JPH1031018A - Reagent for measuring reduced nicotinamide coenzyme, measurement method using the same, and test strip for measurement - Google Patents

Abstract

(57) [Summary] [PROBLEMS] To provide a reagent for measuring reduced nicotinamide coenzyme which is not affected by a coexisting substance having fluorescence or the like in an ultraviolet region and can be measured by dry method. SOLUTION: This is a reagent for measuring a reduced nicotinamide coenzyme containing a specific styryl dye. In the presence of an electron carrier, the styryl dye in the reagent for measuring reduced nicotinamide coenzyme reacts with the reduced nicotinamide coenzyme contained in the liquid sample to cause the styryl dye to fade and absorbance in the visible region By measuring the change in nicotinamide coenzyme, reduced nicotinamide coenzyme can be measured. Specific examples of the styryl dye include 2- [1-cyano-4- (4-dimethylaminophenyl) -1,3-butadienyl] -3-
There is ethylbenzothiazolium iodide, and diaphorase is an electron carrier.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a reagent for measuring reduced nicotinamide coenzyme, a measuring method using the same, and a test strip for measurement.

[0002]

2. Description of the Related Art Reduced nicotinamide coenzyme, that is, reduced nicotinamide adenine dinucleotide (N
ADH) and reduced nicotinamide adenine dinucleotide phosphate (NADPH) are coenzymes involved in redox reactions in vivo. Therefore, by measuring the reduced nicotinamide coenzyme, an enzyme or a substrate related thereto can be measured.

Conventionally, the following methods are known as methods for measuring NADH and NADPH. (1) A method of directly measuring the ultraviolet absorption or fluorescence of NADH and NADPH.

(2) NADH and NA in the presence of a catalyst
A method for measuring the fluorescence of resorufin generated by the reaction between DPH and resazurin. (3) NADH and NADPH in the presence of an electron carrier
A formazan dye by reacting the compound with a tetrazolium salt such as NTB or MTT, and measuring the absorption in the visible region.

[0005]

However, the conventional measuring method has the following problems. First, in the method (1) for measuring ultraviolet absorption and fluorescence, when urine or the like is to be measured, a coexisting substance having ultraviolet absorption or fluorescence, for example, vitamin B such as riboflavin, etc.
Disturbed by _two agents, a fluorescent contrast agent such as sodium fluorescein. This problem is the same in the method (2).

The method (3) in which the tetrazolium salt is used as a coloring agent has a disadvantage that blank coloring is caused by the influence of reducing substances such as ascorbic acid and bilirubin which coexist in the sample.

[0007] As another method for measuring components in a biological sample without using NADH, there is a method using an oxidase. However, similar to the method (3) using a tetrazolium salt, the method coexists in the sample. There is a drawback that coloring is inhibited by the influence of reducing substances such as ascorbic acid and bilirubin.

Further, in the method of measuring NADH and NADPH, realization of dry measurement is required from the viewpoint of measuring operation and the like. The present invention solves the above-mentioned conventional problems,
Provided is a reagent for measuring reduced nicotinamide coenzyme which is not affected by a coexisting substance having ultraviolet absorption or fluorescence and a reducing substance and can be dry-measured, a measurement method using the same, and a test piece for measurement. Aim.

[0009]

In order to achieve the above object, a reagent for measuring a reduced nicotinamide coenzyme of the present invention contains a styryl dye represented by the above formula [Formula 1].

When this special styryl dye is allowed to act on at least one of NADH and NADPH in the presence of an electron carrier, it is not affected by a coexisting substance having fluorescence or the like in the ultraviolet and ascorbic acid, bilirubin, etc. NADH and the like can be measured with high selectivity. Specifically, 10 ^-6
It is possible to measure trace amounts of NADH or less. Furthermore, this special styryl dye does not lose high reactivity even when subjected to dry measurement. As described above, the styryl dye according to the present invention has high reactivity and can be applied to dry measurement.This is presumed to be because an acidic functional group is added to the methine chain in the molecular structure of the styryl dye. Is done.

