JP2008105358A - Pressure-sensitive trigger-type enzyme coloring material, coloring sheet, its manufacturing method and application - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pressure-sensitive trigger-type enzyme coloring material which can develop a color through rapid occurrence of an enzyme-substrate chemical reaction by a simple reaction triggering operation, a color developing ink/a color developing sheet using the coloring material the color developing ink/color developing sheet and its manufacturing method, and a time-temperature indicator/a gas sensor. <P>SOLUTION: This coloring material contains enzyme and a substrate which develop a color through an enzyme-substrate chemical reaction, a water solvent with 100°C or higher boiling point and a microcapsule. In addition, at least either of the enzyme or the substrate is encapsulated in a microcapsule and the color development takes place under the enzyme-substrate chemical reaction in the presence of the water solvent by breaking the microcapsule under pressure. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention is a pressure-sensitive trigger-type enzyme coloring material that initiates a color reaction upon initiation of an enzyme-substrate reaction in a pressure-sensitive manner, an ink using the coloring material, a coloring sheet, a method for producing the same, and a time using the coloring material, etc. The present invention relates to a temperature indicator and a gas sensor.

Materials that develop color by enzyme-substrate reaction are applied in various industrial situations such as immunodiagnosis, trace metal analysis, and bacterial testing.
Recently, a temperature history indicator that displays a time-temperature integration applicable to food distribution and the like using color development by an enzyme-substrate reaction has been studied. For example, Patent Document 1 proposes a temperature history indicator that separates an enzyme and a substrate and presses the enzyme and the substrate so that the enzyme and the substrate come into contact with each other and exhibit a specific color within a certain temperature range. Alternatively, it is also described that one of the enzyme substrates is stored in a microcapsule and pressed at the start of a temperature history to break the microcapsule and mix both reagents to develop color. However, this document does not show any specific examples using the microcapsules.

By the way, a technique for encapsulating an enzyme in a microcapsule has been developed in various applications. For example, Patent Document 2 discloses a technique that can be applied to a biochip or the like by encapsulating a protein such as an enzyme in a microcapsule for stabilization.
On the other hand, Patent Document 3 discloses a technique including a step of attaching a pigment to a receiving layer by applying an enzyme as a receiving layer on a base material and causing an enzyme reaction between a microcapsule having a pigment and a component of the receiving layer. It is disclosed.
As a general technique, Patent Document 4 describes a microencapsulated protein, and Patent Document 5 describes a method of reacting and coloring a coloring substance and an enzyme, and soy protein is pressure-sensitively recorded to reduce coloring stains. Patent Document 6 discloses the use of paper, and Patent Document 7 discloses a method for stabilizing an enzyme using a phosphorylcholine group-containing polymer.

However, there have been no specific examples of coloring materials using enzyme-substrate reaction using microcapsules and various applications using the coloring materials. Sufficient examination has not been made as to whether the reaction occurs effectively.
JP 2001-272283 A JP 2006-223124 A JP-A-11-152848 Japanese Patent Laid-Open No. 7-10771 Japanese Patent Laid-Open No. 6-18521 JP-A-11-165462 Japanese Patent Laid-Open No. 10-45794

An object of the present invention is to provide a pressure-sensitive trigger-type enzyme coloring material that can quickly develop an enzyme-substrate reaction by a simple reaction start operation (trigger), a color-forming ink and a coloring using the coloring material. It is in providing a property sheet and its manufacturing method.
Another object of the present invention is to provide a time-temperature indicator capable of temperature-dependent color display of a temperature history in a low temperature region by a simple reaction start operation.
Still another object of the present invention is to provide a gas sensor that can quickly detect the presence of a specific gas by color change with a simple reaction start operation.

  The present inventors diligently studied to solve the above problems. First, the enzyme and the substrate were separately accommodated in the microcapsule, and then the microcapsule was broken by pressure to cause the enzyme and the substrate to contact with each other. As a result, although color development occurred, the reaction was not rapid, and as a result of repeated improvements, the above problem could be solved by interposing a high-boiling aqueous solvent when the enzyme and the substrate contacted and reacted. The headline and the present invention were completed.

According to the present invention, an enzyme and a substrate that develop color by an enzyme-substrate reaction, an aqueous solvent having a boiling point of 100 ° C. or higher, and a microcapsule are included, and at least one of the enzyme and the substrate is encapsulated in the microcapsule. Disrupting the microcapsules provides a pressure-sensitive trigger type enzyme coloring material characterized in that color develops by an enzyme-substrate reaction in the presence of the aqueous solvent.
Moreover, according to this invention, the color-forming ink containing the said coloring material is provided.
Furthermore, according to this invention, the color development sheet | seat provided with the said coloring material on the surface of a sheet | seat base | substrate is provided.
Furthermore, according to the present invention, the coloring material is divided so that the enzyme and the substrate are separated from each other, applied and fixed to one surface of each of the two sheet bases, and the surfaces on which the coloring material components are fixed overlap. Thus, there is provided a method for producing the color-forming sheet, wherein the sheet base is laminated.
In addition, according to the present invention, the coloring material or coloring sheet containing the microcapsules is provided, and the microcapsules are destroyed by pressure so that the enzyme and the substrate in the coloring material and the like are enzyme-substrate reaction in a specific temperature range. Thus, a time-temperature indicator is provided that is characterized by color development.
Furthermore, according to the present invention, it includes an enzyme, an aqueous solvent having a boiling point of 100 ° C. or higher, a coloring material, and a microcapsule. At least the enzyme is encapsulated in the microcapsule, and the microcapsule is broken and detected by pressure. There is provided a gas sensor comprising a pressure-sensitive coloring material that reacts with an enzyme in the presence of the aqueous solvent, and further reacts with the coloring material to develop color.

The pressure-sensitive trigger type enzyme coloring material, coloring ink and coloring sheet of the present invention include an enzyme, a substrate, a specific solvent and a microcapsule, and at least one of the enzyme and the substrate is encapsulated in the microcapsule, and the presence of the solvent Since the enzyme-substrate reaction is performed below, the enzyme-substrate reaction can be quickly generated and colored by a simple reaction start operation.
Since the time-temperature indicator of the present invention uses the color former or color developable sheet of the present invention, a quick color change depending on the temperature environment occurs due to a simple reaction start operation, depending on the degree of color development after a certain time. The temperature history can be displayed and used for distribution and storage of food.
The gas sensor of the present invention includes an enzyme, a specific solvent, a coloring material, and a microcapsule. At least the enzyme is encapsulated in the microcapsule, and a predetermined gas to be detected reacts with the enzyme in the presence of the aqueous solvent, and further develops color. Since the pressure-sensitive color-developing material that develops color by reacting with the material is provided, a predetermined gas can be quickly detected by color change by a simple reaction start operation.

