JP6985694B2 - Composite composition, its production method, and biosensor - Google Patents

Description

The present invention relates to a composite composition containing cellulose nanofibers and a biomolecule, and a biosensor using the same.

Biosensing is a technology for detecting and measuring various molecules using the recognition mechanism of biomolecules. For example, a biochip is a diagnostic and testing device that utilizes the interaction of biomolecules, and is used for early diagnosis of viral infections of diseases, testing of microorganisms and bacteria in foods and water, and the like. As a biomolecule, a physiologically active substance such as a protein is used, and it is detected and measured by specific binding or interaction of an enzyme-substrate, an antigen-antibody, a hormone-receptor or the like. Since these biochips are used in various places, environments, and conditions, it is desirable to store and use them without being affected by the external environment. Biomolecules such as proteins, enzymes, antibodies, and nucleic acids are susceptible to denaturation and inactivation due to heating, and their activity decreases over time due to oxidation even under non-heating. Therefore, when these biomolecules are used for biosensors such as biochips, there is a need for a storage method that can be stably stored even under heating conditions and does not lose its activity over time.

As a technique for stabilizing a biomolecule, Non-Patent Document 1 reports a technique for conserving and stabilizing acetylcholinesterase (AChE) using pullulan. Non-Patent Document 1 evaluates the heating stability of a tablet in which pullulan and AChE are combined, and shows that the tablet is stable. However, since pullulan is a water-soluble polysaccharide, there may be a problem of water resistance when it is assumed to be used as an inspection device such as a biochip.

As a composition containing cellulose fibers and proteins, Patent Document 1 describes a food composition comprising cellulose nanofibers, proteins and water. However, Patent Document 1 targets a gel containing a large amount of water, is not a technique for stabilizing biomolecules, and the water content described in Examples is as high as 70% by mass or more. ..

Patent Document 2 describes a composite laminated material containing a carbohydrate-based base material and a protein, and describes that cellulose is used as the carbohydrate-based base material. However, Patent Document 2 does not describe the use of cellulose nanofibers having a cellulose type I crystal structure, nor does it describe the stabilization of biomolecules thereby.

Patent Document 3 describes that cellulose nanofibers are obtained by treating cellulose fibers with an enzyme and then defibrating them. However, since the enzyme is inactivated prior to defibration, it can no longer be said that biomolecules are present after defibration, and therefore it is not described that biomolecules and cellulose nanofibers are combined.

Japanese Unexamined Patent Publication No. 2016-6757 Japanese Unexamined Patent Publication No. 2017-12762 Japanese Unexamined Patent Publication No. 2017-2136

Sana Jahanshahi-Anbuhi, 9 others, "PullulanEncapsulation of Labile Biomolecules to Give Stable Bioassay Tablets", Angew.Chem. Int. Ed., 53, 6155-6158 (2014)

It is an object of the present invention to provide a composite composition capable of stabilizing a biomolecule.

The composite composition according to the embodiment of the present invention is a composite composition containing cellulose nanofibers having a cellulose I-type crystal structure and anion-modified, and a biomolecule, and has a water content of 20% by mass or less. A composite composition.

In the method for producing a composite composition according to an embodiment of the present invention, an aqueous dispersion of cellulose nanofibers having a cellulose I-type crystal structure and anion-modified is mixed with a biomolecule to have a water content of 20% by mass or less. It is a method for producing a composite composition, which is dried until it becomes dry.

The biosensor according to the embodiment of the present invention contains the above-mentioned composite composition.

In the composite composition according to the embodiment of the present invention, biomolecules can be stabilized by cellulose nanofibers. Therefore, when the composite composition is used, for example, in a biosensor, the biomolecule can be stabilized and deactivation can be suppressed.

