JPS62291565A - Curing reagent matrix film and manufacture thereof - Google Patents

Abstract

(57) [Summary] This bulletin contains application data before electronic filing, so abstract data is not recorded.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides hardened reagent coatings.
entcoatings) and their manufacturing method.

Reagent coatings are used in so-called dry chemistry test chemicals, also known as test strips. Said reagent coatings are cured by carboxyl group-activated crosslinking agents, hereinafter also referred to as curing agents. Consisting of a matrix based on proteins and/or polymers, the reagent coating according to the invention is so hard that it can be wiped off without damage.

When the test coating according to the invention is brought into contact with the sample to be analyzed, usually a biological fluid (for example blood, plasma, serum or urine), the coating absorbs the sample and swells to a predetermined and checkable extent. .

In recent years, clinical chemistry measurement methods have been greatly revolutionized and simplified with the introduction of "dry chemistry" test chemicals, also called test strips. Mixing solutions and sometimes very expensive A1
A change in color or fluorescence must be measured in a fixed device.
In contrast to wet chemistry methods, dry chemistry methods typically only require the specimen to be brought into contact with the sample.

In some cases it is necessary to pre-dilute the sample.

The literature contains many examples of "dry chemistry" test chemicals.

In each case, the reagents and auxiliaries are incorporated into the carrier matrix.

When a sample containing the substance to be measured is applied, a reaction occurs within the matrix that generates a measurable signal (eg color or fluorescence).

There are a number of materials suitable as carrier matrices, such as paper (US Pat. No. 3,092,463; 4.27
No. 9.993; No. 4,318,985; No. 4,361,6
No. 48, No. 4,273,868-, No. 4,318,709
No. 4,190,419; No. 4,215,157; 4
, 038,031) or natural proteins (U.S. Pat. Nos. 3,983,005, 4,042,335; 4.0
No. 98,594; 4+ No. 166.093; 4,274
, No. 832; No. 4,255,384; 4.452,88
No. 7; No. 4,340,565) is publicly known.

The use of synthetically prepared polymers is also described in US Pat. Nos. 3,630,957 and 4,312,834.

Measuring the blood sugar content in whole blood is particularly important in the treatment of diabetic patients. If patients are able to perform such tests themselves, they will have the opportunity to conduct their own therapeutic tests and drug treatments, and will be able to monitor and better adjust their diet.

Such a test is easy to perform, ie.

It is necessary to apply a sample (eg, whole blood) to the test strip and after a predetermined period of time to wipe or remove excess sample with a cotton swab.

One of the requirements that such a test agent must meet is that the matrix (test coating) absorbs only a specific and reproducible amount of the liquid sample. Additionally, the matrix must be physically hard enough to resist breakage by wiping or swabbing excess sample.

After finding a suitable carrier matrix meeting these conditions, the starting point was a water-soluble polymer that was hardened by a cross-linking agent during the preparation of the test coating, for example gelatin was used as the soluble polymer.

Most known crosslinking agents cannot be used to prepare reagent coatings.

Some react with indicator dyes to produce a color change. Others release mineral acids during the reaction or deactivate enzymes necessary for the detection reaction. Still others provide coatings that are inflexible or that are not sufficiently abrasion resistant. Some reaction rates are too fast or too slow, and some are too unstable in an aqueous environment.

Surprisingly, a crosslinking agent has now been found that has the properties required for the test coating.

The present invention relates to cured test coatings having a matrix based on polymers and/or proteins and cured using carboxyl-activated crosslinkers. A carboxyl group-activating crosslinking agent refers to a crosslinking agent that activates carboxyl groups by reacting with the carboxyl groups of a polymer or protein acting as a binder in the first reaction.

Such activated carboxyl groups can further react with other functional groups, such as amine groups, SH groups or hydroxy groups of the binding agent, thereby crosslinking the matrix.

Suitable binding agents (matrix materials) are proteins and derivatives thereof, which usually have carboxyl groups that can be activated. Proteins also have other functional groups necessary for cross-linking.

Suitable proteins include gelatin, gelatin derivatives, casein or zein, with gelatin and its derivatives being preferred. Polymers such as, for example, amphoteric polymers having the groups mentioned above are likewise suitable as binders (matrilas materials).

