JPS60209175A - Composition for measuring hydrogen peroxide - Google Patents

Abstract

PURPOSE:To enable quantitative analysis over a wide concn. range with high sensitivity by using 3,3',5,5'-tetraalkyl deriv. or the water soluble salt thereof as an original color substance. CONSTITUTION:The sulfoalkyl deriv. of the 3,3',5,5'-tetraalkyl benzidine expressed by the formula (in which R1, R2, R3 and R4 are the same or different and denote respectively straight alkyl groups of 1-6 carbon atoms and n is 1-6 integer, the sulfoalkyl group may be subtd. at least by one hydroxyl group) or the water soluble salt thereof is used as an original color substance in a compsn. for measuring hydrogen peroxide contg. enzyme, buffer material and original color substance. The comps. for measuring hydrogen peroxide which has excellent solubility with water and usable in a wide pH range is thus obtd.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a reagent composition for determining hydrogen peroxide in liquids, particularly body fluids.

(Prior Art) Today, hydrogen peroxide measurement is carried out in a very wide range of fields, including clinical tests in the medical field, the chemical industry, the food industry, and public health. Particularly in clinical testing, methods for analyzing body fluid components using enzymes are rapidly becoming popular, and among these methods, the measurement of hydrogen peroxide occupies an important position. This is because there are a large number of components in body fluids that can be analyzed using relatively simple methods such as allowing oxidation enzymes to act on the components to generate hydrogen peroxide, and then measuring this hydrogen peroxide. . Typical component/oxidase combinations include glucose/glucose oxidase, cholesterol/cholesterol oxidase, uric acid/uricase, amino acid/amino acid oxidase, pyruvate/pyruvate oxidase, and the like.

Now, the hydrogen peroxide produced can be measured in various ways, but
The most commonly used method is to form a pigment by oxidizing a chromogen in the presence of peroxidase, and perform colorimetric determination. Such a chromogen generally has an amino group or a hydroxyl group in its molecule, and a chromogen that is stable and has a large dye-forming ability is selected. The most frequently used chromogen is a combination of 4-aminoantipyrine and phenol known as the Trinder reagent, but in recent years many variations thereof have been reported.

On the other hand, in the field of so-called dry chemistry, in which reagent compositions for measuring components in body fluids are fixed on carriers,
Benzidine derivatives, especially 0-tolidine, have been used with preference as chromogens. This is because when this substance is used as a chromogen, extremely sensitive sensitivity is obtained, and it produces a pigment that has a very thick color on the long wavelength side of the visible light range, so it is difficult to distinguish between the various coloring components that coexist. This is because it has several advantages such as being less susceptible to influence. However, o-
) Lysine is considered carcinogenic and special care must be taken when manufacturing test devices using this substance.

In recent years, chromogens that take advantage of the properties of 0-tolidine and are considered non-carcinogenic have been developed. This is a compound in which the 3.3' and 5.5' positions of pentidine are each substituted with a methyl group.
Tetrahedron (Tetrahedr) et al.
on), Vol. 30, pp. 5299-55Q2 (1974
2007)]. Furthermore, West German Patent No. 2.460,903 states that pentidine substituted with an alkyl group other than methyl at the 3.6' and 5.5' positions can also be used as a chromogen. has been done.

However, all of these benzidine derivatives have extremely poor solubility in water, and therefore can only be used in a limited concentration range in an acidic pH region. This can be considered a serious drawback, considering that most analyzes of components in body fluids using enzymes are carried out near neutrality. The reason why these benzidine derivatives are mainly used in test pieces is in consideration of the above-mentioned drawbacks. However, even in the test pieces using all of these benzidine derivatives, the disadvantages due to the above-mentioned drawbacks cannot be overcome, and they can only be used when the amount of hydrogen peroxide produced is very small.

For this reason, attempts have been made to expand the measurable concentration range of hydrogen peroxide, and a method of using hydrogen peroxide in combination with other chromogens has been reported (Japanese Patent Publication No. 57-5520).

Another disadvantage of the low solubility of s, s', 5,5'-tetraalkylbenzidine in water is also brought about during the production of test pieces. For example, when manufacturing test strips for measuring glucose in body fluids, generally
An impregnating aqueous solution consisting of an enzyme, a buffer substance, and a chromogen is prepared and prepared, but when 5+ 3C5,5'-tetraalkylbenzidine is selected as the chromogen, it is very difficult to obtain a single impregnating aqueous solution. That's funny. For this reason, a method was developed in which a mordant was added to an aqueous solution impregnated with a chromogen (US Pat. ,361,64
Countermeasures such as No. 8) are being considered.