Graph showing thermogravimetric analysis of AChE / TOCN pills according to examples Graph showing thermal stability evaluation results of AChE / TOCN pills according to Examples A graph showing the thermal stability evaluation results of AChE in the TOCN aqueous dispersion according to the reference example. A graph showing the room temperature stability evaluation results of the AChE / TOCN paper device according to the examples.

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

The composite composition according to the present embodiment contains anion-modified cellulose nanofibers having a cellulose type I crystal structure and biomolecules. Since the dry film made of cellulose nanofibers forms a very dense film made of water-insoluble fibers and exhibits excellent oxygen barrier properties, it is possible to stabilize biomolecules that are easily deactivated by oxidation. In addition, biomolecules are stabilized under heating conditions by a dry film of cellulose nanofibers. Further, from the viewpoint of the biochemical reaction field of the composite composition of the present embodiment, since the anion-modified cellulose nanofibers are hydrophilic, water adheres to the composite composition in a dry state and swells in the water. This can contribute to the expression of activity of biomolecules by giving a reaction field by biomolecules again. That is, since the cellulose nanofibers are water-insoluble, they can exhibit an appropriate affinity for water when used as an inspection device such as a biosensor, and can solve the problem of water resistance which has been a problem in the prior art. there is a possibility. Furthermore, since the aqueous dispersion of the cellulose nanofibers has thixotropic properties, it is possible to form a composite composition by, for example, inkjet printing, and there is a possibility that the manufacturing process can be simplified.

In this embodiment, cellulose nanofibers are used as the cellulose fibers. The cellulose nanofibers are cellulose fibers having a fiber diameter on the nanometer level, and the number average fiber diameter thereof may be, for example, 3 to 400 nm, 3 to 150 nm, or 3 to 80 nm.

The number average fiber diameter of the cellulose nanofibers can be measured as follows. That is, an aqueous dispersion of cellulose nanofibers having a solid content of 0.05 to 0.1% by mass is prepared, and the aqueous dispersion is cast onto a hydrophilized carbon film-coated grid to be permeable. Use as an observation sample of an electron microscope (TEM). Then, observation is performed using an electron microscope image at a magnification of 5000 times, 10000 times, or 50,000 times depending on the size of the constituent fibers. At that time, an axis having an arbitrary vertical and horizontal image width is assumed in the obtained image, and the sample and observation conditions (magnification, etc.) are adjusted so that 20 or more fibers intersect the axis. Then, after obtaining an observation image satisfying this condition, two random axes are drawn vertically and horizontally for each image, and the fiber diameters of the fibers intersecting the axes are visually read. In this way, images of at least three non-overlapping surface portions are taken with an electron microscope and the value of the fiber diameter of the fibers intersecting each of the two axes is read (hence, at least 20 fibers × 2 × 3 = 120). Information on the fiber diameter of the book can be obtained). From the fiber diameter data obtained in this way, the number average fiber diameter is calculated.

The average aspect ratio of the cellulose nanofibers may be, for example, 10 to 1000, 50 to 1000, or 100 to 1000. The average aspect ratio can be measured by the following method. That is, from the TEM image (magnification: 10,000 times) in which cellulose nanofibers were cast on a hydrophilicized carbon film-coated grid and then negatively stained with 2% uranyl acetate, the number average of the shorter width of the cellulose nanofibers. Observe the width and the number mean width of the long width, and use these values to calculate the mean aspect ratio according to the following formula.
Mean aspect ratio = number average width (nm) for the long width / number average width (nm) for the short width

Cellulose nanofibers are obtained by performing a defibration treatment. The defibration treatment may be carried out after introducing the anion group described later, or may be carried out before the introduction. As the defibration treatment, for example, a homomixer under high-speed rotation, a high-pressure homogenizer, an ultrasonic disperser, a beater, a disc-type refiner, a conical-type refiner, a double-disc-type refiner, a grinder, or the like is used to water the cellulose fibers. This can be done by treating the dispersion, and an aqueous dispersion of cellulose nanofibers can be obtained.