The present invention will be described in detail below.
The coloring material of the present invention includes an enzyme and a substrate that develop color by an enzyme-substrate reaction, an aqueous solvent having a boiling point of 100 ° C. or higher, and a microcapsule, and at least one of the enzyme and the substrate is encapsulated in the microcapsule. Then, the microcapsules are destroyed by pressure, and color is developed by an enzyme-substrate reaction in the presence of the aqueous solvent.

  The enzyme and the substrate are not particularly limited as long as a coloring system can be constructed. For example, a blue color developing system that generates a tetramethylbenzidine polymer from peroxidase, hydrogen peroxide, and tetramethylbenzidine, and a yellow color that generates p-nitrophenol from alkaline phosphatase, p-nitrophenylphosphate, and hydrogen peroxide. Color development system, red color development system in which naphthol produced from alkaline phosphatase and naphthol phosphate is reacted with diazonium salt, color development system using galactosidase, yellow color development system of protease and pNA-peptide, blue of amylase and blue starch Color development system, yellow color development system of amylase and 2-cyclo-4-nitrophenyl-4-O-β-D-galactopyranosyl maltoside, glucosidase color development system, ammonia is produced from urease and urea to generate alkaline pH Coloring system with indicator, Lipper Examples thereof include a coloring system that generates fatty acid from glycerin and triglyceride and uses an acid pH indicator, and a coloring system that generates gluconic acid from glucose oxidase and glucose and that uses an acid pH indicator.

  In the present invention, the aqueous solvent is a component that increases the reaction rate of the enzyme-substrate reaction and enhances the color developability, and is a solvent that dissolves in water having a boiling point of 100 ° C. or higher, preferably 150 ° C. or higher, more preferably 200 ° C. or higher. is there. The solubility of the aqueous solvent in water is preferably 5% or more, particularly 10% or more, and more preferably a solvent that can be mixed in water at an arbitrary ratio. In addition, when the colorant of the present invention is used at a particularly low temperature, the aqueous solvent preferably has a low melting point, for example, a melting point of −5 ° C. or lower, particularly −20 ° C. or lower. The viscosity of the aqueous solvent in the liquid state may be low or high. However, a relatively high viscosity of 0.01 to 1,000,000 P, particularly 1 to 100,000 P is preferable.

Examples of the aqueous solvent used in the present invention include alcohols such as butanol, pentanol, hexanol, heptanol, octanol, methoxyethanol, ethoxyethanol, methoxyethoxyethanol, dipropylene glycol monomethyl ether, and tripropylene glycol monomethyl ether; ethylene glycol Polyhydric alcohols such as propylene glycol, butylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, tripropylene glycol, glycerin, diglycerin, triglycerin, tetraglycerin, polyglycerin; Polyethylene glycol, polypropylene glycol, polyethylene glycol-polypropylene Polyalkylene glycols such as glycol copolymers; dimethyl sulfoxide, N- methylpyrrolidone, aprotic polar solvents such as dimethylformamide and the like, can be utilized by mixing two or more of these.
More preferable aqueous solvents include, for example, alcohols such as dipropylene glycol monomethyl ether and tripropylene glycol monomethyl ether; ethylene glycol, propylene glycol, butylene glycol, diethylene glycol, triethylene glycol, tetraethylene glycol, dipropylene glycol, and tripropylene. Polyhydric alcohols such as glycol, glycerin, diglycerin, triglycerin, tetraglycerin, and polyglycerin; high-boiling protic solvents such as polyethylene glycol, polypropylene glycol, and polyethylene glycol-polypropylene glycol copolymers having a molecular weight of 1000 or less Can be mentioned.

Depending on the type of the coloring material of the present invention, the activity may decrease depending on the type of the microcapsule, which will be described later, the coating step, and high temperature conditions. It is preferable that the stabilizer is included.
Any stabilizer can be used as long as it has an effect of stably maintaining the activity of the enzyme. For example, a phosphorylcholine group-containing polymer, glucose, sucrose, maltose, xylitol, trehalose, oligosaccharide, cellulose, carboxymethylcellulose, hydroxyethylcellulose, hydroxypropylmethylcellulose, chondroitin sulfate, dextrin, cyclodextrin, etc .; ethylene glycol, propylene glycol , Polyhydric alcohols such as butylene glycol, glycerin, diglycerin and polyglycerin; polyalkylene glycols such as polyethylene glycol, polypropylene glycol, polybutylene glycol and polyethylene glycol-polypropylene glycol copolymer; amino acids such as aspartic acid and arginine Kind. Among these, from the viewpoint of the enzyme stabilizing effect, preferred are sugars such as a phosphorylcholine group-containing polymer, glucose, sucrose, maltose, xylitol, trehalose, oligosaccharide, cellulose, carboxymethylcellulose, chondroitin sulfate, dextrin, and cyclodextrin. . Most preferred is a phosphorylcholine group-containing polymer that has a high enzyme stabilizing effect even under various environments and can enhance the efficiency of the enzyme reaction in the colorant of the present invention.

As the phosphorylcholine group-containing polymer, any polymer containing a phosphorylcholine group can be used. For example, MPC obtained by polymerizing a monomer composition containing 2-methacryloyloxyethyl phosphorylcholine (MPC). Preferred is a containing polymer.
MPC can be produced by a known method. For example, it can be produced according to known methods disclosed in JP-A-54-63025 and JP-A-58-154591.

  The MPC-containing polymer may be a copolymer of MPC and another monomer, and among them, a polymer obtained by copolymerizing a hydrophobic monomer and MPC is desirable. And a copolymer of a hydrophobic monomer and MPC. Further, a terpolymer obtained by copolymerizing a hydroxyl group-containing monomer such as glycerol monomethacrylate (hereinafter abbreviated as GLM), a hydrophobic monomer such as alkyl (meth) acrylate, and MPC is more preferable. . These copolymers can be further copolymerized with other monomers.

