JP2011057819A - Method for measuring concentration of material to be detected by time measurement of chemiluminescence reaction and kit used for the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a novel concentration measurement method of a material to be detected with high sensitivity and high reproducibility by utilizing a chemiluminescence system and to provide a kit used for the concentration measurement method. <P>SOLUTION: The method is to measure concentration of a material to be detected included in a sample liquid by time measurement of a chemiluminescence reaction. Chemiluminescence is generated by mixing the sample liquid, a luminescence substrate, an oxidizing agent, and a reducing agent together in a solvent. The time from immediately after mixing to the time when luminescence intensity of the chemiluminescence becomes a peak is measured. The concentration of a material to be detected is measured by the measured time and a previously obtained calibration curve. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for measuring the concentration of a substance to be detected by measuring the time of a chemiluminescent reaction and a kit used therefor.

Chemiluminescence is a phenomenon in which excess energy generated by a chemical reaction is released as light instead of losing it as heat. This includes the phenomenon in which energy is transferred from an excited species to a coexisting fluorescent substance and light is emitted. (Patent Document 1 and Non-Patent Document 2). Many of the reactions in which chemiluminescence is observed are redox reactions between a luminescent substrate and a redox reagent. Dissolved oxygen or hydrogen peroxide is used as the oxidizer, but when metal ions are used as the catalyst, hydroxy radicals (.OH) and superoxide radicals (.O ₂ ⁻ ) are reacted with these oxidants. Thus, the generation of reactive oxygen species having strong oxidizing power increases the rate of oxidation reaction of the luminescent substrate, and as a result, the luminescence intensity per unit time increases.

In addition, chemiluminescence has been rapidly developed in terms of analytical chemistry, and the reason is that an analytical method using chemiluminescence is very sensitive. For example, a fluorometric method, which is one of the highly sensitive analytical methods, requires a light source such as a xenon lamp to excite fluorescent molecules, which is caused by the stray light phenomenon derived from it or the solvent-derived Raman light. Will increase the background level. Therefore, as the sensitivity of the detector is increased, not only the signal level but also the noise level is increased.
However, in chemiluminescence, since the excitation source of the fluorescent molecule is energy by chemical reaction, the light source becomes unnecessary and the influence thereof need not be considered. Therefore, it is possible to detect even very weak light by increasing the sensitivity of the photomultiplier tube. In addition, there is an advantage that the sample can be downsized due to its high sensitivity. As a result, side reactions that tend to occur at high concentrations are less likely to occur, and the amount of reagent used can be reduced. As a result, there is an advantage that high-sensitivity analysis can be performed while reducing reagent costs. In addition, the chemiluminescence method has many advantages, such as the fact that the excitation source is not required, the device is simple, and the responsiveness is fast and quick.

  However, the absolute number of luminescent substrates that can currently be used for chemiluminescence analysis is about 10, and the number of chemiluminescent reaction systems with a high luminescence yield is limited. Furthermore, there is a problem of reagents such as selection of an organic solvent and a catalyst to be used, and as a result, there is a problem that a component to be analyzed by the chemiluminescence analysis method is limited and the selectivity is low. As described above, for chemiluminescent systems that support chemiluminescence measurement, new reaction systems and luminescent materials have not been easily found, and chemiluminescent analytical methods that can be applied to a wide range of fields. Discovery is desired.

JP 2000-74841 A

T. Nagoshi, O. Ohno, T. Kotake, S. Igarashi, Luminescence, 20, 401-404 (2005)

  Provided is a highly sensitive and reproducible novel method for measuring the concentration of a target substance using a chemiluminescent system, and a kit for use in the concentration measuring method.

  The present invention is a method for measuring the concentration of a substance to be detected contained in a sample solution, the method comprising mixing the sample solution, a luminescent substrate, an oxidizing agent, and a reducing agent in a solvent. A step of generating chemiluminescence, a step of measuring a time from immediately after mixing to a time when the emission intensity of the chemiluminescence reaches a peak, and a step of calculating a concentration of the substance to be detected from the measured time. It is characterized by that.