In the present embodiment, cellulose nanofibers having a cellulose I-type crystal structure are used from the viewpoint of water insolubility. The fact that the cellulose constituting the cellulose nanofibers has an I-type crystal structure means that, for example, in the diffraction profile obtained by wide-angle X-ray diffraction image measurement, 2 theta = 14 ° to 17 ° and 2 theta = 22 ° to 23. It can be identified by having typical peaks at two positions near °.

As the cellulose nanofiber, an anion-modified cellulose nanofiber in which an anionic group is introduced into a glucose unit in a cellulose molecule is used. Examples of the anionic group include at least one selected from the group consisting of a carboxyl group, a phosphoric acid group, a sulfonic acid group, and a sulfate group. As used herein, a carboxyl group is a concept that includes not only an acid type (-COOH) but also a salt type, that is, a carboxylic acid base (-COOX, where X is a cation forming a salt with a carboxylic acid). Similarly, the phosphoric acid group, the sulfonic acid group, and the sulfuric acid group are concepts that include not only the acid type but also the salt type.

In one embodiment, the carboxyl group is preferred as the anion group. Examples of the cellulose nanofibers containing a carboxyl group include oxidized cellulose nanofibers formed by oxidizing the hydroxyl group of a glucose unit in a cellulose molecule and carboxymethylated cellulose formed by carboxymethylating a hydroxyl group of a glucose unit in a cellulose molecule. Examples include nanofibers.

Examples of the oxidized cellulose nanofibers according to the preferred embodiment include those in which the hydroxyl group at the C6 position of the glucose unit in the cellulose molecule is selectively oxidized and modified to a carboxyl group. Oxidized cellulose nanofibers are obtained by oxidizing natural cellulose such as wood pulp with an oxidizer in the presence of an N-oxyl compound and defibrating (refining) the fibers. As the N-oxyl compound, a compound having a nitroxy radical generally used as an oxidation catalyst is used, for example, a piperidine nitroxy oxy radical, and particularly 2,2,6,6-tetramethylpiperidinooxy radical (TEMPO). ) Or 4-acetamide-TEMPO is preferred. Cellulose nanofibers oxidized by TEMPO are generally referred to as TEMPO-oxidized cellulose nanofibers, and can also be used in this embodiment. The cellulose oxide nanofiber may have an aldehyde group or a ketone group together with the carboxyl group, but it is preferable that the aldehyde group and the ketone group are substantially free of the aldehyde group and the ketone group.

The amount of anionic groups in the cellulose nanofibers is not particularly limited and may be, for example, 0.05 to 3.0 mmol / g or 0.5 to 2.5 mmol / g. As for the amount of anionic group, for example, in the case of carboxyl group, 60 mL of 0.5 to 1% by mass slurry was prepared from a cellulose sample whose dry mass was precisely weighed, and the pH was adjusted to about 2.5 with a 0.1 M aqueous hydrochloric acid solution. After that, a 0.05 M aqueous solution of sodium hydroxide was added dropwise, the electric conductivity was measured, and the pH was continued until the pH reached about 11, and the hydroxide consumed in the neutralization step of the weak acid in which the change in the electric conductivity was gradual. It can be obtained from the amount of sodium (V) according to the following formula. The phosphoric acid group can also be measured by the same electric conductivity measurement. Other anion groups may also be measured by a known method.
Anion group amount (mmol / g) = V (mL) x [0.05 / cellulose sample mass (g)]

Examples of the biomolecule in the present embodiment include various biopolymers such as proteins, nucleic acids, lipids, vitamins, hormones, sugars and sugar chains. More specifically, physiologically active substances that are involved in reactions such as specific binding and interaction of enzymes-substrates, antigen-antibodies, hormones-receptors, sugar chains-lectins, etc. can be mentioned, and such activities can be described. Biomolecules having are preferably used. Examples of the biomolecule include at least one selected from the group consisting of enzymes, antibody proteins, receptor proteins, hormones, cytokines, lectins, and nucleic acids. In this embodiment, since cellulose nanofibers have a stabilizing effect on oxidation and heating, biomolecules that can be deactivated by oxidation and / or heat are preferably used. The protein referred to here also includes oligopeptides and polypeptides.