Examples of carboxyl group-activated crosslinking agents suitable for the present invention include the following.

General formula (I): In formula C, R1 is preferably an alkyl group having 1 to 3 carbon atoms; an aryl group optionally substituted with a lower alkyl group or a halogen atom, e.g., optionally,
methyl, ethyl, propyl, phenyl di- or aralkyl groups substituted by C or Br, e.g.
Represents a benzyl group which may be substituted in the same manner as the above aryl group; R2 represents the same as R1, or a divalent optionally substituted alkylene group, arylene group, aralkylene or alkyl-aryl-
represents an alkyl group, such as an ethylene, propylene, phenylene or xylene group; such a group is further bonded by a second bond to a carbamoylammonium group of the above general formula; or R, and R2 become -g, optionally substituted formed an atomic group necessary to complete the piperidine, piperazine or morpholine ring, and the ring is an alkyl group having 1 to 3 carbon atoms or a halogen atom, such as a C1
Or it may be substituted with Br〈;R3 is -
N Ra -G O-Rs C In the formula, R4 represents H or an alkyl group having 1 to 4 carbon atoms; R6 is H5
represents an alkyl group having 1 to 4 carbon atoms or NR6R7; R6 and R7 represent H or an alkyl group having 1 to 4 carbon atoms;
)1-N R6Rg (wherein, R8 is -Co-RII,
R9 represents H or an alkyl group having 1 to 4 carbon atoms, Rlo represents NR, R12; Ru
represents an alkyl group or an aryl group having 1 to 4 carbon atoms; R12 represents H, an alkyl group or an aryl group; m represents 1 to 3; or R3 represents -(CH2), I C0NR*Rsa C In the formula, Ru represents H, an alkyl group having 1 to 4 carbon atoms, or an aryl group; R14 represents H or an alkyl group having 1 to 4 carbon atoms;
represents an alkyl group of 4; or R13 and R14 are
- together form the atomic group necessary to complete a 5- or 6-membered aliphatic ring; n represents O~3; or R3 is -(CH21p-CH- R-engineer.

(In the formula, R + S represents H or an alkyl group having 1 to 4 carbon atoms, optionally substituted with a halogen atom; Y represents 10- or -NRη; RL@ represents H, an alkyl group, -Co Rts or 100-N
HR□; Ra7, R11 and RR9 represent H or an alkyl group having 1 to 4 carbon atoms; p is 2 to
3); Z represents a carbon atom necessary to complete a 5- or 6-membered aromatic heterocyclic ring optionally having a fused benzene ring; xe represents an acid anion; carbamoylammonium compounds.

General formula (II): ! 110 R9χ [wherein R and R2 represent the same as described for the carbamoylammonium compound of formula (I); Rz represents a methylene group, ethylene group, propylene group or a single chemical bond; R21 is hydrogen Represents an atom, methyl group, or ethyl group;
10a - or CR30S O3-; M
ee represents an alkali metal cation group, and carbamoylammonium compounds represented by are preferred.

Furthermore, compounds of general formulas (III) and (IV) are suitable as crosslinking agents.

In the compound of general formula (■):, R1 and R2 (which may be the same or different) are each an alkyl group having 1 to 10 carbon atoms (for example, methyl, ethyl, 2-ethylhexyl, etc.)
, an aryl group having 6 to 15 carbon atoms (e.g. phenyl,
naphthyl, etc.) or an aralkyl group having 7 to 15 carbon atoms (e.g. -benzyl, phenethyl, etc.), or are bonded to each other and together with a nitrogen atom, represent a heterocyclic ring (e.g. pyrrolidine ring, piperazine ring, morpholine ring, etc.) forming; R3 is a hydrogen atom, a halogen atom, a carbamoyl group,
Sulfo group, halogen atom, carbamoyl group, ureido group, alkoxy group having 1 to 10 carbon atoms, 1 to 1 carbon atom
When R3 represents an alkyl group or an alkoxy group as described above, such a group may be further substituted in each case by a halogen atom, a carbamoyl group, a sulfo group, a ureido group, etc. , in the above formula, Xθ represents an anion and acts as a counter ion of N-carbamoyl-pyridinium ion to form a salt; or when R3 contains a sulfo group as a component and an inner salt is formed, xe is Examples of suitable anionic groups represented by xe, which may be absent, include halide ions, sulfonate ions, sulfonate ions, ClO4-, BF4-, PF5-, and the like.