[0011] The measuring reagent of the present invention may contain an electron carrier. It is preferable to use diaphorase as this electron carrier. In the measurement method of the present invention, the reduced nicotinamide coenzyme is usually
It is a coenzyme of at least one of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate.

Next, the method for measuring a reduced nicotinamide coenzyme of the present invention comprises the step of including a styryl dye in a reagent for measuring a reduced nicotinamide coenzyme of the present invention and a liquid sample in the presence of an electron carrier. The styryl dye is discolored by reacting with the reduced nicotinamide coenzyme to be measured, and the change in absorbance in the visible region is measured.

In the measuring method of the present invention, examples of the liquid sample include blood, urine, bone marrow fluid, recall, pleural effusion, ascites, sweat, tears, and saliva. In the measurement method of the present invention, it is preferable to use diaphorase as the electron carrier. In this case, usually, diaphorase 0.12 to styrol dye 0.1 to 10.0 mol is used.
Used at a rate of 1.20 mkat.

In the measuring method of the present invention, the reduced nicotinamide coenzyme is usually at least one coenzyme of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate.

Next, the test piece for measuring reduced nicotinamide coenzyme of the present invention is obtained by adhering the reagent for measuring reduced nicotinamide coenzyme containing the electron carrier of the present invention to a carrier. is there. This test piece for measurement, for example,
It can be produced by a method of impregnating a carrier with the measurement reagent containing the electron carrier of the present invention, or by applying, printing or spraying the reagent on the carrier.

[0016]

BEST MODE FOR CARRYING OUT THE INVENTION In the styryl dye represented by the above formula [1], specific examples of the heterocyclic ring formed by Z include a thiazole ring (for example, thiazole,
4-methylthiazole, 4-phenylthiazole, 4,
5-diphenylthiazole), benzothiazole-based ring (for example, benzothiazole, 5-chlorobenzothiazole, 6-methoxybenzothiazole, 5-trifluoromethylbenzothiazole, 5-carboxybenzothiazole, 5,6-dihydroxybenzothiazole ), Naphthothiazole-based rings (eg, α-naphthothiazole, β-naphthothiazole, 5-hydroxy-β-naphthothiazole), oxazole-based rings (eg, 4-methyloxazole, 4,5-diphenyloxazole), Benzoxazole-based rings (eg, benzoxazole, 6-hydroxybenzoxazole, 5-chlorobenzoxazole, 5-phenylbenzoxazole), naphthoxazole-based rings (eg, α-naphthoxazole, β-naphtho) Oxazole), selenazole ring (eg, 4-methyl selenazole), benzo selenazole ring (eg, benzo selenazole,
5-hydroxybenzoselenazole, 5-chlorobenzoselenazole), thiazoline-based ring (eg, thiazoline), 2-pyridine-based ring (eg, 2-pyridine, 5
-Methoxy-2-pyridine), a 4-pyridine ring (eg, 4-pyridine, 3-chloro-4-pyridine),
-Quinoline-based rings (eg, 2-quinoline, 6-hydroxy-2-quinoline, 6-ethoxy-2-quinoline),
4-quinoline-based rings (eg, 4-quinoline, 6-methoxy-4-quinoline, 6-chloro-4-quinoline), 1
An isoquinoline ring (1-isoquinoline), 3,3-
Dialkyl indolenine ring (for example, 3,3-dimethyl indolenine, 5-chloro-3,3-dimethyl indolenine, 5-carboxy-3,3-dimethyl indolenine, 5-sulfoxy-3,3-dimethyl Indolenine, 3-cyclohexyl indolenine, 5-methylsulfonyl-3,3-dimethylindolenine), 3,3-dialkylbenzoindolenine ring (for example, 3,3-
Dimethyl-α-benzoindolenine, 3,3-dimethyl-β-benzoindolenine), benzimidazole-based ring (for example, benzimidazole, 1-alkyl-5,
6-dichlorobenzimidazole, 1-alkyl-5
5- and 6-membered heterocycles such as a ring containing a nucleus-containing ring of sulfonylbenzimidazole, 1-alkyl-5-trifluoromethylbenzimidazole).