The present inventor conducted extensive research and completed the present invention while taking the above-mentioned drawbacks into consideration.

(Objective of the Invention) The present invention is intended to solve the problems in the above-mentioned prior art, and its objects are as follows: 5. 3', 5
．． The object of the present invention is to provide a chromogen that takes advantage of the characteristics of 5'-tetraalkylbenzidine, has excellent solubility in water, can be used in a wide pH range, and can be used in both test solutions and test pieces. .

(Structure of the Invention) That is, the composition for measuring hydrogen peroxide of the present invention is a composition for measuring hydrogen peroxide comprising an enzyme, a buffer substance, and a chromogen, and as a chromogen, the composition has the general formula I: (in the formula R1, R2, R3 and R4 are the same or different,
each represents a straight-chain alkyl group having 1 to 6 carbon atoms, n is an integer of 1 to 6, and the sulfoalkyl group is at least one of the hydroxyl groups;
may be replaced by ) represented by 3,3
', 5. It is characterized by containing a sulfoalkyl derivative of 5'-tetraalkylpentidine or a water-soluble salt thereof.

The present inventors found that using the compound represented by the above general formula (1) or a water-soluble salt thereof as a chromogen provides good sensitivity;
Furthermore, we have discovered that even high concentrations of hydrogen peroxide can be quantitatively measured.

Compounds preferably used in the present invention are listed below, but are of course not limited to: N, N'-bis(2-sulfoethyl)-S, S'.

5.5'-tetramethylpentidine, N, N'-bis(3-sulfopropyl)-3,3'.

5.5'-tetramethylbenzidine, N, N'-bis(4-sulfobutyl)-s, s'.

5.5'-tetramethylbenzidine, N,N'-bis(3-sulfopropyl)-5,5'.

5.5'-tetraethylpentidine, N,N'-bis(2-hydroxy-2-sulfoethyl)-5,3',5. 5'-Tetramethylbenzidine, N,N'-bis(2-hydroxy-3-sulfopropyl)-3,5',5,5'-tetramethylbenzidine.

When the above-mentioned compounds are used as chromogens, they may be used alone or in combination with several types, or may be used in combination with other chromogens.

Most of these compounds have not been described in the literature, but
It can be easily manufactured by a known method.

Example, tba, 3. 3', 5. Total reaction of sultone corresponding to s'-tetraalkylpentidine [Koen et al., Industrial Chemistry Magazine, Vol. 59, No. 9, pp. 1028-1030 (19
1956] method and 3. Reacting s', s, s'-tetraalkylpentidine with an alkylsulfonic acid or hydroxyalkylsulfonic acid having noherogen at the end of the molecule (N, E, Good et al.
Analytical Biochemistry
cal Biochemistry), vol. 104,
600-610 (1980)] method.

3, 5', 5. used in the composition of the present invention. 5'-Tetraalkylpentidine derivatives can be used in place of all chromogens conventionally used for hydrogen peroxide determination. In particular, it is advantageous to use it for enzymatic determination of body fluid components based on a method in which hydrogen peroxide is generated during the analysis process. This is because the compounds of the present invention exhibit excellent solubility, especially in water, so they can be mixed with buffer substances, enzymes, etc. to form a single stable test solution, and at the same time, they can be used at high concentrations. This is because it has a number of advantages, such as being able to quantify hydrogen peroxide with high sensitivity and being less affected by coexisting coloring components.

Furthermore, the chromogen compound used in the composition of the present invention is included in an absorbent carrier together with an enzyme and a buffer substance, and when used,
When the carrier is conveniently attached to a support such as plastic to form a test piece, a particularly simple test tool with excellent storage stability can be obtained. The most frequently used absorbent carrier is F paper, but nonwoven fabrics, cotton, wood chips, etc. can also be used. Furthermore, it is also possible to apply the test reagent composition to a support such as the above-mentioned plastic sheet along with a suitable adhesive such as gelatin, synthetic resin, etc., if desired, and to fix it by drying. It is clear that something can be done.

The invention will be explained in further detail in the following examples. Note that the present invention is not limited to the following examples.