  Examples of the hydrophobic monomers include methyl (meth) acrylate, ethyl (meth) acrylate, butyl (meth) acrylate, 2-ethylhexyl (meth) acrylate, lauryl (meth) acrylate, stearyl (meth) acrylate, and the like. Chain or branched alkyl (meth) acrylate; Cyclic alkyl (meth) acrylate such as cyclohexyl (meth) acrylate; Aromatic (meth) acrylate such as benzyl (meth) acrylate and phenoxyethyl (meth) acrylate; Polypropylene glycol (meth) acrylate Hydrophobic polyalkylene glycol (meth) acrylates such as styrene; styrene monomers such as styrene, methyl styrene and chloromethyl styrene; vinyl ether monomers such as methyl vinyl ether and butyl vinyl ether; vinyl acetate and vinyl propionate Nyl ester monomers may be mentioned, and one or more of these may be used.

  Other monomers for obtaining the MPC-containing copolymer include, in addition to hydrophobic monomers, for example, 2-hydroxyethyl (meth) acrylate (hereinafter abbreviated as HEMA), 2-hydroxybutyl (meta ) Acrylate, 4-hydroxybutyl (meth) acrylate, hydroxyl group-containing (meth) acrylate such as glycerol (meth) acrylate; acrylic acid, methacrylic acid, styrene sulfonic acid, (meth) acryloyloxyphosphonic acid, 2-hydroxy-3- Ionic group-containing monomers such as (meth) acryloxypropyltrimethylammonium chloride; nitrogen-containing monomers such as (meth) acrylamide, aminoethyl methacrylate, dimethylaminoethyl (meth) acrylate; polyethylene glycol (meth) acrylate, Glycidyl (meth) acrylate, one of these or It used two or more kinds. Among these, use of hydroxyl group-containing (meth) acrylates such as HEMA, 2-hydroxybutyl (meth) acrylate, 4-hydroxybutyl (meth) acrylate, and GLM is most desirable in terms of suppressing enzyme degradation.

The proportion of MPC constituting units constituting the MPC-containing polymer is usually 20 to 95% by weight, preferably 30 to 70% by weight. If the ratio of MPC is less than 20% by weight, the reaction efficiency of the enzyme cannot be sufficiently maintained, and if it is more than 95% by weight, the functions of other monomers to be copolymerized may not be sufficiently exhibited. is there.
The molecular weight of the MPC-containing polymer is preferably in the range of 5000 to 5000000 in weight average molecular weight, and more preferably in the range of 50000 to 1000000.

Examples of the most specific combination of monomers constituting the MPC-containing copolymer in terms of the high enzyme activity and the effect of suppressing deterioration of the enzyme activity in the colorant of the present invention include MPC-GLM- A terpolymer of alkyl methacrylate, particularly a combination of MPC-BMA-GLM may be mentioned.
The composition of each monomer constituting the ternary copolymer of MPC-GLM-alkyl methacrylate is desirably in the range of MPC 20 to 90% by weight, GLM 5 to 70% by weight, alkyl methacrylate 5 to 60% by weight, Desirably, MPC is 40 to 80% by weight, GLM is 10 to 50% by weight, and alkyl methacrylate is 10 to 50% by weight.
These MPC-containing polymers can be obtained by known methods.

  In addition to the enzyme stabilizer described above, other substances may be added to the colorant of the present invention as required, as long as the enzyme activity is not impaired. Examples of other substances include binders, inorganic salts, organic salts, surfactants, preservatives, dyes, cross-linking agents, ultraviolet absorbers, and primers for enhancing adhesion to the substrate.

Examples of the binder include rosin-modified phenol resin, phenol resin, ketone resin, polyamide resin, rosin-modified male resin, male resin, epoxy resin, alkyd resin, melamine resin, urea resin, nitrocellulose, ethyl cellulose, butyral resin, polyvinyl alcohol. Examples thereof include resins, water-soluble resins such as latex, gelatin, and cellulose, and ultraviolet curable resins.
When the coloring material of the present invention is used as a coating liquid, the binder can be contained in a necessary amount in the coating liquid. For example, it can be contained in the coating solution in a proportion of 1 to 50% by weight, preferably 2 to 20% by weight.

  In the coloring material of the present invention, the microcapsules enclosing at least one of the above-described enzyme and substrate are separated by, for example, a chemical method such as an interfacial polymerization method or an in-site polymerization method, a submerged drying method, or a gel conversion. A method prepared by any known method such as a physicochemical method such as a method or a coacervation method, or a mechanical method such as a spray drying method or a dry mixing method can be used. Since the microcapsule encloses an enzyme solution or substrate solution that is an aqueous solution, it can be prepared by a method in which the enzyme solution or substrate solution is emulsified with hydrophobic oil and W / O emulsified, and this emulsion is used as an inclusion. it can. Furthermore, an enzyme solution or a substrate solution can be dispersed in a hydrophobic oil instead of an aqueous medium and used for preparing microcapsules.

Any known material can be used as the microcapsule film material, and examples thereof include gelatin, gum arabic, urea resin, urethane resin, acrylic resin, melamine resin, and polyamide resin.
In preparing the microcapsules, a known dispersant can be used. Examples of the dispersant include anionic surfactants such as alkyl sulfates, alkyl sulfonates, alkyl benzene sulfonates, alkyl naphthalene sulfonates, and dialkyl sulfosuccinates; polyoxyethylene alkyl ethers, polyoxyethylene alkyls Nonionic surfactants such as aryl ethers or polyoxyethylene fatty acid esters; partially saponified polyvinyl alcohol, lower alkyl ether-modified polyvinyl alcohol, acrylamide polymers, acrylamide copolymers, polyacrylates, polystyrene sulfonates, maleic anhydride Water-soluble polymer compounds such as acid / isobutylene copolymer salt, carboxymethyl cellulose salt, hydroxyethyl cellulose, starch, casein, gum arabic or gelatin

  The particle size of the microcapsule can be set to any value according to the purpose, but is preferably in the range of 0.5 to 500 μm, more preferably 1 to 50 μm, and most preferably various printing methods and application to ink. It is in the range of 2 to 20 μm with high possibility.

In the color former of the present invention, the microcapsule can contain only one of the enzyme and the substrate, or the enzyme and the substrate can be contained in another microcapsule so as to be separated from each other.
The microcapsule encapsulating the enzyme preferably encapsulates the enzyme stabilizer, and can also encapsulate other materials than the above-mentioned substrate.
The microcapsules enclosing the substrate can enclose the above-mentioned other materials other than the enzyme and the enzyme stabilizer.

  The aqueous solvent having a boiling point of 100 ° C. or higher can be contained in the enzyme-containing microcapsule, the substrate-containing microcapsule, or both. Moreover, as long as it can be made to exist when destroying a microcapsule and carrying out an enzyme-substrate reaction, it does not need to contain in any microcapsule. For example, when the coloring material of the present invention is fixed to a substrate in the form of a coating liquid described later, a method of containing the coloring material in a dispersion solvent of the coating liquid and remaining on the substrate after coating, a coating liquid not containing the aqueous solvent A method of impregnating the microcapsules and / or the substrate with the aqueous solvent by spraying or coating can be employed after fixing the substrate to the substrate.

