JP3095472B2 - Super oxide sensor - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a sensor for analyzing superoxide in a solution.

[0002]

2. Description of the Related Art One of the defense mechanisms in vivo is phagocytosis by phagocytes (such as polymorphonuclear leukocytes). In this case, it is considered that superoxide is produced, which is further converted into a substance having a strong bactericidal action by the second reaction, and sterilizes. It is also said that superoxide has a carcinogenic effect and is produced in large amounts in cancer tissues. More recently, attention has been focused on the relationship with aging. For these studies, it is essential to measure in vivo because superoxide is an unstable substance.

However, since there is no suitable means for measuring superoxide, these studies have been difficult and have not made much progress. Conventionally, cytochrome c reduction method has been used for measuring superoxide. Superoxide reduces the oxidized form of cytochrome c and becomes itself an oxygen molecule. Reduced cytochrome c causes a change in the absorption spectrum, usually at 550 nm.
It is measured by absorbance. That is, the amount of superoxide is determined by a change in absorbance of a known amount of cytochrome c at 550 nm. This method is complicated in operation, requires skill, and increases the size of the apparatus. Above all, there is a problem that the measurement cannot be performed unless it is in vitro, that is, ex vivo, and it has been difficult to apply it to the above-mentioned research.

[0004]

Therefore, it has been desired to develop a means capable of easily and stably measuring superoxide with high sensitivity.

[0005]

Means for Solving the Problems The present inventor has made intensive studies to develop means for solving such problems.
As a result, they found that a pH-sensitive element in which a matrix in which a compound of a specific structure and a metal complex composed of iron (divalent) were dispersed was fixed became a highly sensitive and stable sensor for superoxide, and completed the present invention. I came to. Incidentally, a sensor capable of directly measuring superoxide has not been known so far.

That is, the present invention provides a compound represented by the general formula [1]:

[0007]

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(However, R ₁ is

[0009]

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A superoxide sensor in which an iron (divalent) complex coordinated with a tetrakis (2-pyridylmethyl) -diamino compound represented by R ₂ is hydrogen or a methyl group is immobilized on a pH-sensitive element. is there.

The diamino compound represented by the general formula [1] is a ligand which coordinates with an iron compound to form an iron (divalent) complex.

In the general formula [1], R ₁ is

[0013]

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Is a group represented by -CH ₂ -CH ₂ -C
A group having 3 or more carbon atoms in the main chain such as an H ₂ -group hardly exhibits sensitivity when used as a sensor. R ₂ is hydrogen or a methyl group.

These tetrakis (2-pyridylmethyl)
Specific examples of the diamino compound include the following.

[0016]

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The method for producing these diamino compounds is not particularly limited, and known methods are employed. For example, The
Jouranal of Biological Chemistry, vo.264,9243-9249
Page (1989), Helvetica Chimica Acta vol.50,23
Examples include the method described on pages 30-2332 (1967). In addition, the present inventors have further found that the use of a phase transfer catalyst in the above synthesis method enables the synthesis to be performed with a high yield. As one iron compound forming an iron (divalent) complex, a water-soluble divalent iron compound is used without any particular limitation. Specific examples of the iron compound include iron sulfate [FeSO ₄ ], ferrous chloride [FeC
l ₂ ], iron lactate [Fe (CH ₃ CHOHCOO) ₂ ], iron oxalate [FeC ₂ O ₄ ], and the like.

The diamino compound is an iron compound of iron (2
Complex with the (valent) ion to form a 1: 1 complex in a molar ratio. However, when R ₁ is a cyclohexenyl group, a 2: 3 complex is formed. The method for forming the complex is not particularly limited, and a known method is used. For example, the general formula [1]
A method of adding an aqueous solution of iron sulfate (FeSO ₄ ) to a methanol solution of a diamino compound represented by the formula ( ₁ ) and stirring at room temperature.

The diamino compound represented by the general formula [1]
The (valent) complex catalyzes the reaction shown below.

[0020]

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During this reaction, the pH changes because hydrogen ions are consumed. That is, since the hydrogen ion consumption is proportional to the amount of the superoxide present, the magnitude of the pH change depends on the amount of the superoxide. Therefore, the diamino compound represented by the general formula [1]
By combining a (valent) complex and a pH-sensitive element, superoxide can be quantified.

The method for immobilizing the above complex on the pH-sensitive element is not particularly limited, and various known methods can be used. For example, a resin thin film can be formed on a pH-sensitive element by a casting method, and the complex can be adsorbed on the thin film. In this case, the resin to be used is preferably an ion exchange resin, and it is desirable to use a cation exchange resin having a carboxyl group, a sulfonic acid group or the like as an ion exchange group.

Further, the above-mentioned resin and the general formula [1]
It is also useful to dissolve an iron (divalent) complex of a diamino compound represented by the following formula in an organic solvent, apply the solution on a pH-sensitive element, and dry the solution to obtain a thin film.

The pH-sensitive element is not particularly limited, and various elements can be used. One example is a pH sensor constituted by a glass electrode. In particular, ISFET (Ion Sequential) is used due to its sensitivity, response speed, and ease of miniaturization when configured as a superoxide sensor.
lective Field Effect Transistor).

[0025]

The superoxide sensor thus constructed has high sensitivity and can perform stable measurement. The sensor of the present invention can be easily miniaturized by using a minute pH-sensitive element, for example, an ISFET having a diameter of about 1 mm. By attaching a lead wire coated with a biocompatible material to this, it is possible to insert it into a living body, take out the output of the sensor outside the living body, and use it. That is, the use of the sensor of the present invention enables measurement in a living body, which has been conventionally difficult. Therefore, the present invention is a very useful measuring means in the fields of medicine and biochemistry, and has great industrial value.