Further, in the carbamoyloxoammonium compound represented by the following formula (■): niote, R,, R2, R, and xe are the same as described in formula (III).

The following formula (V): In the formula, R,, R2, R3 and R4 (which may be the same or different) each represent an alkyl group having 1 to 20 carbon atoms (
For example, methyl, ethyl, butyl, 2-ethylhexyl,
dodecyl, etc.), 7ralkyl groups having 6 to 20 carbon atoms (e.g., phenyl, phenethyl, 3-pyrimidylmethyl, etc.)
or a 7-aryl group having 5 to 20 carbon atoms (e.g., phenyl, naphthyl, pyridyl, etc.);
-20 alkoxy groups, alkoxy groups having 6 to 20 carbon atoms, N,N-disubstituted carbamoyl groups, etc.), and 2 selected from R, , R2, R3 and R4.
Two groups may be bonded to each other to form a ring. For example, R□ and R2 or R3 and R4 may be bonded to each other to form a ring together with a nitrogen atom. Specific examples of the ring formed in such a case include a pyrrolidine ring, a piperazine ring, a perhydroazepine ring, and a morpholine ring. Further, R1 and R3 or R2 and R4 are bonded together with two nitrogen atoms having one carbon atom therebetween to form a ring. Specific examples of the ring formed in this way include an imidacilline ring, a tetrahydropyrimidine ring, a tetrahydrodiazepine ring, etc. In formula (V), represents a group that can be eliminated from the compound, and preferable examples thereof include a halogen atom, a sulfonyloxy group, and a 1-pyridinyramyl group. In the above formula (V), ye represents an anion,
Suitable examples include halide ions, sulfonate ions, sulfate ions, CfL4-, BFa-,
Examples include PFs-. R1, R2, R3 and R4
When any one of the formula (V) has a sulfo group, the crosslinking agent of formula (V) 1+Ve-n壬子吒/I can also form an inner salt.

In the carbodiimide represented by the following formula (■): R1-N=C=NR2(Vl), which is a suitable crosslinking agent, R is an alkyl group having 1 to 10 carbon atoms (e.g. methyl, ethyl, 2- ethylhexyl, etc.), cycloalkyl groups having 5 to 8 carbon atoms (e.g. cyclohexyl, etc.), alkoxyalkyl groups having 3 to 10 carbon atoms (e.g. methoxyethyl, etc.), or aralkyl groups having 7 to 15 carbon atoms (e.g. benzyl, phenethyl, etc.). In the above formula (V), in addition to the group defined for R1, R2 has the following formula:
(propylene, trimethylene, etc.): R4 and R5
(which may be the same or different) each preferably represents an alkyl group having 1 to 6 carbon atoms (e.g. methyl, ethyl, etc.), or is bonded to each other together with a nitrogen atom to form a heterocyclic ring ( For example, it is also preferable to form pyrrolidine, piperazine, morpholine, etc.).

In the above formula, R6 preferably represents an alkyl group having 1 to 6 carbon atoms substituted with a specific substituent. Preferred examples of such substituents include unsubstituted and substituted carbamoyl groups and sulfo groups. It will be done. xe is an anion, such as halide ion, sulfonate ion, sulfate ion, Cf
0 formula (V
) (7) If the curing agent forms an inner salt, the counterion indicated by X is not required.

In the halogenopyridinium compound represented by the following formula (■): RI Y9, R1
is an alkyl group having 1 to 10 carbon atoms (e.g. methyl, ethyl, butyl, etc.), an aryl group having 6 to 15 carbon atoms (
(e.g., phenyl, naphthyl, etc.), an aralkyl group having 7 to 15 carbon atoms (e.g., benzyl, phenethyl, etc.); each of these groups may be substituted with a substituent; preferred examples of such substituents include , carbamoyl group, sulfamoyl group, sulfo group, etc.
), R2 and R3 (which may be the same or different) are hydrogen atoms, halogen atoms, acylamido groups, nitro groups, carbamoyl groups, ureido groups, alkoxy groups, alkylno, (, alkenyl groups, aryl groups), respectively. It is also preferable that R2 and R3 represent a group, an aralkyl group, etc., and bond to each other to form a condensed ring having a pyridinium ring structure.