  In the color former of the present invention, the content ratio of the enzyme and the substrate is not particularly limited as long as it is an amount that can be colored by the enzyme-substrate reaction. For example, when used in a time-temperature indicator described later, the enzyme and the substrate. Depending on the type, temperature and time, the color can be appropriately selected in order to produce a desired color. The content of the aqueous solvent having a boiling point of 100 ° C. or higher can be determined by appropriately selecting the amount that can be present during the enzyme-substrate reaction.

The coloring material of the present invention can be used, for example, by using a suitable liquid medium as a coating liquid and coating and fixing on various substrates.
As the liquid medium, any solvent can be used as long as it can coat a liquid containing microcapsules, and it can be used from the aqueous medium having a boiling point of 100 ° C. or higher used in the present invention. For example, various buffer solutions such as water, acetate buffer, phosphate buffer, Tris-HCl buffer, carbonate buffer, Good's buffer, methanol, ethanol, propanol, methyl ethyl ketone, methyl propyl ketone, methyl butyl A single solution or a mixture of various organic solvents such as ketone, ethyl acetate, propyl acetate, toluene, hexane, and chloroform can be used.

  The substrate for fixing the coloring material of the present invention may be any substrate as long as it can coat a liquid containing microcapsules. For example, paper, nonwoven fabric, fiber, fabric, film, plastic, foamed plastic, wood, metal, rubber, Examples include ceramics and painted surfaces, and not only flat surfaces but also curved substrates. A substrate having a minute and complicated surface such as a fiber or a porous body can also be applied. Among these, those that can be used for packaging materials such as food are preferable, and for example, paper, nonwoven fabric, film, plastic, and foamed plastic are more preferable. Furthermore, a sheet substrate such as paper, non-woven fabric, or film that can be used as a substrate of the color-forming sheet of the present invention described later is preferable in that it can be used for various applications such as food packaging and wallpaper.

Any method can be used for coating the coating liquid containing the color former of the present invention as long as the liquid containing microcapsules can be coated. For example, bar coating, blade coating, roll coating, gravure coating, silk Examples include screen coating, spray coating, dip coating, and spin coating.
The coating conditions are not particularly limited. However, since the enzyme may be deteriorated by heat, the temperature during drying is usually in the range of 5 to 50 ° C., particularly 10 to 30 ° C.

Specific examples of the coating method include, for example, a method of coating a substrate with a coating solution containing a substrate, and coating a coating solution containing enzyme-containing microcapsules thereon, and coating the substrate with an enzyme-containing coating solution, A method of coating a coating solution containing a substrate-containing microcapsule on the substrate, a method of coating a substrate with a coating solution containing a substrate-containing microcapsule, and coating a coating solution containing an enzyme-containing microcapsule thereon, an enzyme in the reverse order A method of coating a substrate with a coating solution containing a substrate containing a coating solution containing microcapsules, a substrate containing a coating solution containing a substrate containing microcapsules, and a coating solution containing an enzyme Coating method, a method of coating a substrate with a coating solution containing enzyme-containing microcapsules, and coating a coating solution containing substrate-containing microcapsules thereon, a coating solution containing enzyme-containing microcapsules and a substrate, and substrate-containing methods A method of coating a substrate with a coating solution containing microcapsules and an enzyme or a coating solution containing enzyme-containing microcapsules and a substrate-containing microcapsule, coating each of the above coating solutions on another substrate, and overlaying the coating surface of each substrate The method of contacting together is mentioned.
At this time, the above-mentioned aqueous solvent having a boiling point of 100 ° C. or higher can be included in one or both of the coating liquids, and another coating liquid containing an aqueous solvent having a boiling point of 100 ° C. or higher is prepared, and in an appropriate order. The substrate can be coated.

The color-forming ink of the present invention contains the above-described color forming material of the present invention.
In order to use the color-forming ink of the present invention as an ink, a resin, a solvent, a drying oil, an antifoaming agent, a film reinforcing agent, a drying accelerator, an anti-set-off agent, a drying inhibitor, a polymer, an oligomer, Monomers, initiators, photoinitiators, sensitizers and the like can be blended in appropriate amounts depending on the purpose.
The color-forming ink of the present invention can be used in known printing methods such as offset printing, letterpress printing, gravure printing, screen printing, and ink jet printing.

The color-forming sheet of the present invention is a sheet comprising the above-described color-forming material of the present invention on the surface of a sheet substrate. In addition, the color-forming sheet of the present invention includes a sheet in which two sheet bases each having an enzyme on the lamination surface of one sheet substrate and a substrate on the other surface of the other sheet substrate are laminated, and the lamination surface of one sheet substrate. In which the enzyme and the substrate are separated from each other, and the other sheet substrate is laminated on the laminated surface of the two sheet substrates having the above-mentioned aqueous solvent having a boiling point of 100 ° C. or higher. Non-carbon paper) is also included.
By using the pressure-sensitive paper, it can be confirmed by the color development of the pressure-sensitive paper that the measurement has been started by the enzyme coloring system in a pressure-sensitive manner, and the convenience is enhanced.

  The color-forming sheet of the present invention is, for example, a method in which a sheet base is coated and fixed with the color-forming material of the present invention by the above-described coating method or the like. A method of coating and fixing the coloring material of the invention, and contacting each other by overlapping each coating surface. Specifically, as an example, the coloring material of the present invention is divided so that the enzyme and the substrate are separated from each other. The sheet substrate is coated and fixed on one surface of the sheet substrate, and the sheet substrate is laminated so that the surface on which the coloring material component is fixed overlaps, and further, these methods are combined by pressure sensitive paper. Can do.

  Examples of methods for combining pressure-sensitive paper include, for example, a method of laminating pressure-sensitive paper and the above-mentioned coating sheet with the coating surfaces thereof laminated, a method of using pressure-sensitive paper as the sheet substrate, and a color developing material solution for pressure-sensitive paper. Examples thereof include a method of coating on the material, and a method of coating the sheet solution with the color developing material solution of the pressure sensitive paper and the color developing material of the present invention at the same time.

