JP2002214231A - Method for measuring concentration of dioxins in sample and method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring the concentration of dioxins which is for highly sensitive analysis by a new measuring system utilizing electroluminescence, does not require reagents such as hydrogen peroxide necessary for chemical luminescence, and is capable of achieving device compactness and easily achieving high sensitivity. SOLUTION: Hapten simulating part of the structure of the dioxins is chemically bonded to a ruthenium complex to form an antigen, and the antigen is incubated with an antibody fixed to an electrode to bring about an antigen- antibody reaction. By impressing a voltage-on the reaction product from the electrode, the ruthenium complex is oxidized and reduced to generate electroluminescence. By observing the electroluminescence, the antigen is determined to measure the concentration of the dioxins.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
Method and apparatus for measuring the concentration of syns
Especially in the incineration of municipal and industrial waste.
Toxic 2,3,7,8-dioxin tetrachloride (T₄C
DD) containing polychlorinated dibenzo-p-dioxin (P
CDD_S) And similarly toxic polychlorinated dibenzofurans
(PCDF _S) For high sensitivity and high selective measurement
A method and a device for measuring the concentration of ioxins
Things.

[0002]

2. Description of the Related Art In recent years, when wastes are incinerated, a high concentration of dioxin is generated, which has become a social problem. The concentration of dioxins contained in the exhaust gas during incineration, because dioxin is extremely toxic dioxin and its homologues and isomers, as well as poly in a total amount of homologs and isomers of chloride dibenzofuran 0.1 ng / Nm ³ It has become mandatory to: However, since dioxin is so toxic, low concentrations that cannot be detected by ordinary analytical methods pose a problem, and because it contains many dusts, mist, and various other contaminants, dioxins can be directly and accurately detected. Complicated pretreatments and special analyzers are required for detection and quantification.

[0003] As a typical method for quantifying and analyzing dioxins, a Soxhlet extractor is used to extract a solvent for 72 hours with a solvent such as toluene or ethylene glycol. There is a method to analyze with an analyzer. In this method, since the steps required for the analysis such as the concentration treatment are complicated, it takes two to three weeks until the analysis result is obtained, and the analysis cost is high.

Japanese Patent Application Laid-Open No. H11-326222 provides an indirect method for quantifying dioxins using a relatively inexpensive apparatus, which can eliminate the need for a sample concentration operation. As a configuration, a sample in which a metal complex of 8-oxyquinoline and a dioxin are to be measured is mixed with an organic solvent containing a halogen, and the mixture is irradiated with excitation light to measure the fluorescence intensity. There is disclosed a method for measuring dioxins in which halophenols having a concentration correlation with dioxins contained therein are quantified, and the concentration of dioxins is estimated from the concentration of the halophenols.

However, in this method, it is necessary to examine in advance the influence of a component that competes with the halophenol on the assumption that the liquid to be measured interacts with the fluorescent complex.
It is necessary to check the correlation between the halophenol concentration and the dioxin concentration in advance, and it is considered that there is a great possibility that the concentration fluctuates due to the type of dioxin. In this respect, the method is inferior in accuracy because the dioxin concentration is indirectly estimated. In addition, the fluorescence method has high sensitivity, but is easily affected by coexisting substances, and the excitation light may inevitably enter the observation system, so that the sensitivity may not be obtained as expected.

Japanese Patent Application Laid-Open No. 11-79719 discloses a reversible adsorption / desorption method of gas instead of an adsorbent such as activated carbon or silica gel. According to this, a pair of electrodes are brought into contact with a solution in which a ruthenium complex capable of absorbing and releasing molecular nitrogen is dissolved, and nitrogen gas is absorbed and desorbed by controlling the electrode potential. It is possible to control gas adsorption and desorption without using heat, to perform gas adsorption and desorption control only by electric potential operation, and to perform gas adsorption and desorption reversibly and repeatedly. However, in the present invention, a solution in which a ruthenium complex capable of absorbing and releasing molecular nitrogen is dissolved is used, but dioxin having a planar structure and homologues and isomers thereof,
In addition, it was unclear whether homologues and isomers of polychlorinated dibenzofuran could be adsorbed and desorbed.

[0007]

SUMMARY OF THE INVENTION In view of the above problems, the present invention has found that the use of a ruthenium complex has high selectivity for the adsorption of dioxins, and that the novel measurement system utilizing electroluminescence has high sensitivity. Provided is a method for measuring the concentration of dioxins, which does analysis, does not require a reagent such as hydrogen peroxide required for chemiluminescence, can be miniaturized, and can easily achieve high sensitivity, and an apparatus therefor. This is a technical issue.