Production of chromogen compound: Production example 1 N, N'-bis(3-sulfopropyl)-s, 375
．． Synthesis of 5'-tetramethylbenzidine sodium salt 24. OjE y, , sr, s, s/-Tetramethylbenzidine is dissolved in 1-propanol 100%. 1°5-propane sultone 24.45! (i7 Dissolved in methanol 100- and added to the above solution, heated under reflux for about 6 hours. Cooled to room temperature, neutralized by adding 1N NaOH aqueous solution, and then evaporated to dryness. Methanol/
Recrystallization from acetone gave white crystals 281. (Yield 5
3 H) Thin layer chromatography [silica gel plate (manufactured by Merck & Co., Ltd.), developing solvent; 2-propanol: acetic acid; water = 50:15:20) UV
Absorption, ninhydrin reaction t-FlR4=0.25 Infrared absorption spectrum νN)l 5400f".

l10=01620 cm-”, νON 1260 (
z-1゜νBO31200,105105O" Production Example 2 Synthesis of N, N'-bis(2-hydroxy-2-sulfoethyl)-5,5',5.5'-tetramethylbenzidine sodium salt 12.02ji-3 .3',5,5'-tetramethylbenzidine' dissolved in 1500 m/2-propatol.

Dissolve 52f of sodium 2-chloro-1-hydroxyethanesulfonate q3oa, g in water and add to the above solution. Then add 4 parts of NaOH and heat to reflux for about 12 hours. After removing the solvent, 500 d of water was added to the residue to dissolve it, and unreacted 3.3', 5,5
'-Tetramethylbenzidine is extracted. The aqueous layer was again evaporated to dryness and then recrystallized from methanol/acetone to obtain 14.8y of white crystals. (Yield 26%) Thin layer chromatography [Silica gel plate (manufactured by Merck & Co., Ltd.), developing solvent, 2-propatol:acetic acid:water = 50
:15:20] UV absorption, ninhydrin reaction ((1
) R4=Q, 27 Infrared absorption spectrum νoH3500crn-1°1/N
H3400crn-”, ν(3=(31620nn
'.

νON 1260cm-”, LIB031200.1
050m'' Preparation of composition for measuring hydrogen peroxide: Example 1 N,N'-bis(3-sulfopropyl)-3,3'.

5.5'-tetramethylbenzidine sodium salt 200
1pi, peroxidase (1001U/”'5+) 10
11 tris(hydroxymethyl)aminomethane 1.2
1. Dissolve citric acid (monohydrate) 2.10jE and sodium hydroxide α85F in purified water to make a total volume of 100M,
Set the reaction solution (pH 6.5).

Example 2 Glucose oxidase (120 IU/P) α96, peroxidase, (1001 U/”p) 115y
1 Polyvinylpyrrolidone (MW 40000) 5.0
9XN, N'-bis(3-sulfopropyl)-3,s;
5.5'-Tetramethylbenzidine sodium salt αsy
, citric acid (monohydrate) 1.98F, trisodium citrate (dihydrate) IQ, 44P, EDTA・2Na[L
ly was dissolved in purified water to make a total amount of 100 d.

Preparation of calibration curve for measuring hydrogen peroxide: Add the reaction solution an- containing the composition of the present invention prepared in Example 1 to 10 μt of hydrogen peroxide aqueous solution Fukaya prepared at 0, 10, 20, 50, 40 mM/l. In addition, in a 67℃ constant temperature bath
Soak for a minute to develop color. After cooling to 25℃, wavelength 66
Absorbance at 5 nm (or optical density: optical
Measure the density, (lD). By subtracting the absorbance of the blank, ΔO, D is determined, the horizontal axis represents the hydrogen peroxide concentration, and the vertical axis represents Δ0. D was taken and a calibration curve was prepared. As shown in Figure 1, the calibration curve shows hydrogen peroxide concentration of 40
A straight line passed through the origin up to mM/l. Therefore, it can be seen that the hydrogen peroxide concentration can be accurately determined by colorimetric analysis.

Manufacture of a test piece for measuring urine glucose: Okishi (grave 2516, manufactured by Schleicher & Schull) was soaked in the solution prepared in Example 2 and dried at 60° C. for 30 minutes.

The obtained test paper was cut into 5 rran x 5 am and adhered to a 5 mm x 60111 m polystyrene sheet using adhesive tape to produce a test piece for measuring urinary glucose.

