JP2019019169A - Composite composition, method for producing the same, and biosensor - Google Patents

Abstract

To provide a composite composition which can stabilize biomolecules.SOLUTION: Provided is a composite composition comprising: cellulose nanofibers having a cellulose I type crystal structure and being anionically modified; and biomolecules, where a water content of the composite composition is 20 mass% or less. The composite composition can be obtained by mixing the biomolecules with an aqueous dispersion of the cellulose nanofibers having a cellulose I type crystal structure and being anionically modified, and drying the mixture until the water becomes 20 mass% or less. The composite composition can be used, for example, for a biosensor.SELECTED DRAWING: None

Description

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

  Biosensing is a technique for detecting and measuring various molecules using a biomolecule recognition mechanism. For example, a biochip is a diagnostic and testing device that utilizes the interaction of biomolecules, and is used for early diagnosis of viral infection of diseases, testing of microorganisms and bacteria in food and water. Bioactive substances such as proteins are used as biomolecules, and they are detected and measured by specific binding or interaction of enzyme-substrate, antigen-antibody, hormone-receptor and 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 by heating, and their activity decreases with time due to oxidation even under non-heating. Therefore, for example, when these biomolecules are used in biosensors such as biochips, there is a need for a storage method that can be stably stored even under heating conditions and that does not lose activity over time.

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

  In addition, as a composition containing cellulose fiber and protein, Patent Document 1 describes a food composition composed of cellulose nanofiber, protein and water. However, Patent Document 1 is intended for gels containing a large amount of water and is not a technique for stabilizing biomolecules, and the water content described in the Examples is as high as 70% by mass or more. .

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

  Patent Document 3 describes that cellulose nanofibers are obtained by treating cellulose fibers with an enzyme and then performing defibration. However, since the enzyme is deactivated prior to defibration, it cannot be said that biomolecules are present anymore after defibration, and thus there is no description of combining biomolecules and cellulose nanofibers.

JP 2006-63757 A JP 2017-12762 A Japanese Patent Laid-Open No. 2017-2136

Sana Jahanshahi-Anbuhi and 9 others, “Pullulan Encapsulation of Labile Biomolecules to Give Stable Bioassay Tablets”, Angew.Chem. Int. Ed., 53, 6155-6158 (2014)

  An embodiment of the present invention aims to provide a composite composition capable of stabilizing a biomolecule.

  A composite composition according to an embodiment of the present invention is a composite composition comprising cellulose nanofibers having a cellulose I-type crystal structure and anion-modified, and a biomolecule, and having a moisture 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 so that the water content is 20% by mass or less. It is the manufacturing method of a composite composition which dries until it becomes.

  A biosensor according to an embodiment of the present invention includes the above 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 in, for example, a biosensor, biomolecules can be stabilized and inactivation can be suppressed.

The graph which shows the thermogravimetric analysis of the AChE / TOCN pill which concerns on an Example The graph which shows the thermal-stability evaluation result of AChE / TOCN pill which concerns on an Example. The graph which shows the thermal stability evaluation result of AChE in the TOCN aqueous dispersion which concerns on a reference example The graph which shows the room temperature stability evaluation result of the AChE / TOCN paper device which concerns on an Example

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

  The composite composition according to the present embodiment includes an anion-modified cellulose nanofiber having a cellulose I-type crystal structure and a biomolecule. A dry film made of cellulose nanofibers forms a very dense film made of water-insoluble fibers and exhibits an excellent oxygen barrier property, so that biomolecules that are easily deactivated by oxidation can be stabilized. Biomolecules are stabilized under heating conditions by a dry film of cellulose nanofibers. Furthermore, from the viewpoint of the biochemical reaction field of the composite composition of the present embodiment, since the anion-modified cellulose nanofiber is hydrophilic, water adheres to the composite composition in a dry state and swells in water. Thus, the reaction field by the biomolecule can be given again and contribute to the expression of the biomolecule activity. That is, since the cellulose nanofibers are insoluble in water, in use as an inspection device such as a biosensor, the water resistance problem that has been a problem in the prior art can be solved by exhibiting an appropriate affinity for water. there is a possibility. Furthermore, when the aqueous dispersion of cellulose nanofibers has thixotropic properties, it becomes possible to form a composite composition by, for example, ink jet printing, and the production process may be simplified.