  In the concentration measurement method of the present invention, the luminescent substrate is an iron porphyrin complex or hemoglobin. In the concentration measurement method of the present invention, the iron porphyrin complex is an iron phthalocyanine complex or an iron chlorophyllin complex. In the concentration measurement method of the present invention, the oxidizing agent is hydrogen peroxide. In the concentration measurement method of the present invention, the reducing agent is L-ascorbic acid or L-cysteine.

  In the concentration measurement method of the present invention, the substance to be detected is one metal ion selected from the group consisting of copper ions, aluminum ions, iron ions, zinc ions, and nickel ions, L-tyrosine, L-tryptophan. , L-cysteine, D-glucose, peroxidase, or an enzyme-labeled antibody.

  According to another aspect of the present invention, there is provided a kit for measuring the concentration of a substance to be detected contained in a sample, the kit comprising a luminescent substrate, an oxidizing agent, and a reducing agent, When the substance to be detected, the luminescent substrate, the oxidizing agent, and the reducing agent are mixed in a solvent, chemiluminescence is generated after a lapse of time according to the concentration of the substance to be detected.

  The kit of the present invention is characterized in that the luminescent substrate is an iron porphyrin complex or hemoglobin. According to the kit of the present invention, the iron porphyrin complex is an iron phthalocyanine complex or an iron chlorophyllin complex. The kit of the present invention is characterized in that the oxidizing agent is hydrogen peroxide. According to the kit of the present invention, the reducing agent is L-ascorbic acid or L-cysteine.

  In the kit of the present invention, the substance to be detected is one metal ion selected from the group consisting of copper ion, aluminum ion, iron ion, zinc ion, and nickel ion, L-tyrosine, L-tryptophan, L -Cysteine, D-glucose, peroxidase, or labeled antibody.

  According to another aspect of the present invention, there is provided an apparatus for measuring the concentration of a substance to be detected, wherein the apparatus comprises the substance to be detected, a luminescent substrate, an oxidizing agent, and a reducing agent in a solvent. A chemiluminescence measuring means for measuring chemiluminescence generated by mixing; a time measuring means for measuring an induction time from immediately after mixing to a time when the emission intensity of the chemiluminescence reaches a peak; and And a concentration calculating means for calculating the concentration of the substance.

  According to the quantification of the substance to be detected by the time measurement of the chemiluminescence reaction according to the present invention, while having the advantages of the conventional chemiluminescence system such as reduction in reagent cost, ease of instrumentation, and quick response, High-sensitivity and reproducible quantification is possible compared to luminescent signal measurement.

It is a flowchart which shows time measurement of the chemiluminescence reaction in one embodiment which concerns on this invention. It is a graph which shows the induction time of the chemiluminescence by L-ascorbic acid non-addition in the chemiluminescent reaction in one embodiment of this invention. It is a graph which shows the induction time of chemiluminescence when L-ascorbic acid is added in the chemiluminescence reaction in one embodiment of the present invention. It is a graph which shows the relationship between a chemiluminescent intensity | strength and L-ascorbic acid density | concentration in the chemiluminescent reaction in one embodiment of this invention. It is a graph which shows the relationship between chemiluminescence intensity | strength and hydrogen peroxide concentration in the chemiluminescent reaction in one embodiment of this invention. It is a graph which shows the relationship between chemiluminescence intensity | strength and pH in the chemiluminescent reaction in one embodiment of this invention. It is a graph which shows the relationship between a chemiluminescent intensity | strength and the volume of DMSO in the chemiluminescent reaction in one embodiment of this invention. It is a graph which shows the relationship between a chemiluminescence intensity | strength and Fe-PTS density | concentration in the chemiluminescent reaction in one embodiment of this invention. In time measurement by one embodiment of the present invention, it is a graph (calibration curve) which shows relation between copper ion concentration and induction time. LUMICOUNTER NU-600 was used for time measurement. In time measurement by one embodiment of the present invention, it is a graph (calibration curve) which shows relation between copper ion concentration and induction time. GENELIGHT GL-200S was used for time measurement. In time measurement by one embodiment of the present invention, it is a graph (calibration curve) which shows relation between peroxidase concentration and induction time. LUMICOUNTER NU-600 was used for time measurement.