The substrate provided with the coloring material or coloring ink of the present invention, or the coloring sheet of the present invention is such that the enzyme and the substrate come into contact with each other by breaking the above-described microcapsules by pressure, and the enzyme-substrate reaction is started and color develops. .
The pressure applied to break the microcapsules is not particularly limited as long as breakage is possible, and examples thereof include a pressure of about 1 g to 100 kg, preferably about 10 g to 1 kg per 1 cm ² . Examples of the method of applying the pressure include a method of tracing the surface of the substrate with a pointed jig such as a pen, and a method of rolling on the surface of the substrate using a metal roller of about 10 cm.

The time-temperature indicator of the present invention comprises the color former or color developable sheet of the present invention containing microcapsules, and the microcapsules are destroyed by pressure so that the enzymes and substrates contained therein are in a specific temperature range. The color develops in the enzyme-substrate reaction.
Such color control can be performed depending on time-temperature by controlling the conditions such as the type of the coloring system of the enzyme and the substrate, the solvent, the concentration and the like in the above-described coloring material of the present invention. . The target time-temperature history can be detected based on the degree of color development. In particular, time-temperature history management in a relatively low temperature region of 5 to 40 ° C. is important for food management and the like, and can be suitably used for this application.
Since the coloring material of the present invention can be controlled to change the degree of coloring depending on substances such as trace gas and moisture in the atmosphere, gas detection and humidity measurement that can start measurement pressure-sensitively Etc. can also be used.

The gas sensor of the present invention includes an enzyme, an aqueous solvent having a boiling point of 100 ° C. or higher, a coloring material, and a microcapsule. At least the enzyme is included in the microcapsule, and the microcapsule is broken and detected by pressure. A gas is provided with a pressure-sensitive coloring material that reacts with an enzyme in the presence of the aqueous solvent and further reacts with the coloring material to develop color.
As the aqueous solvent having a boiling point of 100 ° C. or higher and the microcapsules, those exemplified in the color former of the present invention can be preferably used, and in addition to the above essential components, the above-mentioned other materials can be included. .

  The enzyme and the coloring material used in the gas sensor of the present invention may be those which can be colored by the presence of the gas to be detected. For example, as a measurement system for detecting formaldehyde, formaldehyde by formaldehyde dehydrogenase and coenzyme NAD + is used. Enzyme and color-developing material for coloring pH change due to formic acid generated by decomposing aldehyde, or NADH and phenazine methosulfate (oxidized form) generated by decomposing formaldehyde with formaldehyde dehydrogenase and coenzyme NAD + The phenazine methosulfate (reduced form) is produced by this, and the phenazine methosulfate (reduced form) reacts with nitrotetrazolium blue to produce diformazan dye, which is representative of the enzyme and coloring material for color development in blue. As a result It is.

The gas sensor of the present invention uses a coloring material instead of the substrate in the coloring material or coloring sheet of the present invention. For example, the above-mentioned substrate, particularly the sheet substrate, using the above-described coating method, It can be manufactured by coating the pressure-sensitive color former in the gas sensor.
The gas sensor of the present invention can be implemented by a method similar to the destruction of microcapsules caused by the pressure described above.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated further in detail, this invention is not limited to these.
Production Example 1
Preparation of MPC-BMA copolymer (MPC0.7-BMA0.3)
28.0 g of MPC and 12.0 g of butyl methacrylate (BMA) are dissolved in 160 g of ethanol, put into a four-necked flask, blown with nitrogen for 30 minutes, and then added with 0.82 g of azobisisobutyronitrile (AIBN) at 60 ° C. For 8 hours. The polymerization solution was added dropwise to 3 L of diethyl ether with stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to prepare 32.5 g of a powdered MPC-BMA copolymer. This is abbreviated as polymer 1.

Production Example 2
Preparation of MPC-BMA-GLM copolymer (MPC0.4-BMA0.4-GLM0.2)
MPC 11.4 g, BMA 5.5 g, and GLM 3.1 g were dissolved in ethanol 180 g, put into a four-necked flask, blown with nitrogen for 30 minutes, then AIBN 0.85 g was added at 50 ° C., and a polymerization reaction was carried out for 8 hours. The polymerization solution was added dropwise to 3 L of diethyl ether while stirring, and the deposited precipitate was filtered and vacuum dried at room temperature for 48 hours to prepare 16.1 g of powdered MPC-BMA-GLM copolymer. This is abbreviated as polymer 2.

Production Example 3
Preparation of Enzyme-Containing Microcapsule Coating Liquid (A) A liquid (A-1) was prepared by dissolving 75 g of sorbitan trioleate in a mixed liquid of 250 g of chloroform and 750 g of cyclohexane. A solution (A-2) was prepared by dissolving 5.0 g of hexamethylenediamine and 5.0 g of sodium carbonate in 200 g of pure water, and further dissolving 50 mM magnesium salt and 0.02 g of alkaline phosphatase. Next, the solution (A-2) was added to the solution (A-1) and stirred vigorously, and the solution (A-3) in which 10 g of chloroterephthalic acid was dissolved in a mixed solution of 250 g of chloroform and 750 g of cyclohexane was added. Slowly added and allowed to react for 1 hour. After the reaction, the obtained microcapsules were settled, the supernatant solvent was removed, and 500 g of ethanol was added for washing. This ethanol washing was repeated once more, and after further washing with pure water, water was removed. To the obtained microcapsule slurry, a solution obtained by dissolving 2.5 g of polyvinyl alcohol in 50 g of pure water was added and stirred, 150 g of styrene-butadiene rubber latex was added and stirred, and the enzyme-containing microcapsule coating solution (A) was added. Prepared.

Production Example 4
Preparation of (Enzyme + Glycerin) -Containing Microcapsule Coating Liquid (B) A liquid (B-1) was prepared by dissolving 75 g of sorbitan trioleate in a mixed liquid of 250 g of chloroform and 750 g of cyclohexane. A solution (B-2) was prepared by dissolving 5.0 g of hexamethylenediamine, 5.0 g of sodium carbonate, and 50 g of glycerin in 150 g of pure water, and further dissolving 50 mM magnesium salt and 0.02 g of alkaline phosphatase. Next, the (B-2) solution was added to the (B-1) solution and stirred vigorously, and the (B-3) solution in which 10 g of chloroterephthalic acid was dissolved in a mixed solution of 250 g of chloroform and 750 g of cyclohexane was added thereto. Slowly added and allowed to react for 1 hour. After the reaction, the obtained microcapsules were settled, the supernatant solvent was removed, and 500 g of ethanol was added for washing. This ethanol washing was repeated once more, and after further washing with pure water, water was removed. To the obtained microcapsule slurry, a solution obtained by dissolving 2.5 g of polyvinyl alcohol in 50 g of pure water was added and stirred, 150 g of styrene-butadiene rubber latex was added and stirred, and the enzyme-containing microcapsule coating solution (B) was added. Prepared.