  In the present embodiment, cellulose nanofiber is used as the cellulose fiber. The cellulose nanofiber is a cellulose fiber having a nanometer level fiber diameter, and the number average fiber diameter 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 nanofiber 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 was prepared, and the aqueous dispersion was cast on a carbon film-coated grid that had been subjected to a hydrophilization treatment. A sample for observation with an electron microscope (TEM) is used. Then, observation with an electron microscope image is performed at a magnification of 5000 times, 10000 times, or 50000 times depending on the size of the constituent fibers. At that time, assuming an axis of an arbitrary image width in the obtained image, the sample and observation conditions (magnification, etc.) are adjusted so that 20 or more fibers intersect the axis. Then, after obtaining an observation image that satisfies this condition, two random axes, vertical and horizontal, per image are drawn on this image, and the fiber diameter of the fiber that intersects the axis is visually read. In this way, images of at least three non-overlapping surface portions are taken with an electron microscope, and the fiber diameter values of the fibers intersecting with each of the two axes are read (thus, at least 20 × 2 × 3 = 120). Information on the fiber diameter of the book is obtained). The number average fiber diameter is calculated from the fiber diameter data thus obtained.

The average aspect ratio of the cellulose nanofiber 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: 10000 times) which was negatively stained with 2% uranyl acetate after the cellulose nanofibers were cast on a carbon film-coated grid that had been hydrophilized, the number average of the shorter side of the cellulose nanofibers The number average width of the width and the longer width is observed, and the average aspect ratio is calculated according to the following formula using these values.
Average aspect ratio = Number average width (nm) for longer width / number average width (nm) for shorter width

  Cellulose nanofibers can be obtained by defibrating treatment. The defibrating treatment may be performed after introducing an anionic group described below, or may be performed before the introduction. As the defibrating treatment, for example, using a homomixer under high-speed rotation, a high-pressure homogenizer, an ultrasonic dispersion processor, a beater, a disc type refiner, a conical type refiner, a double disc type refiner, a grinder, etc. It can carry out by processing a dispersion liquid and can obtain the water dispersion liquid of a cellulose nanofiber.

  In the present embodiment, cellulose nanofibers having a cellulose I-type crystal structure are used from the viewpoint of water insolubility. The cellulose constituting the cellulose nanofiber has a type I crystal structure, for example, in the diffraction profile obtained by wide-angle X-ray diffraction image measurement, in the vicinity of 2 theta = 14 ° to 17 ° and 2 theta = 22 ° to 23 °. It can be identified from the fact that there are 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 sulfuric acid group. In the present specification, the carboxyl group is a concept including not only the acid form (—COOH) but also a salt form, that is, a carboxylate group (—COOX, where X is a cation forming a salt with the carboxylic acid). Similarly, the phosphoric acid group, the sulfonic acid group and the sulfuric acid group are concepts including not only the acid type but also the salt type.

  In one embodiment, the anionic group is preferably a carboxyl group. Examples of cellulose nanofibers containing carboxyl groups include oxidized cellulose nanofibers formed by oxidizing the hydroxyl groups of glucose units in cellulose molecules, and carboxymethylated celluloses formed by carboxymethylating the hydroxyl groups of glucose units in cellulose molecules. Nanofiber is mentioned.