The present invention is described below in more detail with reference to Examples, but the present invention is not limited to these Examples.

[0027]

EXAMPLES Production Example 1 0.5 ml of 1.97 g (12 mmol) of 2-picolyl chloride hydrochloride
And 3 ml of a 5 M aqueous sodium hydroxide solution was added thereto with stirring under a nitrogen atmosphere. 0.
18 g (3 mmol) of 1,2-diaminoethane and 20 mg (0.07 mmo
1) Cetyltrimethylammonium chloride was added, and stirring was continued at room temperature for 24 hours. The product was extracted from the solution with dichloromethane, and the dichloromethane solution was washed with water and dried over magnesium sulfate. After filtration of the insoluble matter, a solid was obtained by distillation under reduced pressure. This was purified by a chromatographic method using alumina as a carrier, and 1.1 g of N, N, N ', N'-Te
trakis (2-pyridylmetyl) -1,2-diaminoethane was obtained (yield 68%).

By using various compounds as raw materials in the same operation, four kinds of compounds were obtained. Table 1 together
Shown in

[0029]

[Table 1]

To 30 ml of a methanol solution containing 5 mmol of these compounds was added 0.5 ml of an aqueous solution containing 5 mmol of iron sulfate, and the mixture was stirred at room temperature for 10 minutes. The solvent was distilled off, and 50
It was dissolved in 10 ml of mM potassium phosphate buffer. Thus, aqueous solutions of five types of iron (divalent) complexes shown in Table 2 were obtained (Solutions Nos. 1 to 5). For solution No. 3 only, an aqueous solution containing 7.5 mmol of iron sulfate was used. Example 1 Measurement of SOD (Super Oxidase) Activity The SOD activity {catalytic activity for the reaction of the formula (1)} of each complex synthesized in Production Example was measured. The measurement used the cytochrome c method. The measurement method will be described below.

Xanthine is used as xanthinoxidase (X
Superoxide (O ₂ ⁻ .) Is generated from dissolved oxygen in the measurement solution in the presence of (OD). This superoxide reduces oxidized cytochrome c and becomes itself an oxygen molecule.
Since the cytochrome c changes to a reduced form, the absorbance at 550 nm decreases.

In the presence of compounds having SOD activity, superoxide is quickly trapped and disproportionates to produce oxygen and hydrogen peroxide. Oxidized cytochrome c
Is inhibited.

[0033]

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The unit time of the absorbance at 550 nm of the SOD active substance-added system relative to the SOD active substance-free system (1
(Min) The concentration (mol / l) of the SOD active substance at the time when the rate of change becomes 50% is defined as the SOD activity (IC ₅₀ ), and the measurement results are shown in Table 2. As a comparative example, Bacillus stearot
Hermophilus-derived SOD actives are shown.

[0035]

[Table 2] Table 2 -------------------------------------------- --------------- Solution No. SOD active substance IC ₅₀ (mol / l) ---------------------- ------------------------------------- Comparative Example 3.8 1 Compound No. 1-Iron ( (Divalent) complex 1.1 2 Compound No. 2-iron (divalent) complex 1.1 3 Compound No. 3-iron (divalent) complex 0.56 4 Compound No. 3-iron (divalent) complex 0 .80 5 Compound No.4-iron (divalent) complex 3.8 --------------------------------- ------------------------- It was found that the iron complexes of Solution Nos. 1 to 4 had high SOD activity. The iron complex of solution No. 5 is also S originated from microorganisms.
The activity was comparable to that of the OD active substance. In addition, solution N
The temperature dependence of the SOD activities of o.1 and 4 was measured. The results are shown in FIG. SOD activity was found to be almost independent of temperature.

Further, the time-dependent changes of the SOD activities of the solutions Nos. 1 and 4 were measured. The aqueous solution of the complex was stored at room temperature, and the reduction inhibition rate of cytochrome c in the cytochrome c method was measured. The results are shown in FIG. Solution No. 4 showed high SOD activity for over one month.

Example 2 A thin film of Nafion (a product of DuPont), a kind of cation exchange resin, was formed on an ISFET electrode (available from Shindengen Kogyo Co., Ltd.) by a casting method. This electrode was immersed in the above solution Nos. 1 to 5 to obtain a superoxide sensor in which various complexes were immobilized. (Sensor No.
1 to 5) Various superoxide-generating solutions were prepared with various concentrations of xanthine-xanthine oxidase to prepare sample solutions. Sensor No. 1 superoxide sensor and ISFE with only Nafion membrane immobilized as reference electrode
The differential output potential when the T electrode was immersed in the sample solution was measured. FIG. 3 shows the relationship between the time variation (mV / min) of the differential output potential and the superoxide generation rate (M / min).
(The superoxide generation rate was measured by the cytochrome c reduction method).
At l / min or more, a remarkable increase in potential was observed. This sensor shows that it is possible to quantify trace amounts of superoxide. When the same measurement was performed for Nos. 2 to 5, almost the same results were obtained.

[Brief description of the drawings]

FIG. 1 is a graph showing the temperature dependence of the SOD activity of two kinds of SOD active substances measured in Example 1.

FIG. 2 is a graph showing the change over time of the SOD activity of two kinds of SOD active substances measured in Example 1.

FIG. 3 is a diagram showing a relationship between a time change amount of a differential output potential between a superoxide sensor and a reference electrode measured in Example 2 and a superoxide generation speed.

Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 33/48-33/52 G01N 33/58-33/98 CA (STN) REGISTRY (STN)

Claims (1)

(57) [Claims] 1. A compound of the general formula [1] (However, R ₁ is , R ₂ is hydrogen or a methyl group). A superoxide sensor in which an iron (divalent) complex coordinated with a tetrakis (2-pyridylmethyl) -diamino compound represented by the formula (I) is immobilized on a pH-sensitive element.