installed while stirring. By leaving this petri dish at a constant temperature of 50 ° C. overnight, an aluminum oxide-added cellulose nanofiber composite material having a porosity of 75%, a thickness of 150 μm, and an average pore size of 6.8 μm was obtained.

(3) Development of Polystyrene Particles Containing Fluorophore The aluminum oxide-added cellulose nanofiber composite material obtained in (2) above was cut into a width of 7.5 mm and a length of 75 mm to prepare a sample thin film. 200 μL of a dispersion of polystyrene particles containing a fluorescent dye (a product of Merck) having a particle size of 22 nm prepared to be 1.0% by mass with a 400 mM Tris buffer at a position 10 mm away from one end of this sample thin film. It was dropped and expanded toward the end opposite to the expansion start position. The developed sample thin film was dried at room temperature for 20 minutes.
The fluorescence of the surface of the sample thin film was measured using the fluorescence measuring device shown in FIG. That is, a fluorescence detector (a product of Nippon Sheet Glass Co., Ltd.) is fixed above the unfolding start position of the sample thin film placed on the transport table, a rope is attached to one end in the longitudinal direction of the transfer table, and the rope is wound by a winding device. By moving the carrier at a constant speed by winding the sample, the fluorescence intensity was measured over the entire surface of the sample thin film from the deployment start position to the end on the opposite side (hereinafter referred to as the downstream end). .. The measurement results are shown in FIG. From FIG. 2, in the sample thin film prepared at any of the aluminum oxide addition concentrations, relatively constant weak fluorescence was observed from the development start position to 7.5 mm before the downstream end, and large fluorescence was observed at the downstream end. You can see that. That is, it is considered that the polystyrene particles dropped at the development start position permeated the sample thin film and expanded to the downstream end, and stayed at the downstream end, so that large fluorescence was observed. From this, it was found that the polystyrene particles were developed over the entire area of the sample thin film.

The ratio of the average relative fluorescence intensity of the entire film to the maximum peak was taken and used as the particle mobility (1) shown in the following equation.
Particle mobility (1) = (maximum fluorescence intensity) / (average relative fluorescence intensity of the entire film)
The larger the mobility value, the higher the particle unfolding ability of the membrane sample. The relationship between the aluminum oxide addition concentration and the particle mobility (1) is shown in FIG. The mobility increased with the increase in the concentration of aluminum oxide added, and tended to show a constant value at 30 mg / mL or higher. From this, it was found that the concentration of aluminum oxide added suitable for the carrier for later later chromatography was 30 mg / mL.

(4) Immobilization of antibody The aluminum oxide-added cellulose nanofiber composite material obtained in (2) above was cut into a width of 7.5 mm and a length of 75 mm to prepare a sample thin film. An anti-influenza virus antibody solution of 1.5 μg / mL was applied in a line in the width direction to the center of this sample thin film. After application, it was left in a desiccator for 18 hours to be dried and immobilized. Then, one end of the sample thin film was immersed in phosphate buffered saline (hereinafter referred to as PBS) to develop an excess amount of PBS for washing, and then Coomassie Brilliant Blue (hereinafter referred to as CBB) staining was performed. .. The results are shown in FIG. As shown in FIG. 4, a bluish-purple coloration by CBB staining was observed at the antibody-fixed position. From this, it was found that the antibody was immobilized on the sample thin film.

(5) Preparation of immunochromatographic test strip for detecting influenza A and B viruses Type A in order to evaluate the performance of the aluminum oxide-added cellulose nanofiber composite material prepared in (2) above as a carrier for immunochromatography. It was evaluated using an influenza virus detection system. As a control immunochromatographic test strip for performance comparison, Immunoace Flu (a product of Towns Co., Ltd.) using a membrane carrier made of a nitrocellulose membrane was used. As shown in FIG. 5, for Immuno Ace Flu (a product of Towns Co., Ltd.), a carrier 3 (nitrocellulose membrane) for immunochromatography having a width of 4.0 mm and a length of 35 mm is attached to the middle of the pressure-sensitive adhesive sheet 1. The downstream end of the impregnating member 2 is overlapped and connected on the upstream end of the immunochromatographic carrier 3, and the upstream portion of the impregnating member 2 is attached to the pressure-sensitive adhesive sheet 1 to form the impregnating member 2. The downstream portion of the sample addition member 6 is placed on the upper surface, the upstream portion of the sample addition member 6 is attached to the pressure-sensitive adhesive sheet 1, and further, the downstream portion of the immunochromatographic carrier 3 is placed on the upper surface of the downstream portion. The upstream portion of the absorbing member 5 is placed, and the downstream portion of the absorbing member 5 is attached to the adhesive sheet 1. Then, a capture site 41a in which the anti-influenza A virus antibody was immobilized was formed at a position 5.0 mm from the end on the chromatographic expansion start point side of the immunochromatographic carrier 3, and at a position 8.0 mm from the same end. A capture site 41b on which the anti-influenza B virus antibody was immobilized was formed, and a control line 42 for confirming that the reaction was performed regardless of the presence or absence of the substance to be analyzed was provided at a position 15.0 mm from the same end. It is provided. The impregnating member 2 is impregnated with a mixture of platinum-colloidal gold-labeled anti-influenza A virus antibody and platinum-colloidal gold-labeled anti-influenza B virus antibody.