[0008]

Means for Solving the Problems The inventor of the present invention has made intensive studies to solve the above-mentioned problems, and as a result, completed the present invention.

That is, according to the present invention, there is provided a method for measuring the concentration of dioxins in a sample, the method comprising forming a hapten, which mimics a part of the structure of dioxins, with a ruthenium complex to form an antigen. A step of incubating a sample considered to contain the antigen and dioxins with an antibody to the dioxins immobilized on an electrode to cause an antigen-antibody reaction, by applying a voltage to the reaction product from the electrode. A step of oxidizing and reducing the ruthenium complex to generate electroluminescence, and a step of measuring the amount of luminescence by the electroluminescence to determine the amount of the antigen to determine the amount of dioxins in the sample, Taking the technical measures of that.

As a function of dioxins, when a chemical substance having a planar structure (such as dioxin, dibenzofuran, and coplanar PCB) is taken into cells, it exhibits a high affinity for a protein called an Ah receptor in the cytoplasm. It is known to combine. Utilizing the properties of these dioxins, they form a foreign small molecule that mimics only a part of the structure of dioxin, and when this small molecule binds to a polymer, it becomes a hapten that forms a specific antibody. Use A hapten that mimics only part of the structure of dioxin is chemically bonded to a ruthenium complex that can initiate an electroluminescent reaction simply by oxidation / reduction to form an antigen, and this antigen is incubated with an antibody immobilized on an electrode. Then, an antigen-antibody reaction is performed, and by applying a voltage from the electrode to the reaction product, electroluminescence is generated. That is, by observing the luminescence intensity or luminescence extinction of the electroluminescence, the antigen can be quantified and the concentration of dioxins can be measured. This eliminates the need for a reagent such as hydrogen peroxide required for chemiluminescence, allows the apparatus to be downsized, and facilitates high sensitivity.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, the dioxin hapten site 2 (see FIG. 1) and the ruthenium complex 3 (see FIG. 1) are chemically bonded to form an antigen 1 (FIG. 1) which becomes an electroluminescent reagent.
Will be described. First, as the synthesis of the dioxin hapten, for example, the following bonds can be considered.

Embedded image

The above

On the left-hand side of the formula, for example, a phthalic ester having a part of the structure of 2,3-dibenzodioxin: 4,5-dichlorophthalic anhydride (A) is present and binds to the antibody. Is reacted with 4- (p-aminophenyl) butanoic acid (B), which serves as a spacer in the reaction. Then, N- (4 ', 5'-dichlorophthaloyl) -4- (p-aminophenyl) phthalic acid (C) is produced. The portion (C) is a hapten site, and the ruthenium complex is bound by the carboxyl group of the portion (D).

On the other hand, it is necessary to provide a spacer for binding to the antibody on the ruthenium complex 3 side.

From

This will be described with reference to the following formula.

[0014]

Embedded image On the left side of the above (2), 1- (N-phthaloylamino) -3-fluoromomide which serves as a spacer when the phthalimitopotassium (A) is allowed to react with 1,3-chlorofluorofuran (B) to bind to the antibody. Generates a flow (C).

[0015]

Embedded image Next, on the left side of the above (3), 4,4′-dimethyl-2,2′-divinyl (A) and the above

1- (N-phthaloylamino) -3-fluorofluorobenzene (B) produced by the reaction is reacted with 4- (4- (N-phthaloylamino) butyl) -4'-methyl
-2,2'-Phiverse (C) is generated.

[0016]

Embedded image Further, on the left side of the above (4), 4- (4- (N-phthaloylamino) butyl) -4'-methyl-2,2'-divinyl (A) is reduced by human paraffin H ₂ NNH ₂ and the right side is obtained. 4- (4-aminobutyl) -4'-methyl-2,2'-divinyl (B).

[0017]

Embedded image And, on the left side of the above (5), 2,2'-Phi-PhD (A)
Is reacted with ruthenium trichloride to give dichlorobis (2,2'-dihydroxy) ruthenium (C).

[0018]

Embedded image Finally, (5) dichlorobis (2,2'-dihydroxy) ruthenium (A) and (4) 4- (4-aminobutyl) -4'-methyl-2,2'-dihydroxy (B) ), When bound to an antibody, the antibody having a spacer (2,2'-dihydroxy) {4- (4-aminobutyl) -4'-
A methyl-2,2'-hydroxypyridine ruthenium complex (C) is formed.

Next, a spacer is provided.