Production Example 5
Preparation of (enzyme + glycerin + polymer 1) -containing microcapsule coating solution (C) In a mixed solution of 250 g of chloroform and 750 g of cyclohexane, 75 g of sorbitan trioleate was dissolved to prepare a solution (C-1), which was ice-cooled. did. Hexamethylenediamine 5.0 g, sodium carbonate 5.0 g, and glycerin 50 g are dissolved in 150 g of pure water. Further, 1.5 g of the polymer 1 produced in Production Example 1, 50 mM magnesium salt, and 0.02 g of alkaline phosphatase are sequentially added. Dissolved (C-2) solution was prepared. Next, the (C-2) solution was added to the (C-1) solution and stirred vigorously, and the (C-3) solution in which 10 g of chloroterephthalic acid was dissolved in a mixed solution of 250 g of chloroform and 750 g of cyclohexane was added thereto. Slowly added and allowed to react for 1 hour. After the reaction, the obtained microcapsules were settled, the supernatant solvent was removed, and 500 g of ethanol was added for washing. This ethanol washing was repeated once more, and after further washing with pure water, water was removed. To the obtained microcapsule slurry, a solution obtained by dissolving 2.5 g of polyvinyl alcohol in 50 g of pure water was added and stirred, 150 g of styrene-butadiene rubber latex was added and stirred, and (enzyme + glycerin + polymer 1) -containing microcapsules A coating solution (C) was prepared.

Production Example 6
Preparation of microencapsule coating solution (D) containing (enzyme + glycerin + polymer 2) In the same manner as in Production Example 5 except that Polymer 2 produced in Production Example 2 was used instead of Polymer 1, (Enzyme + A coating liquid (D) of microcapsules containing glycerin + polymer 2) was prepared.

Production Example 7
Preparation of (enzyme + polyglycerin) -containing microcapsule coating liquid (E) Except that glycerin was changed to polyglycerin, the same operation as in Production Example 4 was performed, and (enzyme + polyglycerin) -containing microcapsule coating liquid (E ) Was prepared.

Production Example 8
Preparation of (enzyme + diethylene glycol) -containing microcapsule coating liquid (F) The same procedure as in Production Example 4 was repeated except that glycerin was changed to diethylene glycol to prepare (enzyme + diethylene glycol) -containing microcapsule coating liquid (F). did.

Production Example 9
Preparation of (enzyme + isopropanol) -containing microcapsule coating liquid (G) The same procedure as in Production Example 4 was repeated except that glycerin was changed to low-boiling isopropanol. ) Was prepared.

Production Example 10
Preparation of coating liquid (H) for (capsule + glycerin) -containing microcapsules In a mixed liquid of 250 g of chloroform and 750 g of cyclohexane, 75 g of sorbitan trioleate was dissolved to prepare (H-1) liquid and ice-cooled. Hexamethylenediamine 5.0 g, sodium carbonate 5.0 g, and glycerin 50 g are dissolved in 150 g of water containing 50 mM magnesium salt, and further 80 mg of nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate A solution (G-2) in which 40 mg of toluidine salt was dissolved was prepared. Next, (H-2) solution was added to (H-1) solution and stirred vigorously, and (H-3) solution in which 10 g of chloroterephthalic acid was dissolved in a mixed solution of 250 g of chloroform and 750 g of cyclohexane was added thereto. Slowly added and allowed to react for 1 hour. After the reaction, the obtained microcapsules were settled, the supernatant solvent was removed, and 500 g of ethanol was added for washing. After removing the supernatant solvent once more and adding ethanol for washing, the solvent ethanol was removed. To the obtained microcapsules, 50 g of pure water in which 2.5 g of polyvinyl alcohol was dissolved was added and stirred, and then 150 g of styrene-butadiene rubber latex was added and stirred to prepare a substrate-containing microcapsule coating liquid (H). .

Production Example 11
Preparation of enzyme coating solution (I) 0.01 g of alkaline phosphatase was dissolved in 39.95 g of 0.1 M Tris buffer (pH 9.5) containing 50 mM magnesium salt, mixed with 50 g of styrene-butadiene rubber latex, and enzyme coating solution ( I) was prepared.

Production Example 12
Preparation of Substrate Coating Solution (J) 80 mg of nitroblue tetrazolium salt and 40 mg of 5-bromo-4-chloro-3-indolyl phosphate toluidine salt were added to 0.1 M Tris buffer (pH 9.5) 40 containing 50 mM magnesium salt. Dissolved in 0.0 g, mixed with 50 g of styrene-butadiene rubber latex to prepare a substrate coating solution (J).

Production Example 13
Preparation of enzyme-containing microcapsule coating sheet The enzyme-containing microcapsule coating solution (A) prepared in Production Example 3 was applied to a base paper of 100 g / m ^{2 at} a thickness of 20 μm, and 6 times with a 35 ° C. circulation dryer. The enzyme-containing microcapsule coated sheet was prepared by drying for a period of time. This sheet is abbreviated as EMA.

Production Examples 11 to 19
A sheet was prepared using each coating solution in the same manner as in Production Example 13 except that each of the coating solutions (B) to (J) prepared in Production Examples 4 to 12 was used instead of the coating solution (A). did. Of the enzyme, substrate, microcapsule, glycerin, polymer 1 and polymer 2, the material fixed to the prepared sheet, the abbreviation of the sheet, and the type of coating liquid used are shown in Table 1.

Reference example 1
Coloring test of pressure-sensitive trigger type enzyme color-forming sheet Each coating liquid application surface in sheets EMA and SCJ shown in Table 1 was overlapped to prepare a pressure-sensitive trigger type enzyme-coloring sheet. Next, the surface of the sheet was slowly dragged while applying a load of 50 g to a jig having a 2 mm diameter stainless steel sphere attached to the tip, and left in an environment at a temperature of 30 ° C. After 0 minutes, 5 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 3 hours, 6 hours, 12 hours, and 24 hours, the color development status (shown as a decrease in lightness) is shown as a simple spectral color difference meter NF333. (Nippon Denshoku Industries Co., Ltd.) evaluated. The results are shown in Table 2. At this time, the time required for the brightness to be 70 or less was 1 hour.

Example 1
Coloring test of pressure-sensitive trigger type enzyme coloring sheet A coloring sheet was prepared in the same manner as in Reference Example 1 except that the EMB and SCJ shown in Table 1 were used as the sheet, and the coloring state was evaluated. The results are shown in Table 3. At this time, the time required for the brightness to be 70 or less was 10 minutes.