The enzyme which is a biomolecule according to one embodiment is not particularly limited, and examples thereof include a hydrolase, an oxidoreductase, a transferase, a lyase, an isomerase, and a ligase. As the hydrolase according to one embodiment, cholinesterase (for example, acetylcholinesterase, butyrylcholinesterase, etc.), protease, amylase, or the like may be used. As the antibody protein, immunoglobulin is used. The nucleic acid may be single-stranded or double-stranded, and examples thereof include RNA, DNA, and DNA / RNA hybrids, whether artificial or natural.

In the composite composition according to one embodiment, the biomolecule is wrapped in the cellulose nanofibers and stabilized thereby. That is, the composite composition according to one embodiment is a composite containing the cellulose nanofibers and biomolecules encapsulated or embedded in the cellulose nanofibers. The biomolecule is retained in the cellulose nanofibers in an uninactivated state, and by being wrapped by the cellulose nanofibers, the deactivation due to oxidation and / or heat is suppressed, that is, stabilized.

In the composite composition according to the embodiment, the content ratio of the cellulose nanofibers and the biomolecule is not particularly limited, and for example, the biomolecule may be 0.001 to 30 parts by mass with respect to 100 parts by mass of the cellulose nanofibers. It may be 01 to 5 parts by mass.

In the composite composition according to the embodiment, the content of the cellulose nanofibers is not particularly limited and may be 75 to 95% by mass or 80 to 90% by mass.

The composite composition according to the embodiment has a water content of 20% by mass or less. Due to the low water content, cellulose nanofibers develop an oxygen barrier property, can stabilize biomolecules that are easily deactivated by oxidation, and enhance the heat stabilizing effect of biomolecules. can. The water content of the composite composition is preferably 5 to 20% by mass, more preferably 10 to 15% by mass. The water content of the composite composition is the water content of the composite composition itself excluding the support even when the composite composition is formed in a state of being integrated with a support such as paper as described later.

In addition to the above-mentioned cellulose nanofibers, biomolecules and water, the composite composition according to the embodiment includes, for example, inorganic and / or organic fillers, clay minerals, as long as the effects of the present invention are not impaired. Contains dry residues of colorants such as pigments, salts, pH adjusters, oils, water-soluble polymers, surfactants, and hydrophilic solvents added to improve the film-forming properties of dry coatings. You may stay. For example, examples of the hydrophilic solvent for improving the film-forming property include alcohols, cellosolves, carbitols, glycols, glycol monophenyl ethers, phenyl alcohols, phenylcarboxylic acid esters, oxycarboxylic acid phenyl esters and the like. May include. In addition, it may contain a light-resistant agent, an antioxidant, a preservative, an inorganic substance and the like.

The composite composition according to the embodiment preferably has a stable biomolecule under heating conditions. Specifically, the heating condition refers to a state in which heat is applied to the composite composition from the outside, but in the present embodiment, i) heating from the initial temperature T1 to the temperature T2. There are two conditions, ii) a constant temperature heating condition in which the temperature T3 is maintained for a predetermined time after the temperature is raised from the initial temperature T1 to reach the temperature T3. In this embodiment, the assumed handling conditions are as follows.

There is an operation from a temperature of −15 ° C. or lower to −20 ° C. or lower, which is generally called a freezing temperature, that is, a state in which a biomolecule is usually frozen or frozen, to an ice melting temperature (0 ° C.). Further, there is an operation of exceeding the melting temperature of ice to reach normal temperature (20 ° C to 25 ° C). Further, it is conceivable that the operation is handled at a higher temperature regardless of intentional or unintentional. As operations handled at high temperatures, specifically, whether intentionally or unintentionally, the operation of the summer indoor, summer outdoor, outdoor vehicle, direct sunlight irradiation, freezer, refrigerator or air conditioner is stopped. It is expected to be handled in. It is also expected to be used in the event of a power outage, disaster, or battlefield.