Specific examples of R ₁ include alkyl groups such as methyl group, ethyl group, isopropyl group, butyl group, isoamyl group, octyl group, 2-ethylhexyl group and dodecyl group, and substituted alkyl groups thereof. Specific examples of the substituent of the substituted alkyl group include a halogen atom such as chlorine, bromine, fluorine and iodine, an aryl group (for example, a phenyl group or a naphthyl group), an alkoxy group, a hydroxyl group, a carboxylic acid group, a sulfonic acid group and the like. No.

Specific examples of the benzene ring substituted by R ′ and R ″ include 4-dimethylaminobenzene,
1,2,3,4-tetrahydroquinoline, julolidine and the like.

Specific examples of X include chloride, bromide,
There are halides such as fluorides and iodides,
Examples thereof include sulfonic acid ions such as methanesulfonic acid ion and p-toluenesulfonic acid ion, and sulfate ions such as methyl sulfate ion and ethyl sulfate ion, and also include ions such as perchlorate ion and nitrate ion. When the substituent of R ₁ contains an acid group such as a carboxylic acid group or a sulfonic acid group, and the styryl dye forms an inner salt, X is not necessary.

As a specific example of the styryl dye according to the present invention, 2- [1-cyano-4-
(4-dimethylaminophenyl) -1,3-butadienyl] -3-ethylbenzothiazolium iodide.

[0021]

Embedded image

Next, an example of the production of the styryl dye according to the present invention will be described. As styryl dyes, 2-
[4- (4-Dimethylamino) phenyl-1,3-butadienyl-1-cyano] -3-ethylbenzothiazolium iodide was synthesized.

That is, first, 2-cyanomethyl-3-
1.2 g of ethylbenzothiazolium iodide and 0.7 g of 4-dimethylaminocinnamaldehyde in 1
Add to 0 ml of acetic anhydride and mix. This mixture is
When heated in a water bath at a temperature of 2 to 3 minutes, acicular crystals precipitate. After heating for 20 minutes, the precipitated needle crystals were separated by suction filtration, washed with methanol, and then recrystallized from methanol to obtain 1 g of the styryl dye.
can get.

In the present invention, examples of the electron carrier include Meldora Blue, 1-methoxy-5-methylphenazinium methyl sulfate (1-mPMS), and diaphorase, with diaphorase being preferred.

The styryl dye according to the present invention is NADH
And preferably 0.1 to 10.0 moles, more preferably 0.5 to 2.0 moles per mole of NADPH.
It may be used in moles. Since electron carriers such as diaphorase act as catalysts, their amounts may be catalytic.
As mentioned earlier, typically 0.1-1.0 mkat / l
It is used in the ratio as shown below.

The measurement reagent of the present invention can arbitrarily select a measurement wavelength from 400 to 900 nm by selecting the styryl dye, for example, selecting n in the above formula (Formula 1).

Since other measurement parameters are mainly restricted by the electron carrier, for example, when the electron carrier is diaphorase, the usual pH range is 5 to 9, preferably pH 7 to 8, and the temperature is usually 20 to 20. ~ 40 ° C,
Preferably it is 30-37 degreeC. Since the styryl dye according to the present invention has high reactivity, the time required for the reaction is an extremely short time that is almost instantaneous.

The liquid sample targeted by the present invention is usually a body fluid such as blood or its components (plasma, serum, etc.), urine,
Cerebrospinal fluid or cell extract, but NADH and NA
Any liquid sample containing at least one of DPH is an object of the present invention.

When it is intended to measure a component such as an enzyme or an enzyme substrate contained in these samples, a substance produced by the enzyme and the substrate is reacted with a reductase and an oxidized nicotinamide coenzyme to produce a reduced enzyme. The amount of type nicotinamide coenzyme is determined by the method of the present invention. Thereby, the amount and concentration of the component in the target liquid sample can be measured. The following formula (Formula 3) shows an example of the reaction of the reagent for measurement of the present invention.