Examples 2-8
A color forming sheet was prepared in the same manner as in Example 1 except that the combination of sheets shown in Table 4 and the temperature were changed, and the state of color development was evaluated. The time when the lightness became 70 or less and the lightness after 1 hour are shown in Table 4 together with the results of Reference Example 1 and Example 1.
In Examples 1 to 4, the color development situation could be controlled by the difference in temperature environment with the same material, and the temperature environment in which the material was placed could be known from the brightness value after 1 hour. Moreover, in Reference Example 1, since an aqueous solvent having a boiling point of 100 ° C. or higher was not included, it took a long time for the lightness to be 70 or less compared to Example 1 in which the test was performed at the same temperature.

Example 9
Coloring test of pressure-sensitive trigger type enzyme coloring sheet A coloring sheet was prepared in the same manner as in Example 1 except that EME and SCJ shown in Table 1 were used as the sheet, and the coloring state was evaluated. The results are shown in Table 5. At this time, the time required for the brightness to be 70 or less was 10 minutes.

Example 10
Coloring test of pressure-sensitive trigger type enzyme coloring sheet A coloring sheet was prepared in the same manner as in Example 1 except that the EMF and SCJ shown in Table 1 were used as the sheet, and the coloring condition was evaluated. The results are shown in Table 6. At this time, the time required for the brightness to be 70 or less was 10 minutes.

Comparative Example 1
When microcapsules are not used A coated sheet of sheets ECI and SCJ shown in Table 1 was overlapped to produce a pressure sensitive trigger type enzyme coloring sheet. The color was developed immediately after the application surfaces were stacked, and the reaction could not be started at any time in a pressure sensitive manner.

Comparative Example 2
A color forming sheet was prepared in the same manner as in Example 1 except that EMG and SCJ shown in Table 1 were used as the sheet, and the state of color development was evaluated. The results are shown in Table 7. At this time, the time required for the lightness to be 70 or less required 2 hours because an aqueous solvent having a boiling point of 100 ° C. or higher was not used.

Examples 11-13
Durability test (50 ° C, 4 hours)
Each sheet shown in Table 8 was stored at 50 ° C. for 4 hours, and then a color-forming sheet was prepared in the same manner as in Example 1 with the combination of sheets shown in Table 8, and the color development status was evaluated. Table 8 shows the time when the lightness became 70 or less and the lightness after 1 hour.

Comparative Example 3
Durability test when microcapsules are not used (50 ° C, 4 hours)
The sheets ECI and SCJ shown in Table 1 were stored at 50 ° C. for 4 hours, and then the coated surfaces were stacked to prepare a pressure-sensitive trigger type enzyme color-forming sheet. As a result, the color developed weakly immediately after the application surfaces were overlapped, and the reaction could not be initiated at an arbitrary time in a pressure sensitive manner.

Examples 14-16
Durability test (60 ° C, 4 hours)
After each sheet shown in Table 9 was stored at 60 ° C. for 4 hours, a color-forming sheet was prepared in the same manner as in Example 1 with the combination of sheets shown in Table 9, and the state of color development was evaluated. Table 9 shows the time when the lightness became 70 or less and the lightness after 1 hour.

Comparative Example 4
Durability test when microcapsules are not used (60 ° C, 4 hours)
The sheets ECI and SCJ shown in Table 1 were stored at 60 ° C. for 4 hours, and then the coated surfaces were stacked to prepare a pressure-sensitive trigger type enzyme coloring sheet. As a result, the brightness immediately after the application surfaces were overlapped was 89, and after that, there was no change, no color was developed even after pressure was applied, and there was no change in brightness.

Example 17
Pressure Sensitive Trigger Type Enzyme Coloring Material + Polyhydric Alcohol Coloring Test An ethanol solution containing 5% by mass of diethylene glycol was sprayed on the coated surface of the sheet EMA shown in Table 1, and ethanol was dried with hot air at 40 ° C. Next, the coated surface of the sheet SCJ shown in Table 1 was overlapped, and the state of color development was examined by the same operation as in Example 1. The results are shown in Table 10. The time when the lightness became 70 or less was 10 minutes, and the lightness after 1 hour was 61.

Example 18
Pressure Sensitive Trigger Type Enzyme Coloring Material + Polyhydric Alcohol Coloring Test An ethanol solution containing 5% by mass of diglycerin was sprayed on the coated surface of the sheet EMA shown in Table 1, and ethanol was dried with hot air at 40 ° C. Next, the coated surface of the sheet SCJ shown in Table 1 was overlapped, and the state of color development was examined by the same operation as in Example 1. The results are shown in Table 11. The time when the lightness became 70 or less was 10 minutes, and the lightness after 1 hour was 55.

Example 19
Coloring test of color developing sheet combining pressure sensitive trigger type enzyme coloring material and pressure sensitive paper. The application surface of sheets EMB and SCJ shown in Table 1 is overlapped, and commercially available carbonless paper is overlapped on the back side, pressure sensitive trigger type An enzyme coloring sheet was prepared. As in Example 1, a 50 mm load was applied to a jig with a 2 mm diameter stainless steel sphere attached to the tip, and the surface was slowly dragged over the chromogenic sheet to start the enzyme reaction in an environment at a temperature of 30 ° C. The black color of carbonless paper was recorded on the back side of the chromogenic sheet. Table 12 shows the color development on the front side. The time when the lightness became 70 or less was 10 minutes, and the lightness after 1 hour was 60. The black record clearly indicated that the enzyme reaction was started.

Example 20
Ink made of pressure-sensitive trigger type enzyme coloring material 10 g of coating solution (D) of microcapsule containing enzyme + glycerin + MPC-BMA-GLM copolymer prepared in Production Example 6 was taken, diluted with 10 g of pure water, and further styrene -The maleic anhydride copolymer 1.5g and isopropanol 40g were added and stirred. Next, 5 g of the substrate coating liquid (J) prepared in Production Example 12 was added and mixed to prepare an ink made of a pressure-sensitive trigger enzyme coloring material. This ink was printed on paper with a porous ink penetration mark. After the printed part was dried, the enzyme reaction was started by tracing under the same method as in Example 1 in an environment of 30 ° C. After 30 minutes, the color developed clearly only in the part traced by applying pressure.