In formula (■), X represents a group that is eliminated from the compound represented by formula (1) by reaction with a nucleophilic reagent, and preferable examples include a halogen atom, a sulfonyloxy group, -
OF (OR4)2 or -OF (OR4)2
(wherein R4 represents an alkyl group, an alkyl group, or an aryl group). When X is a sulfonyloxy group, it is also preferable that X is bonded to R1.

In formula (■), ye represents an anion 1, such as halide ion, sulfonate ion, sulfate ion, C04
-, BF4- or PF, -, etc. are preferred. However, if R is a group substituted with a sulfo group, Ye may not be present since the above compound forms an inner salt.

For sulfonylaminopiperidinium represented by the following formula (■): R1 and R2 represent the same as defined for R1 and R2 in formula (III); R3 has 1 carbon atom
~10 alkyl groups (e.g. methyl, ethyl, butyl, etc.), aryl groups having 6 to 15 carbon atoms (e.g. phenyl, naphthyl, etc.) or aralkyl groups having 7 to 15 carbon atoms (e.g. benzyl, phenylethyl, etc.) Representation;
Xe represents an anion, and preferable examples thereof include halide ion, sulfonate ion, sulfate ion, C1o
4-, BF4-1PF6-, and the like.

In addition to the curing agents represented by formulas (I) to (■) above, JP-A-Sho (JP-O5) No. 50-38.540; t552-
No. 93,470, No. 56-43.353 and No. 58-
No. 113,929 and U.S. Patent Publication Section 3,321,3
The compounds described in No. 13 can also be used as carboxy group-activated curing agents.

Specific examples of curing agents of the above-mentioned type that are particularly advantageously used are shown below, without being restricted thereto.

a) Compounds of formulas (I) to (I[r) °”° to 8-1°-: n 9 to! m, p-108-110C H3 Highly hygroscopic syrup 2H5 m, 0.105- 107 C syrup m, p, 103-105 C m, p, 75-77 C m, p, 110-112 C CH3 m, p, 95-96 C 舅 0 Mystery The above compound can be prepared by simple methods known from the literature. The second carbamoyl chloride is reacted under dark conditions with an aromatic heterocyclic nitrogen-containing compound.
Prepared from amines, for example using phosgene. The method for producing compound 3 is Chemishen Bericht
En, 40 (1907), p. 1831.

Further information regarding the above synthesis is also available from JP-O
3) No. 49-51.945 and JP-A-Sho (JP-OS
) t552-59.625.

Niri Inu Yu 11 Mamehoku Cup Bureau ClO4 I le Cv The synthesis method of these compounds is Che■1str Le
tters (Chemical Society of Japan), pp. 1891-1894 (
1982).

(CH3)2CH-N=C=N-(CH2 Synthesis of these compounds-05) No. 57-44.

No. 46.538 and Special Publication Show 1 described in more detail.

The CH3 method is described in JP-A-Sho (JP No. 140, No. 57-58-50,669 5゜1o4 CH30 C2H5
) No. 52-54,427.

, JP-O350-38540! ”
Kaisei 1go bY monolith I"13h, JP-O3+ 52-93
To 470? 111 Noshii, I JP-O3-56
-43353 Danba” Miki 1dbY monoro OO ° JP-OS 58-11
3 929 "Danba" L solution 1 Colored material OC10 0C10 ΩTo' = 1L material A method for synthesizing the crosslinking agent will be described in the following example together with an example of the above compound.

49.5 g of phenylmethylcarbamoylphosgene chloride was carefully passed into anhydrous toluene 400C113 under cooling (22-25°C). To this solution, while stirring vigorously, add 1 liter of distilled N-methylaniline.
A solution of 0.7 g of anhydrous toluene in 450 cm3 was added dropwise.
The mixture was then heated at 80-90° C. for 30 minutes, then cooled and the precipitated hydrochloride salt was filtered off with suction. Finally, the toluene solution was evaporated to dryness in vacuum while removing water. The residue crystallized upon cooling.

