Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2001-10-30
2004-03-09
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C435S007920, C436S518000, C436S524000, C436S544000, C436S543000, C530S388900
Reexamination Certificate
active
06703210
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to a method and a device for determining the concentration of dioxins present in a sample. More particularly, the invention relates to a method and a device that permit the highly sensitive and highly selective determination of the concentration of polychlorinated dibenzo-p-dioxins (PCDDs) including virulently poisonous 2,3,7,8-tetrachloro-dibenzo-p-dioxin (T
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CDD) and likewise virulently poisonous polychlorinated dibenzofurans (PCDFs), which are generated during the incineration of general waste and industrial waste.
Dioxins are generated in a high concentration when the waste is incinerated and this has recently constituted a serious problem. Accordingly, one has been obligated to control the concentration of dioxins included in the exhaust gas generated during the incineration of the foregoing waste to a level of not higher than 0.1 ng/Nm
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as expressed in terms of the total amount of the dioxins and homologues and isomers thereof as well as the homologues and isomers of polychlorinated dibenzofuran, because the dioxins are virulently poisonous. However; it is needed to detect dioxins present in such a low concentration, which is beyond the detection limit of the usual analysis methods, because of the virulent toxicity of the dioxins and the exhaust gas to be analyzed contains a great variety of impurities and contaminants such as dust and mist in a large quantity. For this reason, the direct and precise determination and/or quantification of dioxins require a complicated pre-treatment of the exhaust gas and the use of a specially designed analysis device.
A typical quantitative analysis method comprises the steps of extracting an exhaust gas with a solvent such as toluene or ethylene glycol for 72 hours using a Soxhlet's extractor, cleaning up the extract, concentrating the extract and then analyzing it using a gas chromatography-mass spectrometer. This method includes the use of complicated steps required for the analysis such as a concentration step. Therefore, it takes two or three weeks till the analysis is completed or desired results are obtained and the analysis is quite expensive.
Japanese Un-Examined Patent Publication (hereunder simply referred to as “J.P. KOKAI”) No. Hei 11-326222 proposes an indirect method for quantitatively analyzing dioxins, which can eliminate the use of any operation for concentrating a sample containing dioxins and which employs a relatively cheap device, and this method comprises the steps of mixing a halogen atom-containing organic solvent, metal complexes of 8-oxyquinoline and a sample whose concentration of dioxins is to be determined; irradiating the resulting mixture with excited light rays to determine the intensity of the emitted fluorescent light rays and to thus quantitatively analyze halophenols whose concentration is well correlated with that of the dioxins present in the sample; and estimating the concentration of the dioxins on the basis of the concentration of the halophenols thus determined.
In this method, however, (1) the liquid to be analyzed may interact with the fluorescent complexes and therefore, any influence of components present in the liquid, which may compete with the halophenols, should be examined in advance and (2) the correlation between the concentrations of the halophenols and dioxins should be examined in advance. The results obtained by the method may greatly be influenced by the factors concerning the kinds of dioxins. Accordingly, this method may be a means considerably inferior in the precision since the concentration of the dioxins present in the liquid is indirectly evaluated. In addition, the fluorescent method is highly sensitive, but the method is quite susceptible to coexisting substances and the excited light rays are inevitably incident upon the system for observation (or a detection system) and therefore, the method does not always ensure desired high sensitivity.
Incidentally, J.P. KOKAI No. Hei 11-79719 discloses a method for reversibly absorbing and desorbing a gas without using conventionally used absorbent such as activated carbon and silica gel. This method comprises bringing a pair of electrodes into contact with a solution containing a ruthenium complex capable of absorbing and releasing molecular nitrogen and simultaneously controlling the electric voltage applied to the electrodes to thus absorb and desorb nitrogen gas and therefore, this method permits (1) the control of the absorption and desorption of the gas at ordinary temperature and pressure without application of any heat; (2) the control of the gas absorption and desorption simply by adjusting the electric voltage to be applied to the electrodes; and (3) the reversible and repeated absorption and desorption of the gas. In this method, which makes use of a solution containing a ruthenium complex capable of absorbing and releasing molecular nitrogen, however, there are some problems remaining unsolved, for instance, a problem as to whether, or not, the method permits the absorption and desorption of substances having planar structures such as dioxins and homologues and isomers thereof as well as homologues and isomers of polychlorinated dibenzofuran.
SUMMARY OF THE INVENTION
The inventors of this invention have found that the use of a ruthenium complex permits the highly selective determination of dioxins, while taking into consideration the foregoing problems associated with the conventional techniques. Accordingly, it is an object of the present invention to provide a method and a device for determining the concentration of dioxins present in a sample, which make use of a novel measurement system utilizing electrolytic light emission to thus ensure highly sensitive analysis of dioxins, which do not require the use of any reagent such as hydrogen peroxide required for the chemical luminescence, which permits the miniaturization of the device and which can increase the sensitivity of the analysis.
The inventors of this invention have conducted various studies to solve the foregoing problems and as a result, have completed the present invention.
According to a first aspect of the present invention, there is provided a method for determining the concentration of dioxins present in a sample, which comprises the steps of chemically binding a hapten having a structure mimic to a part of the structures of dioxins with a ruthenium complex to thus form an antigen; incubating the resulting antigen together with an antibody fixed to or immobilized on an electrode to thus induce an antigen-antibody reaction; oxidizing or reducing the ruthenium complex by applying an electric voltage to the reaction product through the electrode to thus induce electrolytic light emission; and observing the electrolytically emitted light rays to quantitatively determine the amount of the antigen and to thus evaluate the concentration of the dioxins present in the sample.
According to a second aspect of the present invention, there is provided a method for determining the concentration of a dioxin present in a sample, which comprises the steps of providing a first electrode onto which an antibody to the dioxin is immobilized; immersing the first electrode in a first liquid containing the sample and an antigen, said antigen being formed by chemically binding a hapten having a structure mimic to a part of the structure of the dioxin with a ruthenium complex; incubating the liquid together with the first electrode to thus induce a competitive antigen-antibody reaction between the antibody and the antigen (dioxin) and the hapten; taking out the first electrode and washing it to remove the antigen and the hapten that are not reacted with the antibody; immersing the first electrode and a second electrode in a second liquid containing a reducing agent and an electrolyte; applying to the second liquid an electric voltage through the first and the second electrodes to oxidize the ruthenium complex; allowing the oxidized ruthenium complex to be reduced by the reducing agent to thus induce electrolyti
Counts Gary
Le Long V.
Satake Corporation
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