  Examples of the oxidized cellulose nanofiber according to a 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 can be obtained by oxidizing natural cellulose such as wood pulp using a co-oxidant in the presence of an N-oxyl compound, and performing a defibration (miniaturization) treatment. As the N-oxyl compound, a compound having a nitroxy radical generally used as an oxidation catalyst is used, for example, piperidine nitroxyoxy radical, particularly 2,2,6,6-tetramethylpiperidinooxy radical (TEMPO). ) Or 4-acetamide-TEMPO. Cellulose nanofibers oxidized with TEMPO are generally called TEMPO-oxidized cellulose nanofibers, and can be used in this embodiment. In addition, although the oxycellulose nanofiber may have an aldehyde group or a ketone group with a carboxyl group, it is preferable not to have an aldehyde group and a ketone group substantially.

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

  Examples of biomolecules in the present embodiment include various biopolymers such as proteins, nucleic acids, lipids, vitamins, hormones, sugars, and sugar chains. More specifically, bioactive substances that are involved in reactions such as specific binding and interaction such as enzyme-substrate, antigen-antibody, hormone-receptor, sugar chain-lectin and the like can be mentioned. The biomolecule possessed is 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 the present embodiment, since cellulose nanofibers provide a stabilizing effect against oxidation or heating, biomolecules that can be deactivated by oxidation and / or heat are preferably used. The protein referred to here includes oligopeptides and polypeptides.

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

  In the composite composition according to one embodiment, the biomolecule is encapsulated by the cellulose nanofiber and stabilized thereby. That is, the composite composition according to one embodiment is a composite containing the cellulose nanofiber and a biomolecule encapsulated, that is, embedded in the cellulose nanofiber. The biomolecules are held in the cellulose nanofibers in a non-deactivated state, and the deactivation due to oxidation and / or heat is suppressed, that is, stabilized by being encapsulated by the cellulose nanofibers.

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

  In the composite composition according to the embodiment, the content of the cellulose nanofiber 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. Thus, the low content of water enables cellulose nanofibers to exhibit oxygen barrier properties, stabilize biomolecules that are easily deactivated by oxidation, and enhance the stabilization effect of biomolecules by heat. it can. The water content of the composite composition is preferably 5 to 20% by mass, more preferably 10 to 15% by mass. The moisture content of the composite composition is the moisture content of the composite composition itself excluding the support even when the composite composition is formed on a support such as paper as described later.

  In the composite composition according to the embodiment, in addition to the above-described cellulose nanofibers, biomolecules, and water, in the range that does not inhibit the effects of the present invention, for example, inorganic and / or organic fillers, clay minerals, Contains dry residues of coloring agents such as pigments, salts, pH adjusters, oils, water-soluble polymers, surfactants, hydrophilic solvents added for the purpose of improving the film-forming properties of dry coatings May be. For example, hydrophilic solvents for improving film-forming properties include alcohols, cellosolves, carbitols, glycols, glycol monophenyl ethers, phenyl alcohols, phenylcarboxylic esters, oxycarboxylic acid phenyl esters, etc. May be included. In addition, a light-resistant agent, an antioxidant, an antiseptic, an inorganic substance, and the like may be included.

  In the composite composition according to the embodiment, the biomolecule is preferably stable 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 this embodiment, i) temperature rising heating from the initial temperature T1 to the temperature T2. Conditions and ii) There are two constant temperature heating conditions in which the temperature T3 is maintained for a predetermined time after the temperature is increased from the initial temperature T1 to reach the temperature T3. In the present embodiment, assumed handling conditions are as follows.

  Generally, there is an operation from a temperature of −15 ° C. or lower to −20 ° C. or lower, generally referred to as a freezing temperature, that is, from a state where a biomolecule is frozen or frozen to an ice melting temperature (0 ° C.). Furthermore, there is an operation that exceeds the melting temperature of ice and reaches room temperature (20 ° C. to 25 ° C.). Further, an operation that is handled at a higher temperature is possible regardless of intentional or unintentional. Specifically, the operation handled at high temperatures, whether intentional or unintentional, is indoors in summer, outdoors in summer, in outdoor cars, direct sunlight, freezer, refrigerator or air conditioner is stopped Is assumed to be handled in In addition, during power outages, disasters, battlefield use, etc. are also assumed.