The temperature change from the initial temperature T1 as the starting point to the temperature T2 is defined as ΔT. For example, in the present embodiment, when the temperature of the biomolecule is raised from −20 ° C. to 0 ° C., ΔT = 20 ° C. Similarly, when the temperature is raised from −20 ° C. to 25 ° C., ΔT = 45 ° C. Further, when the temperature is raised from 40 ° C. to 80 ° C., ΔT = 40 ° C.

Regarding the thermal stability of the biomolecule in the composite composition, the specific activity after the heat treatment when the heat treatment for the composite composition is 80 ° C. for 60 minutes and the activity of the biomolecule in the unheated composite composition is 100%. Is preferably 70% or more, more preferably 80% or more.

When producing the composite composition according to the present embodiment, the aqueous dispersion of the cellulose nanofibers and the biomolecule may be mixed and dried until the water content becomes 20% by mass or less.

As described above, the aqueous dispersion of cellulose nanofibers is prepared by introducing an anionic group into natural cellulose and then performing a defibration treatment, or by defibrating natural cellulose and then introducing an anionic group. be able to. The concentration of the cellulose nanofibers in the aqueous dispersion is not particularly limited, and may be, for example, 0.01 to 5% by mass, 0.05 to 3% by mass, or 0.1 to 1% by mass.

Biomolecules are added to the aqueous dispersion of cellulose nanofibers and mixed. The biomolecule may be added as it is to the aqueous dispersion of cellulose nanofibers, or a biomolecule solution previously dissolved in a buffer solution may be added.

After mixing the aqueous dispersion of cellulose nanofibers and biomolecules, the mixture is dried to obtain the composite composition according to the embodiment.

The composite composition may be formed by itself by applying the mixed solution on a releasable substrate such as a fluororesin sheet, drying the composite composition, and removing the dried product from the releasable substrate. The form is not particularly limited, and may be in the form of a sheet or in the form of tablets such as tablets and pills.

Alternatively, the composite composition may be formed in a state of being integrated with the support by applying the mixed solution onto a support such as paper and drying it. The method of applying to the support is not particularly limited, but the composite composition may be formed on the support by, for example, inkjet printing by utilizing the thixotropic property of the cellulose nanofiber aqueous dispersion.

As the drying method, for example, a heat drying method, a vacuum drying method, a blast drying method, a microwave drying method, an infrared drying method, a freeze drying method, a filtration dehydration method and the like are used. A plurality of methods may be combined and dried. The drying temperature is not particularly limited, but if the case of the heat drying method is exemplified, it is preferably 40 ° C. or lower, more preferably 5 to 35 ° C. in order to suppress the deactivation of biomolecules. Further, the drying time is not particularly limited, and the heating temperature may be always constant or may be gradually increased.

The composite composition according to the embodiment can be used, for example, in a biosensor. The biosensor is a sensing device that detects and measures various molecules and the like by utilizing the recognition mechanism of biomolecules, and the composite composition can be used as a component thereof. A preferred example of a biosensor is a biochip in which a large number of biomolecules are immobilized on a substrate (that is, a support), and one embodiment is performed by immobilizing a large number of the above-mentioned composite compositions as the biomolecules on the substrate. The biochip according to the above can be configured. Further, since inkjet printing is possible as described above, it can also be used for a microfluidic paper analysis device (μPAD).

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited thereto.