Having N- (4 ', 5'-dichlorophthaloyl) -4- (p-aminophenyl) phthalic acid of the formula and a spacer

A dioxin hapten and a ruthenium complex are reacted by reacting a bis (2,2'-dihydroxy) {4- (4-aminobutyl) -4'-methyl-2,2'-dipyridin} ruthenium complex of the formula And an antigen 1 as an electroluminescent reagent chemically bonded to is generated.

Embedded image At this time, if the {4- (4-aminobutyl) -4′-methyl-2,2′-dihydroxy} ruthenium complex can be quantified, the hapten site of dioxins can also be quantified.

FIG. 1 is a schematic diagram showing a state in which dioxin 20 and ruthenium complex 3 are bound to dioxin antibody 4 competitively between dioxin hapten sites 2 of antigen 1. The antigen-antibody reaction will be described with reference to this. The dioxin hapten site 2 simulating a part of the structure of dioxin 20 is chemically bonded to the ruthenium complex 3 to form the antigen 1 (see above).

From

## STR5 ## and this antigen 1 becomes an electroluminescent reagent. The antigen 1 and the dioxin 20 are incubated with the antibody 4 immobilized on the electrode 5 to perform an antigen-antibody reaction. This antigen-antibody reaction is not due to a chemical bond but to a weak interaction including a hydrogen bond. However, since the antibody 4 has many places for interaction, the ability to recognize the dioxin hapten site 2 is high, and the dioxin hapten site 2 and dioxin 20 are strongly bound. As an image, as shown in FIG. 1, the antibody 4 has a dioxin hapten site 2 and a groove 4 ′ having the same shape as the dioxin 20, and is completely accommodated therein.

Next, the action of the antigen-antibody reaction will be described in detail. First, as shown in FIG. 1, the antibody 4 is fixed to the gold electrode 5 by chemical bonding. First of all about this fixing method
Acid treatment of the gold electrode 5 with sulfuric acid etc. to clean the surface,
3'-dithiodipropanoic acid solution (H00CCH ₂ CH ₂ S-SCH ₂ CH ₂ COO
H) The gold electrode 5 is immersed in (10 mM) for 30 minutes, and 3,3'-dithiodipropanoic acid is chemically adsorbed on the surface of the gold electrode 5 (3,3'-
Chemical bonding by the interaction between the sulfur atom S of the diotidipropanoic acid and the gold atom Au). Then, the gold electrode 5 is taken out and washed with alcohol such as methanol. Further, a gold electrode (30 mg of hydroxysuccinimide + water-soluble carbodiimide) was used to activate the carboxylic acid of 3,3′-dithiodipropanoic acid immobilized on the surface of the gold electrode 5.
(1-ethyl-3-dimethylaminopropyl carbodiimide hydroc
immersion in 20 ml of 90% aqueous solution of dioxane containing 50 mg) of hloride for 15 minutes Then, a gold electrode 5 having the activated 3,3′-dithiodipropanoic acid was added to the antibody 4 solution at room temperature for 1 to 10 minutes.
Minutes immersion, the reaction by forming an amide bond -CONH- with the amino group NH ₂ antibodies 4. Thus, the antibody 4 is fixed on the gold electrode 5. Thereafter, the gold electrode 5 is washed with a phosphate buffer to remove the antibody 4 not chemically bonded.

On the other hand, a certain amount of antigen 1 in which dioxin hapten site 2 and ruthenium complex 3 are chemically bound is added to a measurement solution prepared with a phosphate buffer containing 3% DMSO (dimethyl sulfoxide). DMSO is used to sufficiently dissolve dioxin. 0.2M phosphate buffer containing 3% DMSO (PH =
When the measurement solution prepared in 7.0) is used for analyzing a sample, dioxin can be dissolved to the ppb level only by adding a small amount of DMSO. Then, the gold electrode 5 on which the antibody 4 is immobilized is immersed in the measurement solution and incubated for 10 minutes to perform an antigen-antibody reaction.
Thereafter, the gold electrode 5 is taken out of the measurement solution and washed lightly with a phosphate buffer (PH 7.0).

FIG. 2 is an action diagram showing the electroluminescence of the antigen 1 in which the dioxin hapten site 2 and the ruthenium complex 3 are chemically bonded. As shown in FIG. 2, an electrode 5 (anode) and a cathode For example, an electrode made of stainless steel (not shown) and a reference electrode such as a silver electrode or a silver chloride electrode (not shown) are made of, for example, trimethylamine (TMA), triethylamine, tripropylamine,
It is immersed in a phosphate buffer containing a reducing agent with a tertiary amine such as tributylamine, proline as an amino acid or oxalic acid as another organic acid. And
When the temperature is between 15 ° C and 30 ° C and the time is between 20 seconds and 2 minutes,
When a voltage of 0.9 to 1.3 V is applied to the electrode 5, electroluminescence is generated. By observing the luminescence intensity, the antigen can be quantified and the concentration of dioxins can be measured.