  A temperature change from the initial temperature T1 as a starting point to the temperature T2 is defined as ΔT. For example, in this embodiment, ΔT = 20 ° C. when the biomolecule is heated from −20 ° C. to 0 ° C. Similarly, when the temperature is raised from −20 ° C. to 25 ° C., ΔT = 45 ° C. Furthermore, 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 biomolecule activity in the composite composition not subjected to heat treatment is 100%. Is preferably 70% or more, and more preferably 80% or more.

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

  As described above, an aqueous dispersion of cellulose nanofibers is prepared by introducing an anionic group into natural cellulose and then performing a defibrating treatment, or by defibrating natural cellulose and then introducing an anionic group. be able to. The density | concentration of the cellulose nanofiber in the said aqueous dispersion liquid is not specifically limited, For example, 0.01-5 mass% may be sufficient, 0.05-3 mass% may be sufficient, and 0.1-1 mass% may be sufficient.

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

  The composite composition which concerns on embodiment is obtained by drying a liquid mixture after mixing the aqueous dispersion of a cellulose nanofiber, and a biomolecule.

  The composite composition may be formed alone by applying the mixed solution onto a releasable substrate such as a fluororesin sheet and drying, and taking out the dried product from the releasable substrate. The form is not particularly limited, and may be a sheet form or a tablet form such as a tablet or a pill.

  Or a composite composition may be formed in the state integrated with the support body by providing a liquid mixture on support bodies, such as paper, and drying. Although it does not specifically limit as a method to provide to a support body, You may form a composite composition on a support body by inkjet printing, for example using the thixotropic property which a cellulose nanofiber aqueous dispersion has.

  As the drying method, for example, a heat drying method, a vacuum drying method, a blow drying method, a microwave drying method, an infrared drying method, a freeze drying method, a filtration dehydration method, or the like is used. A plurality of methods may be combined and dried. The drying temperature is not particularly limited. If the heat drying method is exemplified, the drying temperature is preferably 40 ° C. or less, 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 increased stepwise.

  The composite composition according to the embodiment can be used for, for example, a biosensor. A biosensor is a sensing device that detects and measures various molecules using a biomolecule recognition mechanism, and the composite composition can be used as a constituent element 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 achieved by immobilizing a large number of the composite composition on the substrate as the biomolecules. The biochip according to the above can be configured. Moreover, since inkjet printing is possible as mentioned above, it can also be utilized for a microfluidic paper analysis device (μPAD).

  Hereinafter, although an Example demonstrates further in detail, this invention is not limited to these.

(1) Reagent / TOCN: TEMPO-oxidized cellulose nanofiber having cellulose I type crystal structure, 1.1% by mass aqueous dispersion, “Reocrista” manufactured by Daiichi Kogyo Seiyaku Co., Ltd., number average fiber diameter = 4 nm, average aspect Ratio = 280, anionic group amount = 1.9 mmol / g
Tris: Trishydroxymethylaminomethane, manufactured by SIGMA-ALDRICHI IDA: Indoxyl acetate, manufactured by SIGMA-ALDRICHI AChE: Acetylcholinesterase, derived from Denki eel, 200-1000 Unit / mg protein, manufactured by SIGMA-ALDRICHI Methanol: Kanto Chemical ( 1M NaOH: Sodium hydroxide aqueous solution (1M, manufactured by Wako Pure Chemical Industries, Ltd.)
・ 6M HCl: 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 into 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. When the pH greatly deviated, 1M NaOH and 6M HCl were added dropwise to prepare again.

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

(4) Preparation of AChE / TOCN pill 400 μL of 0.5 mass% TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution per pill were placed in a centrifuge tube and mixed with a vortex mixer. 401 μL of this was dispensed onto a Teflon (registered trademark) sheet with a micropipette and allowed to air dry at room temperature for 2 days to prepare AChE / TOCN pills as a composite composition according to the example. The resulting pill was placed in a sample tube 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 pill The AChE / TOCN pill prepared in (4) above was subjected to thermogravimetric analysis using a thermogravimetric analyzer (manufactured by Seiko Instruments Inc., TGDTA6300). The TG (thermogravimetric curve) and DTG (differential thermogravimetric curve) of AChE / TOCN pill are as shown in FIG. 1, and the water content of AChE / TOCN pill was 13% by mass.