(1) Reagent / TOCN: TEMPO oxidized cellulose nanofiber having cellulose type I crystal structure, 1.1% by mass aqueous dispersion, "Leocrysta" manufactured by Daiichi Kogyo Seiyaku Co., Ltd., number average fiber diameter = 4 nm, average aspect Ratio = 280, amount of anion group = 1.9 mmol / g
・ Tris: Tris hydroxymethylaminomethane, manufactured by SIGMA-ALDRICHI ・ IDA: Indoxyl acetate, manufactured by SIGMA-ALDRICHI ・ AChE: Acetylcholine esterase, derived from electrophorus, 200-1000Unit / mg protein, manufactured by SIGMA-ALDRICHI ・ Methanol: Kanto Chemical ( 1M NaOH: Sodium hydroxide aqueous solution (1M, manufactured by Wako Pure Chemical Industries, Ltd.)
・ 6MHCl: Concentrated hydrochloric acid (6M, manufactured by Wako Pure Chemical Industries, Ltd.)
-Sodium chloride: manufactured by Nacalai Tesque Co., Ltd. These were used without any particular purification.

(2) Preparation of 100 mM Tris-HCl buffer (pH 8.0) Tris was weighed in a 6.057 g beaker, 400 mL of MilliQ water was added, and Tris was dissolved using a magnetic stirrer. 6M HCl was added dropwise thereto to adjust the pH to 8.0. The solution was transferred to a 500 mL volumetric flask and diluted with MilliQ water to prepare 100 mM Tris-HCl buffer. If the pH deviated significantly, 1M NaOH and 6M HCl were added dropwise to reprepare.

(3) Preparation of enzyme reaction solution The IDA solution was prepared on the day of use. Methanol was added to IDA in an Eppen tube so as to be 80 mM, and the mixture was mixed with a vortex mixer. AChE used was stored at −20 ° C. AChE stored at −20 ° C. was weighed to 250 U / mL in an Eppen tube at room temperature, 100 mM Tris-HCl buffer was added, mixed with a vortex mixer, and stored frozen.

(4) Preparation of AChE / TOCN pills 400 μL of 0.5 mass% TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution per pill were taken into a centrifuge tube and mixed with a vortex mixer. Each 401 μL of this was discharged onto a Teflon® sheet with a micropipette and air-dried at room temperature for 2 days to prepare AChE / TOCN pills as the composite composition according to the examples. The resulting pills were placed in sample tubes and stored at room temperature. The AChE / TOCN pill contained 0.06 parts by mass of AChE with respect to 100 parts by mass of TOCN.

(5) Thermogravimetric analysis of AChE / TOCN pills The AChE / TOCN pills prepared in (4) above were subjected to thermogravimetric analysis using a thermogravimetric analyzer (TGDTA6300, manufactured by Seiko Instruments Co., Ltd.). The TG (thermogravimetric curve) and DTG (differential thermogravimetric curve) of the AChE / TOCN pills are as shown in FIG. 1, and the water content of the AChE / TOCN pills was 13% by mass.

(6) Evaluation of Thermal Stability of AChE in TOCN Pill The AChE / TOCN pill 2 days after drying at room temperature according to the example was used. The heat treatment was performed by heating the AChE / TOCN pill from room temperature to 80 ° C. and heat-treating at 80 ° C. for 15, 30, 45, 60 minutes. As the AChE / TOCN pills that had been heat-treated for 0 minutes, AChE / TOCN pills 2 days after drying at room temperature (AChE / TOCN pills before the temperature was raised from room temperature to 80 ° C.) were used. As a control, 1 μL of 250 U / mL AChE solution was dissolved in 195 μL of 100 mM Tris-HCl buffer, and the same heat treatment was performed. AChE / TOCN pills after heat treatment and AChE solutions of controls were placed in well plates, respectively, and an enzymatic reaction (hydrolysis reaction using IDA as a substrate) was carried out. Specifically, 195 μL of 100 mM Tris-HCl buffer and 5 μL of 80 mM IDA solution were added to the AChE / TOCN pill, and 5 μL of 80 mM IDA solution was added to the control, and the mixture was shaken for 30 seconds and subjected to an enzymatic reaction for 1 hour. This well plate was placed in a microplate reader (Tekan, Spark 10M) and the absorbance at the absorption peak position (605 nm) of indigo was measured. The activity of the sample not subjected to the heat treatment was set to 100%, and the specific activity of AChE after the heat treatment was calculated. The measurement was performed in triplets.