Example 21
Preparation of Microcapsules Containing Enzyme + Glycerin + Polymer 1 for Gas Sensor A solution (K-1) was prepared by dissolving 75 g of sorbitan trioleate in a mixed solution of 250 g of chloroform and 750 g of cyclohexane, and ice-cooled. A solution (K-2) in which 5.0 g of hexamethylenediamine, 5.0 g of sodium carbonate and 50 g of glycerin were dissolved in 150 g of pure water, 1.5 g of polymer 1 and 0.02 g of formaldehyde dehydrogenase were further dissolved in order. did. The (K-2) solution was added to the (K-1) solution and stirred vigorously, and the (K-3) solution in which 10 g of chloroterephthalic acid was dissolved in a mixed solution of 250 g of chloroform and 750 g of cyclohexane was gradually added. The mixture was further reacted for 1 hour. After the reaction, the obtained microcapsules were settled, the supernatant solvent was removed, and 500 g of ethanol was added for washing. This ethanol washing was repeated once more, and after further washing with pure water, water was removed. A solution obtained by dissolving 2.5 g of polyvinyl alcohol in 50 g of pure water is added to the microcapsule slurry and stirred, 150 g of styrene-butadiene rubber latex is added and stirred, and a coating liquid for enzyme + glycerin + polymer 1 containing microcapsules is added. (K) was prepared.

Preparation of enzyme-containing microcapsule coating sheet for gas sensor The coating solution (K) was applied to a base paper of 100 g / m ^{2 at} a thickness of 20 μm, and dried for 6 hours with a circulation dryer at 35 ° C. for 6 hours. A capsule-coated sheet was prepared. This sheet is abbreviated as EMK.

Preparation of coloring material coating sheet for gas sensor Coenzyme NAD + 160mg, phenazine methosulphate (oxidized) 4mg and nitrotetrazolium blue 40mg are dissolved in 40.0g of 50mM phosphate buffer pH7.5, and styrene-butadiene rubber latex A coloring material coating liquid (L) was prepared by mixing with 50 g. This coating solution (L) was applied to a base paper of 100 g / m ^{2 at} a thickness of 20 μm, and dried for 6 hours with a circulation dryer at 35 ° C. to prepare a coloring material coating sheet. This sheet is abbreviated as SCL.

Production of gas sensor provided with pressure-sensitive color former A gas sensor provided with a pressure-sensitive color former was produced by overlapping the coated surfaces of the sheets EMK and SCL. This was first put in a desiccator maintained at a concentration of 1 to 5 ppm of formaldehyde gas and allowed to stand for 10 minutes, but there was no change. Then, this sensor is taken out, dried in a vacuum dryer for 24 hours to remove the adsorbed formaldehyde gas, and then slowly dragged on the surface while applying a load of 50 g to a jig with a 2 mm diameter stainless steel ball attached to the tip. The enzyme reaction was started. It was left in the room for 10 minutes, but there was no change. Next, when this was put into a desiccator of 1 to 5 ppm of formaldehyde gas for 10 minutes, it colored blue and functioned as a gas sensor after applying pressure.

Comparative Example 5
Preparation of Enzyme Coating Sheet for Gas Sensor 0.01 g of formaldehyde dehydrogenase was dissolved in 39.95 g of 50 mM phosphate buffer having a pH of 7.5 and mixed with 50 g of styrene-butadiene rubber latex to obtain an enzyme coating solution (M). This coating solution (M) was applied to a base paper of 100 g / m ^{2 at} a thickness of 20 μm, and dried for 6 hours with a circulation dryer at 35 ° C. to prepare an enzyme-coated sheet. This sheet is abbreviated as ECM.

Production of Gas Sensor A gas sensor was produced by overlapping the coated surfaces of the sheets ECM and SCL. This was first placed in a desiccator maintained at a concentration of 1 to 5 ppm of formaldehyde gas and allowed to stand for 10 minutes. As a result, blue color was developed, and color development due to enzyme reaction was confirmed before applying pressure.

Claims (15)

  It contains an enzyme and a substrate that develop color by an enzyme-substrate reaction, an aqueous solvent having a boiling point of 100 ° C. or higher, and a microcapsule. At least one of the enzyme and the substrate is included in the microcapsule, and the microcapsule is destroyed by pressure. Thus, a pressure-sensitive trigger-type enzyme coloring material, which develops color by an enzyme-substrate reaction in the presence of the aqueous solvent.   The coloring material according to claim 1, wherein the coloring material is encapsulated in separate microcapsules so that the enzyme and the substrate do not contact each other.   The coloring material according to claim 1 or 2, wherein the aqueous solvent is encapsulated in all or part of the microcapsule.   The color former according to any one of claims 1 to 3, comprising an enzyme stabilizer in a microcapsule encapsulating an enzyme.   The color former according to claim 4, wherein the enzyme stabilizer is a phosphorylcholine group-containing polymer.   A color-forming ink comprising the color-forming material according to claim 1.   A color-forming sheet comprising the color-forming material according to claim 1 on the surface of a sheet substrate.   The color-forming sheet according to claim 7, comprising a sheet obtained by laminating two sheet bases, an enzyme provided on a laminated surface of one sheet base, and a substrate provided on a laminated surface of the other sheet base.   The color-forming sheet according to claim 7 or 8, further comprising pressure-sensitive paper.   The coloring material according to any one of claims 1 to 5 is divided so that the enzyme and the substrate are separated from each other, applied and fixed to one surface of each of the two sheet bases, and the coloring material component is fixed. 9. The method for producing a color forming sheet according to claim 8, wherein the sheet base is laminated so that the surfaces overlap each other.   A coloring material comprising the microcapsule according to any one of claims 1 to 5, wherein the enzyme and substrate in the coloring material undergo an enzyme-substrate reaction in a specific temperature range by breaking the microcapsule with pressure. Time-temperature indicator characterized by color development.   A chromogenic sheet comprising the microcapsule according to any one of claims 7 to 9, wherein the enzyme and substrate in the chromogenic sheet are enzyme-substrate in a specific temperature range by breaking the microcapsule by pressure. Time-temperature indicator characterized by color development in response.   A predetermined gas that includes an enzyme, an aqueous solvent having a boiling point of 100 ° C. or higher, a coloring material, and a microcapsule, at least the enzyme is included in the microcapsule, breaks the microcapsule by pressure, and is detected by the aqueous solvent A gas sensor comprising a pressure-sensitive coloring material that reacts with an enzyme in the presence and further reacts with a coloring material to develop color.   The gas sensor according to claim 13, comprising an enzyme stabilizer in the microcapsule encapsulating the enzyme.   The gas sensor according to claim 14, wherein the enzyme stabilizer is a phosphorylcholine group-containing polymer.