(6) Thermal stability evaluation of AChE in TOCN pill The AChE / TOCN pill after 2 days of room temperature drying according to the example was used. The heat treatment was performed by raising the temperature of the AChE / TOCN pill from room temperature to 80 ° C., and heat treatment was performed at 80 ° C. for 15, 30, 45, and 60 minutes. As the AChE / TOCN pill for heat treatment of 0 minutes, AChE / TOCN pill after 2 days of room temperature drying (AChE / TOCN pill before raising the temperature from room temperature to 80 ° C.) was 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. The AChE / TOCN pill after the heat treatment and the control AChE solution were placed in a well plate, and an enzyme reaction (hydrolysis reaction using IDA as a substrate) was performed. 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 enzyme reaction for 1 hour. This well plate was placed in a microplate reader (Tecan, Spark 10M), and the absorbance at the absorption peak position (605 nm) of indigo was measured. The specific activity of AChE after heat treatment was calculated with the activity of the sample not subjected to heat treatment as 100%. Measurements were performed in triplicate.

  The result of thermal stability evaluation is shown in FIG. In the control, AChE was inactivated by the 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. Thereby, it is considered that the AChE / TOCN pill can be stably stored and transported even at high temperatures.

(7) Thermal stability evaluation of AChE in TOCN aqueous dispersion (reference example)
400 μL of 0.5 mass% TOCN aqueous dispersion and 1 μL of 250 U / mL AChE solution were placed in an Eppendorf tube and mixed with a vortex mixer (0.5% TOCN / AChE). Moreover, 400 microliters of 1 mass% TOCN aqueous dispersion and 1 microliter of 250 U / mL AChE solution were taken to the Eppendorf tube, and were mixed with the vortex mixer (1% TOCN / AChE). Furthermore, as a control, 400 μL of 100 mM Tris-HCl buffer and 1 μL of 250 U / mL AChE solution were placed in an Eppendorf tube and mixed with a vortex mixer.

  These three types of mixed solutions are heat-treated at 60 ° C. for 0, 5, 10, and 15 minutes, put the mixed solutions after the heat treatment into well plates, add 5 μL of 80 mM IDA solution, and evaluate the thermal stability of (6) above. In the same manner as described above, the enzyme reaction was performed to measure the absorbance. The specific activity of AChE after heat treatment was calculated with the activity of the sample not subjected to heat treatment as 100%. Measurements were performed in triplicate.

  The results are as shown in FIG. In the TOCN dispersion, AChE was easily deactivated by heat treatment. This was the same even when the TOCN concentration was increased. Therefore, it is thought that the deactivation suppression effect by TOCN is expressed in a dry state with a low moisture content.

(8) Evaluation of room temperature stability of AChE in TOCN pill AChE / TOCN pills according to the examples prepared in (4) above were stored in a sample tube at room temperature for 1 month, and AChE enzyme activity after 1 month Was examined in the same manner as in (6) above. As a result, the activity of AChE in the TOCN pill was retained at room temperature for at least one month. In view of the fact that AChE that is not complexed with TOCN is left in air at room temperature, the enzyme activity is deactivated in a few hours, and the activity is lost in a buffer solution usually in about 3 days. It can be seen that inactivation 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 placed in a centrifuge tube and mixed with a vortex mixer. 401 μL of the solution was dropped on a filter paper with a micropipette and allowed to air dry at room temperature for 2 days to prepare an AChE / TOCN paper device according to the example. The obtained paper device was put in a plastic bag with a chuck 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 the support was 18% by mass.