Figure 2 shows the test piece. In the figure, 1 is a test paper and 2 is a polystyrene sheet.

Manufacture of comparative test piece: N,N'-bis(3
-sulfopropyl) -3,3'; 5. The same F paper as above was immersed in a solution excluding 5'-tetramethylbenzidine sodium salt and dried at 60°C for 30 minutes.
5% 3. The sample was soaked again in a 5,5'-tetramethylbenzidine-acetone solution and dried under vacuum. The obtained test paper was made into a test piece by the same operation as above.

Measurement of glucose concentration: Test pieces produced using the composition of the present invention and comparative test pieces were each instantaneously immersed in glucose standard solutions with different concentrations, and after removing excess liquid, they were allowed to react for exactly 20 seconds. Color difference meter (color difference computer E3 pieces 1
11 ND-504DE)'e was used to measure the reflectance at a wavelength of 660 nm. If the horizontal axis is the glucose concentration and the vertical axis is the reflectance, a calibration curve as shown in FIG. 6 is obtained.

As can be seen from the figure, since the test piece using tetramethylbenzidine as a chromogen shows the reflectance as determined by the present invention, the measurable glucose concentration range of this test piece is 0 to 250. p/di.

On the other hand, the test piece based on the present invention has higher sensitivity than the comparative test piece, and a clear change in reflectance is observed up to a glucose concentration of 2ooy/at. Therefore, the measurable glucose concentration range of this test strip is 0 to 2,000"9
/di, the test piece produced using the composition for measuring hydrogen peroxide of the present invention has a wider glucose concentration range than the comparative test piece produced using the conventional composition for measuring hydrogen peroxide. It turns out that it is applicable to. Also, Guru.

Of course, by using suitable enzymes and buffer substances, the composition of the present invention and test strips prepared using the composition can be used for quantifying various components contained in body fluids. be.

(Effects of the Invention) As described above, the composition for measuring hydrogen peroxide of the present invention uses a s, s7 s, s'-tetraalkylbenzidine sulfoalkyl derivative or a water-soluble salt thereof as a chromogen. Therefore, the conventional 3, 3'; 5. Compared to a composition for measuring hydrogen peroxide that uses 5'-tetraalkylbenzidine as a chromogen, the solubility in water is significantly improved and it can be used in a wider pH range.

In addition, since it has excellent solubility in water, it has become possible to quantitatively analyze components in an aqueous solution with higher sensitivity and over a wider concentration range than when using conventional compositions.

Furthermore, when producing test strips for measuring various components in body fluids, the composition for measuring hydrogen peroxide can be loaded onto the carrier from an aqueous solution in one step, which simplifies the test strip production process and makes it easier to use conventional compositions. Compared to the test piece used, it has high sensitivity and c
It is possible to obtain test pieces that can be used over a wide concentration range.

In this way, the composition for measuring hydrogen peroxide of the present invention can be used in the form of an aqueous solution or a test piece, and can be modified in various ways, so it can be used to accurately, easily and quickly quantify various components in body fluids. It has excellent effects on analysis.

[Brief explanation of the drawing]

FIG. 1 is a graph showing a calibration curve prepared using the composition for measuring hydrogen peroxide of the present invention, and FIG. 2 is a graph showing a test piece for measuring urine glucose produced using the composition of the present invention. A perspective view of an example. Figure 3 shows the relationship between the glucose concentration and the reflectance at a constant wavelength of the test paper surface read from a colorimeter, measured using a test piece using the composition of the present invention and a comparative test piece. It is a graph showing a relationship. In the figure, 1...Test paper 2...Polystyrene sheet Patent applicant Sakae, Kengaku Co., Ltd. (and 1 other person) Figure 1 Hydrogen peroxide water source M<mFJA) Figure 2

Claims (1)

[Claims] A composition for measuring hydrogen peroxide comprising an enzyme, a buffer substance and a chromogen, the chromogen having the general formula l: (wherein R1, R2, R3 and R4 are the same or different). C,
each represents a straight-chain alkyl group having 1 to 6 carbon atoms, n is an integer of 1 to 6, and the sulfoalkyl group represents at least one of the hydroxyl groups.
may be replaced by ) represented by 3, 5
', 5. 1. A composition for measuring hydrogen peroxide, comprising a sulfoalkyl derivative of 5'-tetraalkylpentidine or a water-soluble salt of 5'-tetraalkylpentidine.