[0030]

Embedded image

In the reaction formula on the right side of the above formula (Chem. 3), N in the presence of diaphorase (Diaphorase)
The styryl dye (blue) according to the present invention is produced by ADH.
(dye) is discolored (discolor), and the NADH becomes NAD. In addition, the reaction formula on the left side of the above formula (Formula 3) shows that in the presence of β-hydroxysteroid dehydrogenase (β-HSD), NAD converts 3β-hydroxyvalic acid into a 3-oxosteroid, NADH. That is, this reaction is an enzyme reaction of β-HSD using NAD as a coenzyme. Therefore, in this case, the measurement of β-HSD and the like can be performed by the measurement of NADH according to the present invention.

Other objects of the present invention are, for example, α-hydroxysteroid dehydrogenase, alcohol dehydrogenase, glycerol dehydrogenase, pyruvate dehydrogenase, glucose dehydrogenase and the like.

The carrier used for the test piece for measuring reduced nicotinamide coenzyme of the present invention includes, for example, filter paper, cloth, nonwoven fabric, polyethylene terephthalate film, polyethylene film, glass, ceramic and the like. Is appropriately selected according to the type of test piece for use. For example, when a test piece for measurement is prepared by impregnating the carrier with the reagent for measurement of the present invention, a filter paper is suitable as an absorbable carrier, and a polyethylene terephthalate film is preferred as a general carrier. It is.

The measuring method using the test piece for measurement is as follows. That is, first, a liquid sample such as blood is attached to the test piece for measurement. Then, since the test piece fades, the degree of the fading is measured by a spectrophotometer or a densitometer or the like, whereby the N in the liquid sample is measured.
ADH and the like can be measured.

[0035]

Next, an embodiment will be described. As a styryl dye used in Examples, 2- [1-cyano-4- (4-dimethylaminophenyl) -1,3-butadienyl] -3-ethylbenzothiazolium represented by the above formula (Formula 2) is used. Iodide was used. The composition of the reaction solution and the measurement sample (liquid sample) are shown below. The compositions of the following reaction solutions and measurement samples all indicate final concentrations.

Example 1 Composition of Reaction Solution Dye 0.05 mmol / L Diaphorase 0.83 mkat / L Tris-HCl (pH 8.0) 0.20 mol / L Specimen for Measurement β-NADH 0 to 50 μmol / L Example 2) Composition of reaction solution Dye 0.05 mmol / L Diaphorase 0.83 mkat / L Tris-HCl (pH 8.0) 0.20 mol / L Sample for measurement β-NADH 0 to 50 μmol / L Ascorbic acid 1.0 g / L Next, in Examples 1 and 2, measurement was performed according to the following procedure to prepare a calibration curve. (1) Add Tris-HCl buffer to the reaction cell. This amount is 2.65 ml in Example 1 and 2.55 ml in Example 2. (2) 0.1 ml of the dye solution and 0.15 ml of diaphorase are added to the reaction cell, stirred, and incubated at 25 ° C. for 5 minutes. (3) Add 0.1 ml of each concentration of the sample to the reaction cell in Example 1 and 0.2 ml in Example 2, and measure the absorbance at 640 nm immediately after stirring to prepare a calibration curve.

The calibration curve of Example 1 is shown in the graph of FIG. 1, and the calibration curve of Example 2 is shown in the graph of FIG. As described above, in the examples of the present invention, measurement can be performed immediately after the reaction, so that quick measurement was possible. Example 2
From the results, it can be said that even when ascorbic acid as a reducing substance coexists, NADH can be measured without being affected by this.

Example 3 An impregnating solution having the following composition was prepared and impregnated with a filter paper (3MM, Chr, Whatman) having a size of 10 × 10 cm, and then dried (YAM).
It was dried at 50 ° C. for 15 minutes using DF62 (ATO). Then, this filter paper is cut into a width of 5 mm, and is adhered to a polyethylene terephthalate film using a double-sided tape.
A test piece for measuring m was prepared.