(10) Room temperature stability evaluation of AChE in TOCN paper device The AChE / TOCN paper device according to the example was stored at room temperature for 28 days to evaluate the stability at room temperature. As a control, only 1 μL of 250 U / mL AChE solution was dropped on the filter paper and air dried to prepare a paper device, which was stored in a plastic bag with a chuck and stored at room temperature. evaluated.

  The enzyme activity was determined by adding 195 μL of 100 mM Tris-HCl buffer and 5 μL of 80 mM IDA solution to both AChE / TOCN paper device and control paper device. After drying for 1 hour or more, the image of the paper device is taken 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), so that the sample before storage at room temperature And the specific activity of AChE after storage was calculated.

  The results are as shown in FIG. 4. In the control paper device, the specific activity greatly decreased to about 15% after 28 days of storage, whereas in the AChE / TOCN paper device according to the example, the specific activity was 90%. The degree was held. Therefore, even on paper, deactivation of unstable molecules could be suppressed by TOCN.

(11) Formation of composite composition by inkjet printing To 949.76 μL of 0.5 mass% TOCN aqueous dispersion, 50 μL of 250 U / mL AChE solution was added and mixed, and the resulting AChE / TOCN aqueous dispersion was A nonionic surfactant (octylphenol ethoxylate, Triton X-100) 0.24 μL was added as a surface tension adjusting agent to prepare an ink jet ink. As a 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 ink jet apparatus ("Labojet-1000" manufactured by Microjet Co., Ltd.), printed on the filter paper is a Gifu University logo mark (vertical length of about 5 mm x width of about 15 mm), and dried on the filter paper. The composite composition according to the example was formed in a logo mark shape. After drying, appropriate amounts of 100 mM Tris-HCl buffer and 80 mM IDA solution were dropped on the printing section, and the appearance of indigo formation and coloration was observed. Ink-jet printing was similarly performed on the ink not containing TOCN, and the appearance after the enzyme reaction was compared.

  As a result, in the case of printing using an ink containing TOCN (Example), the ink was discharged well and a fine logo mark was formed. Ink containing no TOCN oozes indigo and the enzyme easily flows out by passing water, whereas the ink printed with the TOCN-containing ink according to the example generates indigo in a logo mark shape. Since the dry solidified product of TOCN forms hydrogen bonds between nanofibers, it has a feature that it is difficult to redisperse even when water is added. For this reason, it is considered that the TOCN layer swells during water flow, and the reaction solution gradually permeates to become an 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 the microfluidic paper analysis device, it is important to immobilize molecules involved in the reaction at the detection site. From the above results, it was found that TOCN not only stably stores biomolecules, but also has a function of suppressing the outflow of biomolecules from the detection site, and is therefore suitable for a microfluidic paper analysis device.

  As mentioned above, although some embodiment of this invention was described, these embodiment is shown as an example and is not intending limiting the range of invention. These embodiments can be implemented in various other forms, and various omissions, replacements, and changes can be made without departing from the spirit of the invention. These embodiments and their omissions, replacements, changes, and the like are included in the inventions described in the claims and their equivalents as well as included in the scope and gist of the invention.

Claims (8)

  A composite composition comprising cellulose nanofibers having a cellulose I-type crystal structure and anion-modified, and a biomolecule, wherein the water content is 20% by mass or less.   The composite composition according to claim 1, wherein the biomolecule is encapsulated by the cellulose nanofiber.   The composite composition according to claim 1 or 2, wherein the cellulose nanofiber contains a carboxyl group as an anionic group.   The composite composition according to claim 3, wherein the cellulose nanofibers are those in which a hydroxyl group at the C6 position of a glucose unit in a cellulose molecule is selectively oxidized and modified to a carboxyl group.   The composite composition according to claim 1, wherein the biomolecule is a protein.   The composite composition according to claim 5, wherein the protein is an enzyme.   A method for producing a composite composition, comprising mixing an aqueous dispersion of cellulose nanofibers having a cellulose I-type crystal structure and anion-modified and a biomolecule, and drying the mixture until the water content becomes 20% by mass or less.   The biosensor containing the composite composition of any one of Claims 1-6.

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