The results of the thermal stability evaluation are shown in FIG. In the control, AChE was inactivated by heat treatment at 80 ° C., whereas AChE in the TOCN pill according to the example maintained the enzyme activity even after heating for 1 hour. As a result, it is considered that AChE / TOCN pills can be stably stored and transported even at high temperatures.

(7) Evaluation of thermal stability of AChE in TOCN aqueous dispersion (reference example)
400 μL of 0.5% by mass TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution were taken in an Eppen tube and mixed with a vortex mixer (0.5% TOCN / AChE). Further, 400 μL of 1 mass% TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution were taken in an Eppen tube and mixed with a vortex mixer (1% TOCN / AChE). Further, as a control, 400 μL of 100 mM Tris-HCl buffer and 1 μL of 250 U / mL AChE solution were taken in an Eppen tube and mixed with a vortex mixer.

Heat-treat these three kinds of mixed solutions at 60 ° C. for 0, 5, 10, and 15 minutes, put the heat-treated mixed solutions in well plates, add 5 μL of 80 mM IDA solution, and evaluate the thermal stability of (6) above. Similarly, an enzymatic reaction was carried out and the absorbance was measured. The activity of the sample not subjected to the heat treatment was set to 100%, and the specific activity of AChE after the heat treatment was calculated. The measurement was performed in triplets.

The results are as shown in FIG. In the TOCN dispersion, AChE was easily deactivated by heat treatment. This was the same even if the TOCN concentration was increased. Therefore, it is considered that the deactivation inhibitory effect of TOCN is exhibited in a dry state with a low water content.

(8) Evaluation of room temperature stability of AChE in TOCN pills The AChE / TOCN pills according to the example prepared in (4) above are stored in a sample tube at room temperature for 1 month, and the enzyme activity of AChE after 1 month has passed. Was examined in the same manner as in (6) above. As a result, the activity of AChE in TOCN pills was retained at room temperature for at least 1 month. Considering that the enzyme activity is inactivated in a few hours when AChE not complexed with TOCN is left in the air or at room temperature, and the activity is usually lost in about 3 days even in a buffer solution, encapsulation with TOCN is unstable. It can be seen that deactivation due to oxidation of various biomolecules can be effectively suppressed.

(9) Preparation of AChE / TOCN Paper Device 400 μL of 0.5 mass% TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution were taken into a centrifuge tube and mixed with a vortex mixer. This was dropped onto a filter paper in an amount of 401 μL with a micropipette and air-dried at room temperature for 2 days to prepare an AChE / TOCN paper device according to an example. The obtained paper device was placed in a plastic bag with a zipper and stored at room temperature. When this paper device was subjected to thermogravimetric analysis in the same manner as in (5) above, the water content in the composite composition excluding the filter paper as a support was 18% by mass.

(10) Evaluation of Room Temperature Stability of AChE in TOCN Paper Device The AChE / TOCN paper device according to the example was stored at room temperature for 28 days, and the stability at room temperature was evaluated. As a control, only 1 μL of 250 U / mL AChE solution is dropped onto the filter paper and air-dried to prepare a paper device, which is placed in a plastic bag with a zipper and stored at room temperature for stability at room temperature as well. evaluated.

For the enzyme activity, 195 μL of 100 mM Tris-HCl buffer and 5 μL of 80 mM IDA solution were added to both the AChE / TOCN paper device and the control paper device, and the enzyme reaction was carried out. After drying for 1 hour or more, the image of the paper device is scanned into a personal computer with a scanner (24 bit color, resolution 800 dpi), and the blue intensity of the generated indigo is measured with image processing software (ImageJ) to obtain a sample before storage at room temperature. The specific activity of AChE after storage was calculated with the activity of 100%.