Yield: 81g, melting point = 85°C 49.5g of morpholinocarbonylphosgene chloride in 400% of anhydrous toluene 1=^-
/ +1 Shizl “Spider Day Kozuki Stock The jso 111
While stirring, distill 87 g of morpholine and 45 g of anhydrous toluene.
○-The solution was added dropwise at room temperature.

The mixture was then heated at 80-90° C. for 30 minutes, cooled and the precipitated hydrochloride salt was filtered off with suction.

The product was washed with anhydrous toluene.

The furnace liquor was evaporated in vacuo while removing moisture.

The residual oil was then distilled.

Boiling point = 72°C, yield: 50 g Compound 6 phenylmethylcarbamoyl chloride 33, 9 g was stirred and mixed with 400 cm3 of anhydrous pyridine in a dark room while removing moisture. After 3 hours, 0 equivalents of precipitated ether were added to the resulting carbamoylpyridinium chloride, and the crystals were filtered with suction. This was taken up in a small amount of absolute ethanol and precipitated again using ether. The compound was filtered off with suction, washed with ether and then dried in a desiccator.

Yield: 41 g, melting point 108-110 °C 10.5 g of 10.000) were dissolved in 150 g of absolute ethanol and 10.8 g of dimethylcarbamoyl chloride were added with vigorous stirring.

The mixture was stirred at room temperature for 5 hours and concentrated in vacuo. The above mixture was passed through ether with stirring, and the precipitated product was filtered with suction and washed thoroughly with ether. 'rl It's L.

The compound was dried in a desiccator.

Yield: 18g 3-acetaminoviridine 1/20 mole of morpholinocarbonyl chloride was added to a 60 sl solution of 1/20 mole of chloroform with stirring. The mixture was stirred for a further 2 hours with removal of water. The chloroform was then removed in vacuo and the thick residue was triturated with a small amount of anhydrous acetone.

The crystalline product was filtered off with suction and washed with a small amount of anhydrous ether.The product was then dried in a desiccator.

Yield: 10g, melting point: 118-123℃ (Compound 41) e e Na Cl 15.9 g of pyridine sulfonic acid and 200 rn of anhydrous dimethylformamide! suspended in it. Add to this 2.6 g of sodium and 150 g of methanol. / solution was added dropwise (pH
7). The above solution was filtered and washed with N chloride.

13 g of N-dimethylcaroctamoyl was added. The mixture was left at room temperature overnight. The reaction product precipitated after adding ether 400-. This was filtered with suction and washed thoroughly with ether.

Yield: 19.5 g, decomposition point: 250° C. (Compound 44) ee CI Na 18 g of sodium pyridine-3-sulfonic acid was dissolved in 1000 g of dimethylformamide (dry) and 1 g of methanol.
00- and added 15 g of piperidinocarbonyl chloride. The mixture was allowed to stand overnight and the reaction product was precipitated with ether 200-. This was filtered with suction and washed with ether.

Yield: 13 g, decomposition point: 250°C or above.
The 8-I solution was added dropwise to the I^l, 9 Cgm Confucian Bottle Jay No. 1 (pH 7).

The resulting solution was filtered to remove small amounts of insoluble components and then reacted with 18.9 g of morpholifcarbonyl chloride. The mixture was allowed to stand at room temperature for 24 hours; the reaction product partially precipitated. The yield was improved by adding anhydrous ether.

The residue was filtered with suction and washed with anhydrous ether.

Yield: 18 g, melting point: 236-237 °C Transfer 1 (Compound 46) Preparation of Compound 46 e Na Cl 18.2 g of the sodium salt of pyridine-3-sulfonic acid was dissolved in a mixture of 100% dimethylformamide and 100% methanol. 18.5 g of N-methyl-N-methyllipenyl chloride was added. After standing for 3 days, a small amount of crystals precipitated. This is suction filtered,
Washed with ether.

Yield = 3g, melting point = 300°C or higher Branch 1Lu (Compound 55) Na'i' C1゜Sodium salt of pyridine-4-ethanesulfonic acid 18.
9 g was dissolved in a mixture of 400 g of dimethylformamide (dry) and 400 g of methanol (dry) and 15 g of morpholinocarbonyl chloride was added. - After standing overnight, remove the reaction product.

Precipitation was performed using ether 1.34Q. The crystals were filtered off with suction and washed with ether.