Although the above procedure is derived in relation to the type and concentration of dioxin, the concentration of antigen 1 is preferably, for example, 20 μg / L. FIG. 4 shows a calibration curve created by plotting the data.

The material required for electroluminescence is a phosphate buffer aqueous solution containing ruthenium complex 3 and trimethylamine (TMA) 6 as a reducing agent. The ruthenium complex 3 is oxidized by the gold electrode 5 and becomes an active reaction from divalent to trivalent. At this time, the ruthenium complex 3 reacts with trimethylamine (TMA) 6 and is oxidized to obtain trimethylamine oxide (TMA _OX ) 7, and trimethylamine (TMA) 6
When the active reaction by the ruthenium complex 3 decreases (at this time, the charge decreases from trivalent to divalent), light is emitted. Phosphate buffer solutions are important for optimal luminescence conditions. In particular,
The electrolysis environment is prepared by providing conductivity necessary for electrolysis and controlling the pH value to (pH 7.0). The ruthenium complex 3 binds to the antibody 4 immobilized on the gold electrode 5 depending on the dioxin concentration of the aqueous solution. When the dioxin concentration is high, the amount of the ruthenium complex 3 decreases.
Thereafter, in order to minimize the effect of the interfering substance (which causes electroluminescence) in the dioxin solution, the gold electrode 5 to which the ruthenium complex 3 is bonded is lightly washed, and then phosphoric acid containing separately prepared trimethylamine 6 (TMA) Put in a buffered aqueous solution and observe the electroluminescence.

In order to observe the electroluminescence, it is preferable to install a detecting device such as a condenser lens 8, a mirror 9, an optical filter 10 and a measuring sensor 11 as shown in FIG. By monitoring the luminescence intensity and luminescence time detected by the measurement sensor 11, the ruthenium complex in the sample is quantified, and the quantification of this ruthenium complex and the quantification of the dioxin hapten site are linked, so that dioxin Quantitation becomes possible. In addition, it is desirable to store a calibration curve as shown in FIG. 4 in advance and provide an arithmetic unit (not shown) such as a personal computer or the like, which can quantify dioxin based on emission intensity.

More specifically, a first measurement solution containing antigen 1 is added to dioxin in a sample, and an electrode (anode) 5 on which an antibody 4 is immobilized is immersed in the measurement solution.
Antibody 4 immobilized on electrode 5 and dioxin 20
Causes an antigen-antibody reaction with the antigen 1. Then, the antigen-antibody reaction between dioxin and antigen 1 in the sample occurs competitively with antibody 4. If the dioxin concentration in the sample is high, the reaction ratio between dioxin and antibody 4 increases, and the reaction ratio between antigen 1 and antibody 4 decreases. As a result, the emission intensity becomes weak. On the other hand, if the dioxin concentration in the sample is low, the reaction ratio between dioxin and antibody 4 will be low, and the reaction ratio between antigen 1 and antibody 4 will be high.

The present invention will be described in detail with reference to the following examples, but the present invention is not limited to these examples.

EXAMPLE As an example of the present invention, FIG. 4 is a graph showing the relationship (calibration curve) between the dioxin concentration in a sample and the luminescence intensity. As a measurement solution, the concentration is 0.02, 0.06, 0.1, 0.2, 0.5, 1.0,
Contains 3.0 μg / L of 2,3,7,8-tetrachloro-p-dibenzopentadioxin, 0.2 M with 3% DMSO (dimethyl sulfoxide)
What was prepared with the phosphate buffer of the density | concentration was prepared. Then, the concentration prepared by the method described above is 20
μg / L of antigen 1 and 1 mmol / L of trimethylamine were mixed. The antibody (EWVATI) prepared by the above method
TGGGTYTYYPDSVRGC) 4 immobilized gold electrode (anode) 5,
Stainless steel electrode (cathode) and reference electrode (silver / silver chloride)
Was immersed in a measurement solution containing an antigen, and incubated at room temperature for 2 minutes. The light emission amount of electroluminescence obtained by applying a voltage of 1.1 V to the electrode at room temperature for 1 minute was detected by the detection device shown in FIG. The detection result is plotted on FIG. 4 and shown in a graph. The data in FIG. 4 clearly show the emission intensity due to electroluminescence, and the method and apparatus of the present invention show that the dioxin concentration is extremely low (for example, 0.01 to 0.1 pp).
It can be recognized even in the case of b), and the measurement of the concentration of dioxins can be easily performed with high sensitivity, and the high sensitivity is considerably improved.