The aluminum oxide-added cellulose nanofiber composite material prepared in (2) above was cut into a width of 4.0 mm and a length of 35 mm, and prepared as a carrier for immunochromatography of a chromatographic medium. 0.5 μL of a solution containing 1.0 mg / mL of anti-influenza virus antibody was applied in a line at a position 5.0 mm from the end on the chromatographic development start point side of this carrier for later chromatography. This was dried at room temperature to form a capture site 41a of a complex of influenza A virus antigen and platinum-gold colloidal labeled antibody. Further, 0.5 μL of a solution impregnated with 1.0 mg / mL of anti-influenza virus antibody was applied in a line at a position 8.0 mm from the end on the chromatographic development start point side of this carrier for later later chromatography. This was dried at room temperature to form a capture site 41b of a complex of influenza B virus antigen and platinum-gold colloid-labeled antibody. The obtained immunochromatographic carrier is the same as in FIG. 5 except that the immunochromatographic carrier 3 (nitrocellulose membrane) of Immunoace Flu (a product of Towns Co., Ltd.) shown in FIG. 5 is replaced. Was produced.

(6) Detection of influenza A virus The antigen solution of influenza A virus was diluted with a sample extract to prepare a predetermined concentration, which was used as a test sample. The test sample was chromatographically developed by dropping 100 μL with a micropipette onto the sample addition member of the immunochromatography test strip obtained in (5) above, left at room temperature for 15 minutes, and then captured at the capture site 41a. The amount of capture of the complex of platinum-gold colloid-labeled antibody and influenza A virus antigen was observed with the naked eye. The amount of capture increases or decreases in proportion to the amount of black color in four stages:-(no coloring), ± (slight coloring), + (clear coloring), and ++ (significant coloring). Judgment was made separately. As a control, Immuno Ace Flu (a product of Towns Co., Ltd.) was used, the same operation as described above was performed, and the observation was made in the same manner as described above.

The results are shown in Table 1. As is clear from Table 1, when the aluminum oxide-added cellulose nanofiber composite material is used as the carrier for the immunochromatography, when the test sample having the antigen concentration of 1 × 10 ⁴ TCID ₅₀ is provided, it is clear at the capture site 41a. Coloring was confirmed. On the other hand, in the control, remarkable coloring was confirmed in the test sample having the same concentration. Therefore, when the aluminum oxide-added cellulose nanofiber composite material is used, although the detection sensitivity is slightly inferior to that of the control nitrocellulose membrane, it has the performance that can be carried out as a carrier for the later later chromatography of the later. Shown.

Based on the above results, the aluminum oxide-added cellulose nanofiber composite material has a function of immobilizing proteins such as antibodies, and as a carrier for immunochromatography, a labeling substance is developed and an antibody antigen reaction is carried out on a membrane. It was shown to be a capable material.

Since the porous cellulose nanofiber composite material of the present invention has excellent protein adsorption ability and can satisfactorily develop a labeling substance such as a solvent and polystyrene latex particles even when used as an immunochromatographic carrier, conventional nitro in the field of immunoassay. It can be used as an alternative to cellulose membranes.

1 Adhesive sheet 2 Impregnated member 3 Immunochromatographic carrier 41a Capture site 41b Capture site 42 Control line 5 Absorption member 6 Sample addition member 10 Immunochromatography test strip

Claims (3)

It contains at least a cellulose nanofiber in which at least a part of the hydroxyl group on the surface is oxidized to a carboxyl group and an inorganic oxide having a particle size of 0.1 to 300 μm, and the content of the inorganic oxide is 100 parts by mass of the cellulose nanofiber. A porous cellulose nanofiber composite material having an amount of 800 to 6000 parts by mass. A slurry containing 800 to 6000 parts by mass of an inorganic oxide having a particle size of 0.1 to 300 μm is formed into a film with respect to 100 parts by mass of cellulose nanofibers in which at least a part of the hydroxyl groups on the surface is oxidized to a carboxyl group. A method for producing a porous cellulose nanofiber composite, including drying. An immunochromatographic carrier comprising the porous cellulose nanofiber composite material of claim 1.

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