Yield = 22g, melting point: 152-153℃ Min.
7.8% 7.2%5 8.9%
9.3%Na 6.4% 6.2
% pyridine odor 121 (odor thresol
d) is about 0.0004 g/rn' in air, so pyridine is perceived by the sense of smell in extremely low amounts. In contrast, the maximum workplace concentration (MWC: m
aximum workplace concert
+tration) is quite high at 15mg/rnj,
Odor testing is therefore an extremely suitable method for determining extremely low pyridine concentrations in workplaces. The workplace concentration of pyridine must not exceed the limit of 15 mg/m'' in air [Solvent Effects in Organic Chemistry (Rei
chard.

Losun 5m1tteleNeket in de
r or anischenChemie), Ve
rlag Chemie 1 7 2 pages].

According to the present invention, the compound used as a curing agent is odorless because it is fixed in the coating.

This can be readily seen by evaporating an aqueous solution of compound 1a7 to dryness at a 5% concentration and testing this process by an odor test for a pyridine-like odor. When used in the above-mentioned compound, the same conditions apply.

The fact that the carbamoyl compounds containing sulfonic acid groups are virtually odorless under the above conditions also means that they can be processed without the risk of exceeding maximum workplace concentrations.

The compounds used in the method according to the invention are conveniently added, preferably in the form of an aqueous or alcoholic solution, to the bend in which the reagent coating to be cured is cast. The compounds described above are conveniently added to gelatin or other proteins conventionally used in diagnostic chemistry. After addition of the above compounds, the casting solution should be cast within a few minutes. The rate at which the curing reaction proceeds depends primarily on the concentration of protein in the casting solution.

In contrast to derivatives of pyridine, either unsubstituted or substituted with lower alkyl groups, which lead to the evolution of unpleasant odors during the casting and drying process, the compounds of the invention can be used without the evolution of gaseous products. Crosslink gelatin.

MWC value (in the United States, it is called the threshold 1
1m1t) is a gaseous-vaporous substance or dust-like substance in the air in a workroom that does not pose a health hazard even if employees work in the workroom for 8 hours a day for more than a year. Indicates the maximum workplace concentration of the substance.

A further possibility for using the abovementioned compounds is firstly the uncured casting solution 7. After casting the violet, the coating thus obtained is coated with a solution of a hardener compound.

)νIIQ I-)-)7 Uetsu So 49 moves/Paban dome
In addition to the Houkiyama-button protein derivatives, water-soluble polymeric compounds, especially polyvinyl alcohol, sodium polyacrylate and other carboxyl group-containing homopolymers or copolymers; furthermore, polyvinylpyrrolidone, polyacrylamide or high molecular weight natural substances, For example, it may contain dextran, dextrin, starch ether, alginic acid or alginic acid derivatives.

The concentrations used for the curing agents of the present invention can vary widely and depend substantially on the curing agent compound used.

0.1 to 10% by weight based on the dry weight of the binder, preferably
An amount of 0.2 to 6% by weight gives good results.

As mentioned above, the reaction between the compounds of the invention and the gelatin or protein begins immediately, so that optimum hardening conditions can be obtained almost simultaneously with drying of the coating following casting or processing thereof.

The action of the curing agent compound depends on the melting point of the coating;
The melting point is measured as follows.

Half of the coating cast on the support was immersed in water and
Heat continuously to ℃. The temperature at which the coating runs off the support (streaks are formed) is called the melting point or melting point;
The melting point of pure protein or gelatin coatings without hardeners was not increased by this measurement method. Under these conditions the melting point is 30-35°C.

Since the compounds according to the invention react with proteins with surprising speed, it is possible to cure protein-containing substances to the optimal state in a very short time. The unexpected action of the above compounds is of particular importance for the curing of diagnostic materials containing proteins or protein derivatives as binders. The desired cure state can be easily determined in a manner that is practically incurable during the manufacturing process of the above-mentioned materials, which are subject to uncertainties associated with relatively long shelf life and the need for uncontrollable post-curing. It can be set without any need.