[0030]

As described above, according to the present invention, there is provided a method for measuring the concentration of dioxins in a sample, wherein a hapten imitating a part of the structure of dioxins is chemically bonded to a ruthenium complex to form an antigen. Forming a sample, and incubating a sample considered to contain the antigen and dioxins with an antibody against the dioxins immobilized on an electrode to cause an antigen-antibody reaction, applying a voltage to the reaction product from the electrode. A step of oxidizing and reducing the ruthenium complex to cause electroluminescence by applying, and measuring the amount of luminescence by the electroluminescence to quantify the antigen, so that a reagent such as hydrogen peroxide necessary for chemiluminescence is used. No need was made, the device could be downsized, the sensitivity could be easily increased, and the sensitivity increased considerably.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a state in which dioxin and a dioxin hapten site of an electroluminescent reagent are competitively bound to a dioxin antibody.

FIG. 2 is a schematic diagram showing a state of electroluminescence at a hapten site.

FIG. 3 is a schematic diagram showing a detection device for observing electroluminescence.

FIG. 4 is a graph showing the relationship (calibration curve) between dioxin concentration and luminescence intensity.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Antigen 2 Dioxin hapten site 3 Ruthenium complex 4 Antibody 5 Gold electrode 6 Trimethylamine 7 Trimethylamine oxide 8 Condensing lens 9 Mirror 10 Optical filter 11 Measurement sensor 20 Dioxin

Claims (7)

[Claims] 1. A method for measuring the concentration of dioxins in a sample, the method comprising the step of chemically binding a hapten, which mimics a part of the structure of dioxins, to a ruthenium complex to form an antigen, the antigen and the dioxins Incubating a sample supposed to contain an antibody against the dioxins immobilized on an electrode to cause an antigen-antibody reaction, and oxidizing the ruthenium complex by applying a voltage to the reaction product from the electrode. Reducing the amount of dioxins in the sample by reducing the amount of dioxins in the sample by measuring the amount of light emitted by the electroluminescence and quantifying the antigen. A method for measuring the concentration of dioxins in water. 2. The electroluminescence of the ruthenium complex is carried out by an oxidation reaction from divalent to trivalent by receiving a charge from the electrode and a phosphate buffer aqueous solution containing trimethylamine.
2. The method for measuring the concentration of dioxins in a sample according to claim 1, which utilizes a reduction reaction from valence to divalent. 3. The antigen-antibody reaction according to claim 1, wherein the antigen is incubated with an antibody immobilized on the electrode using a measurement solution prepared with a phosphate buffer containing 3% dimethyl sulfoxide. A method for measuring the concentration of dioxins in a sample. 4. A method for measuring the concentration of dioxins in a sample, comprising the steps of: preparing a first electrode on which an antibody against dioxins is immobilized; A first electrode, on which an antibody against the dioxins is immobilized, is immersed in a first measurement solution containing an antigen obtained by chemically bonding a hapten that mimics a part of the structure to a ruthenium complex, and the mixture is incubated. A step of competitively performing an antigen-antibody reaction between the antibody immobilized on the first electrode and an antigen obtained by chemically bonding a ruthenium complex to the dioxins and a hapten simulating a part of the structure of the dioxins , First
Removing the unreacted antigen from the first liquid, washing the unreacted antigen, immersing the first electrode and the second electrode in a second measurement liquid containing a reducing agent, A voltage is applied to oxidize the ruthenium complex bound by an antigen-antibody reaction with an antibody against dioxins immobilized on the first electrode, and reduce the oxidized ruthenium complex with the reducing agent. From the step of measuring the amount of electroluminescence to be performed, and the calibration curve obtained in advance,
Determining the amount of the dioxins in the sample. A method for measuring the concentration of dioxins in a sample. 5. The method for measuring the concentration of dioxins in a sample according to claim 4, wherein said first electrode comprises a gold electrode. 6. The method for measuring the concentration of dioxins in a sample according to claim 5, wherein the reducing agent is selected from the group consisting of trimethylamine, triethylamine, tripropylamine, tributylamine, proline and oxalic acid. . 7. An apparatus used in the measurement method according to claim 1, wherein the measurement solution is dissolved in a phosphate buffer containing the ruthenium complex and trimethylamine, and A gold electrode for applying a voltage, an antibody immobilized on the gold electrode, a luminescence detector for measuring the amount of luminescence by electroluminescence, and a luminescence intensity detected from the luminescence detector An apparatus for measuring the concentration of dioxins in a sample, comprising: an arithmetic device for quantifying the concentration of dioxins.