Impregnating Solution Composition Dye 0.05 mmol / L Diaphorase 0.83 mkat / L Tris-HCl (pH 7.2) 0.2 mol / L On the other hand, four types of NADH aqueous solutions (concentration: 0 μmol / L) were used as measurement samples. , 20 μmol / L, 50 μm
ol / L, 100 μmol / L).

Then, the above NADH aqueous solution was spotted on the test piece at a spotting amount of 8 μl, and a measuring time of 120 seconds (end point) and a measuring temperature of 25 ° C. were measured using a color difference meter (# 90, manufactured by Nippon Denshoku). Under the conditions, the reflectance at 640 nm was measured. The measurement results are shown in the graph of FIG.

As shown in the figure, a calibration curve corresponding to the concentration of NADH was obtained. In addition, this example is an example of dry measurement, and high reactivity was confirmed in the measurement.

[0042]

As described above, the reagent for measuring reduced nicotinamide coenzyme of the present invention contains the styryl dye represented by the above formula [Formula 1]. This particular styryl dye, in the presence of an electron carrier, NADH and NADPH
NAD with high selectivity
H etc. can be measured. Moreover, this measurement is not affected by coexisting substances having fluorescence or the like in the ultraviolet, ascorbic acid, bilirubin, and the like. Therefore, by using the measuring reagent of the present invention, NADH or the like in a sample can be easily measured with high accuracy without performing pretreatment or the like. In addition, since this special styryl dye does not lose high reactivity even when subjected to dry measurement, a test piece for measurement can be prepared by using this. By using this test piece for measurement, measurement of NADH and the like is further facilitated.

[Brief description of the drawings]

FIG. 1 is a graph showing a calibration curve of NADH in one example of the present invention.

FIG. 2 is a graph showing a calibration curve of NADH in another example of the present invention.

FIG. 3 shows a NAD according to still another embodiment of the present invention.
It is a graph which shows the calibration curve of H.

Claims (10)

[Claims] 1. A reagent for measuring reduced nicotinamide coenzyme containing a styryl dye represented by the following formula [Chemical Formula 1]. Embedded image 2. The reagent for measuring reduced nicotinamide coenzyme according to claim 1, which comprises an electron mediator. 3. The reagent for measuring reduced nicotinamide coenzyme according to claim 1, wherein the electron carrier is diaphorase. 4. The reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate coenzyme according to claim 1, wherein the reduced nicotinamide coenzyme is a coenzyme of at least one of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. Reagent for measuring reduced nicotinamide coenzyme. 5. A reaction between a styryl dye in the reagent for measuring reduced nicotinamide coenzyme according to claim 1 and a reduced nicotinamide coenzyme contained in a liquid sample in the presence of an electron carrier. A method for measuring reduced nicotinamide coenzyme, in which a styryl dye is faded and the change in absorbance in the visible region is measured. 6. The method for measuring reduced nicotinamide coenzyme according to claim 5, wherein the liquid sample is blood, urine, bone marrow fluid, recall, pleural effusion, ascites, sweat, tears or saliva. 7. The method for measuring reduced nicotinamide coenzyme according to claim 5, wherein diaphorase is used as the electron carrier. 8. The method for measuring reduced nicotinamide coenzyme according to claim 7, wherein the ratio of diaphorase is 0.12 to 1.20 mkat with respect to 0.1 to 10.0 mol of the styryl dye. 9. The reduced nicotinamide adenine dinucleotide and / or reduced nicotinamide adenine dinucleotide phosphate coenzyme according to any one of claims 5 to 8, wherein the reduced nicotinamide coenzyme is a coenzyme of at least one of reduced nicotinamide adenine dinucleotide and reduced nicotinamide adenine dinucleotide phosphate. Method for measuring reduced nicotinamide coenzyme. 10. A test piece for measuring reduced nicotinamide coenzyme, wherein the reagent for measuring reduced nicotinamide coenzyme according to claim 2 is attached to a carrier.