Soden 1'' A 10% casein solution was prepared by adding an aqueous solution of sodium hydroxide. Taldrazine 0 to 100 of the above solution
．． 1 g was added as filterdy63tuff. Before carrying out the casting, compound 22.19.20
．． 21.24.25.30.31.37.41.44.
Various samples of 3% by weight aqueous solutions of 45, 50, 52, 53, 55 and 56, respectively, were added at pH 7. The above mixture was cast onto a glass plate and after drying a cured filter foil was obtained which no longer dissolves in alkaline water.

It is clear that various modifications and variations can be made to the present invention as described above without departing from the spirit and scope of the invention. Accordingly, only such limitations are indicated in the claims.

Claims (1)

[Scope of Claims] 1. A hardened reagent matrix film, characterized in that it contains a binder made of a protein or a polymer and is hardened using a carboxyl group-activated crosslinking agent. 2.a) General formula (I): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(I) [In the formula, R_1 is preferably an alkyl group having 1 to 3 carbon atoms; such as methyl, ethyl or propyl. a lower alkyl group or an aryl group optionally substituted with a halogen atom such as Cl or Br; or,
Represents an aralkyl group which may be substituted in the same way as the above aryl group; R_2 represents the same as R_1, or a divalent alkylene group, arylene group, aralkylene group, alkyl-aryl-alkyl group, phenylene group, or represents a xylylene group; these groups are further bonded by a second bond to a carbamoylammonium group of the above general formula; or R_1 and R_2 together form an atomic group to complete a piperidine, piperazine or morpholine ring. and the ring may be substituted with an alkyl group having 1 to 3 carbon atoms or a halogen atom: R_3 is -NR_4-CO-R_5 (wherein R_4 is H or an alkyl group having 1 to 4 carbon atoms) represents a group; R_5 represents H, an alkyl group having 1 to 4 carbon atoms, or NR_6R_7; R_5 and R_
7 represents H or an alkyl group having 1 to 4 carbon atoms; or R_3 is -(CH_2)_m-NR_5R_9 (in the formula,
R_5 represents -CO-R_1_0; R_9 represents H or an alkyl group having 1 to 4 carbon atoms; R_1_
0 represents NR_1_1R_1_2; R_1_1 represents an alkyl group or aryl group having 1 to 4 carbon atoms; R_1_2 represents H, an alkyl group or an aryl group; -(CH_2)_n-CONR_1_3R_1
_4 (wherein R_1_3 represents H, an alkyl group having 1 to 4 carbon atoms or an aryl group; R_1_4 represents H or an alkyl group having 1 to 4 carbon atoms; or R_
1_3 and R_1_4 together represent 5- or 6-
or R_3 is a formula: ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (In the formula, R_1_5 is H or optionally represents an alkyl group having 1 to 4 carbon atoms substituted with a halogen atom; Y represents -O- or -NR_1_7;
R_1_8 represents H, an alkyl group, -CO-R_1_8 or -CO-NHR_1_9; R_1_7,
R_1_8 and R_1_9 are H or carbon atoms 1 to
4 represents an alkyl group; p represents 2 to 3); Z represents a carbon atom that completes a 5- or 6-membered aromatic heterocyclic ring; X^■ represents an acid anion] or b) General formula (II): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (II) [In the formula, R_1 and R_2 are the same as those described for the carbamoylammonium compound of formula (I). Represents the same thing; R_2_0 represents a methylene group, ethylene group, propylene group, or a single chemical bond; R_2_1 represents a hydrogen atom, methyl group, or ethyl group; X^■ represents an acid anion; Me^■ represents an alkali or c) General formula (III): ▲ Numerical formula, chemical formula, table, etc. ▼ [In the formula, R_1 and R_2 may be the same or different] , an alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 15 carbon atoms, or an aryl group having 7 to 15 carbon atoms, respectively.
15 aralkyl groups or bonded to each other,
forms a heterocyclic ring with the nitrogen atom; or R_3 is a hydrogen atom, a halogen atom, a carbamoyl group, a sulfo group, a halogen atom, a carbamoyl group, a ureido group,
Alkoxy group having 1 to 10 carbon atoms, 1 to 1 carbon atoms
0 alkyl group; or if R_3 represents an alkyl group or an alkoxy group as mentioned above, these groups are in each case further substituted by a halogen atom, a carbamoyl group, a sulfo group or a ureido group; X^■ in the above formula (III) represents an anion and acts as a counter ion of N-carbamoyl-pyridinium ion to form a salt; or R_3 contains a sulfo group as a component and an inner salt is formed. or d) the following formula (IV): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(IV) [In the formula, R_1, R_2, R_3 and X^■ are expressed by the formula (III