The results are as shown in FIG. 4. In the control paper device, the specific activity was significantly reduced to about 15% after storage for 28 days, whereas in the AChE / TOCN paper device according to the example, the specific activity was 90%. It was held to a certain extent. Therefore, even on paper, TOCN was able to suppress the deactivation of unstable molecules.

(11) Formation of Composite Composition by Ink Printing To 0.5 mass% of TOCN aqueous dispersion 949.76 μL, 50 μL of 250 U / mL AChE solution was added and mixed, and the obtained AChE / TOCN aqueous dispersion was mixed. 0.24 μL of a nonionic surfactant (octylphenol ethoxylate, Triton X-100) was added as a surface tension adjusting agent to prepare an ink for inkjet. For comparison, AChE ink was also prepared by adding 950 μL of 100 mM Tris-HCl buffer to 50 μL of 250 U / mL AChE solution. This is filled in an inkjet device (“Labojet-1000” manufactured by Microjet Co., Ltd.), and the Gifu University logo mark (vertical approx. 5 mm × horizontal approx. 15 mm) is printed on the filter paper and dried to form it on the filter paper. The composite composition according to the example was formed in the shape of a logo mark. After drying, an appropriate amount of 100 mM Tris-HCl buffer and 80 mM IDA solution were dropped onto the printed portion, and the appearance of indigo generation and color development was observed. The inks containing no TOCN were also subjected to inkjet printing in the same manner, and the appearance after the enzyme reaction was compared.

As a result, in the one printed using the ink containing TOCN (Example), the ink was ejected well and a fine logo mark was formed. Further, in the ink containing no TOCN, the indigo bleeds and the enzyme easily flows out due to water flow, whereas in the ink printed with the TOCN-containing ink according to the example, the indigo is generated in the form of a logo mark. Since the dried solidified product of TOCN forms hydrogen bonds between nanofibers, it is difficult to redisperse even if water is added. Therefore, it is conceivable that the TOCN layer swells when water is passed and the reaction solution gradually permeates into the enzyme reaction field. From this, it was shown that the TOCN layer did not flow even when water was passed, and the enzyme was mainly retained in the TOCN layer. In microfluidic paper analysis devices, it is important to immobilize the molecules involved in the reaction at the detection site. From the above results, it was found that TOCN is suitable for a microfluidic paper analysis device because it not only stably stores biomolecules but also has a function of suppressing the outflow of biomolecules from the detection site.

Although some embodiments of the present invention have been described above, these embodiments are presented as examples and are not intended to limit the scope of the invention. These embodiments can be implemented in various other embodiments, and various omissions, replacements, and changes can be made without departing from the gist of the invention. These embodiments, omissions, replacements, changes, etc. thereof are included in the scope and gist of the invention, as well as in the scope of the invention described in the claims and the equivalent scope thereof.

Claims (5)

Cellulose nanofibers anionically modified have a cellulose type I crystal structure, a composite composition comprising an enzyme, a state, and are water content of 20 mass% or less, the cellulose nanofibers carboxyl group as an anionic group A composite composition containing. The composite composition according to claim 1, wherein the enzyme is wrapped in the cellulose nanofibers. The composite composition according to claim 1 or 2 , wherein the cellulose nanofibers are those in which the hydroxyl group at the C6 position of the glucose unit in the cellulose molecule is selectively oxidized and modified to a carboxyl group. A composite composition in which an aqueous dispersion of anion-modified cellulose nanofibers having a cellulose I-type crystal structure and containing a carboxyl group as an anionic group is mixed with an enzyme and dried until the water content becomes 20% by mass or less. Manufacturing method. A biosensor comprising the composite composition according to any one of claims 1 to 3.

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