) A carbamoyl oxo ammonium compound represented by the following formula (V): ▲There are mathematical formulas, chemical formulas, tables, etc.▼(V) R_4 may be the same or different and each represents an alkyl group having 1 to 20 carbon atoms, an aralkyl group having 6 to 20 carbon atoms, or an aryl group having 5 to 20 carbon atoms; such a group may be further substituted. or 2 selected from R_1, R_2, R_3 and R_4.
two groups can also be bonded to each other to form a ring; X represents a group that can be eliminated from the compound of formula (V) by reaction with a nucleophilic reagent; Y^■ represents an anion] or f) the following formula (VI): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (VI) [In the formula, R_1 is an alkyl group having 1 to 10 carbon atoms, a cycloalkyl group having 5 to 8 carbon atoms group, 3 to 1 carbon atoms
0 alkoxyalkyl group or 7 to 15 carbon atoms
In addition to the group defined for R_1, R_2 represents the following formula: ▲Mathematical formula, chemical formula, table, etc.▼ (In the formula, R_3 represents an alkylene group having 2 to 4 carbon atoms; R_4 and R_5 may be the same or different and each represents an alkyl group having 1 to 6 carbon atoms or is bonded to each other to form a heterocyclic ring with the nitrogen atom; R_6 is preferably a non-carbon group; represents an alkyl group having 1 to 6 carbon atoms substituted with a substituent selected from the group consisting essentially of substituted and substituted carbamoyl groups, or sulfo groups; ion, ClO_4^-, BF_4^- or P
or g) The following formula (VII): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (VII): [In the formula, R_1 is the number of carbon atoms Represents an alkyl group having 1 to 10 carbon atoms, an aryl group having 6 to 15 carbon atoms, and an aralkyl group having 7 to 15 carbon atoms; each of these may be substituted; R_2 and R_3 may be the same or different. You can also
Hydrogen atom, halogen atom, acylamide group, nitro group,
represents a carbamoyl group, ureido group, alkoxy group, alkyl group, alkenyl group, aryl group or aralkyl group; or R_2 and R_3 are bonded to each other to form a condensed ring having a pyridinium ring structure; X is a nucleophile Y^■ represents a group that is eliminated from the compound represented by formula (VII) by reaction with;
or PF_6^-; or h) a halogenopyridinium compound represented by the following formula (
VIII): ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (VIII) [In the formula, R_1 and R_2 represent the same as defined for R_1 and R_2 in formula (I); R_3 has a carbon atom number of 1 to 10 alkyl group, an aryl group having 6 to 15 carbon atoms, or an aralkyl group having 7 to 15 carbon atoms; X^■ represents an anion] A cured reagent matrix coating according to claim 1, wherein the matrix is cured using a carboxyl-activated crosslinking agent. 3. The hardening reagent matrix coating according to claim 2, which contains gelatin, a gelatin derivative, casein or zein as a binder. 4. The amount of crosslinking agent is 0.2 to 4 based on the weight of the binder.
4. The cured reagent matrix coating of claim 3, wherein the cured reagent matrix coating is 0% by weight. 5. The cured reagent matrix coating of claim 4, comprising a reagent selected from the group consisting of enzymes, coenzymes, effectors, indicators, substrates, antigens, antibodies, stabilizers, wetting agents, and buffers. 6. Manufacturing a cured reagent matrix coating according to claim 1, by adding a crosslinking agent to a casting solution containing a binder; casting the resulting solution to form a coating; and then drying it. Method. 7. a) forming a binder film by casting a casting solution containing a binder; b) then bringing a crosslinking agent in solution into contact with the binder film to cure it; A method for producing a cured reagent matrix coating as described in Section 1. 8. The method of claim 7, wherein the binder coating is coated with a crosslinker solution having a concentration of 0.2 to 5%. 9. The method according to claim 8, wherein the crosslinking agent is an alcoholic solution, an aqueous solution, or an aqueous alcoholic solution.