  The present inventors have found that by adding a reducing agent to the chemiluminescent reaction between the luminescent substrate and the oxidant, there is a time difference from when the solution is mixed until the chemiluminescent signal is observed. Furthermore, by adding a substance functioning as a catalyst to this reaction system, the time until the chemiluminescence signal was observed changed. From this, as a result of investigating the time measurement of the chemiluminescent system rather than the luminescence intensity, it was found that the quantification of a specific substance to be detected is highly sensitive and reproducible.

  Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

  FIG. 1 is a flowchart showing time measurement of a chemiluminescence reaction in one embodiment of the present invention. As shown in FIG. 1, the chemiluminescence reaction time measurement of the present invention includes a chemiluminescence cell 1, an organic solvent 2 and a pH buffer solution 3 mixed to produce chemiluminescence in the chemiluminescence cell 1. , Reducing agent 4, sample solution 5, luminescent substrate 6, and oxidizing agent 7 are sequentially added.

  The chemiluminescence cell 1 is prepared by mixing an organic solvent 2, a pH buffer 3, a reducing agent 4, a sample solution 5, a luminescent substrate 6, and an oxidant 7, and setting them in a chemiluminescence measuring device as they are. Conventionally used ones can be used as long as the intensity of luminescence generated in the liquid can be measured. Although not particularly limited, for example, a quartz cell, a polystyrene cell, or the like can be given. Moreover, it is preferable that the temperature of the liquid mixture which measures chemiluminescence is maintained at 15 to 40 degreeC, and it is more preferable that it is maintained at 20 to 30 degreeC.

  As the organic solvent 2 used in the present invention, DMSO or the like can be used. Moreover, distilled water can also be used instead of the organic solvent. The volume when DMSO is used as the organic solvent 2 is preferably in the range of 15% by volume to 25% by volume in the final mixed solution, and more preferably in the range of 18% by volume to 22% by volume. About 20% by volume is preferred and most preferred.

  The pH buffer solution 3 used in the present invention is not particularly limited as long as the pH of the mixed solution can be kept constant. For example, Borax buffer solution, sodium hydroxide-acetic acid system, ammonia-acetic acid A system buffer or the like can be used. The pH maintained by the pH buffer 3 is preferably 8 to 12, more preferably 9 to 11, and most preferably about pH 10.

The reducing agent 4 can be used as long as it can suppress the oxidation of the luminescent substrate 6 by the oxidant 7 and cause induction time for chemiluminescence. Although not particularly limited, for example, L-ascorbic acid, L-cysteine and the like can be used, and L-ascorbic acid is particularly preferable. L- ascorbic acid concentration when used is preferably in the range of ^{0.5 × 10 -3 M~2.0 × 10 -3} M in the final mixed solution, 1.0 × 10 ^{- 3} is more preferably in the range of M~2.0 × ^{10 -3} M, and most preferably about 1.6 × ^{10 -3} M.

  The sample solution 5 contains a substance whose concentration is to be measured. Examples of substances that can be measured by the concentration measurement method using the chemiluminescence time measurement of the present invention include copper ions, aluminum ions, iron ions, zinc ions, and Mention may be made of one metal ion selected from the group consisting of nickel ions, L-tyrosine, L-tryptophan, L-cysteine, D-glucose, peroxidase, or enzyme-labeled antibodies. These substances affect a reducing agent such as L-ascorbic acid and influence the induction time of chemiluminescence. Although it differs depending on the measurement object, the detection limit of this concentration measurement method can measure a very small amount of several ppt level. When an enzyme-labeled antibody is used, since an enzyme is added as a label to the antibody, the labeled antibody that has been specifically bound to the substance to be detected and purified can be used in this chemiluminescence reaction system. The enzyme-labeled antibody is, for example, an enzyme-labeled antibody added by a conventional method (see Bull. Natl. Inst. Anim. Health No. 111. 37-42 (March 2005) etc.) using HRP (Horse Radish Peroxidase) as a label. Such enzyme-labeled antibodies can be used to measure a very small amount of component concentration. As the enzyme-labeled antibody, a peroxidase-labeled antibody is particularly preferable. In addition, when using a sample containing a large amount of protein such as serum, it is desirable to perform deproteinization before using it in the chemiluminescence reaction system.

The luminescent substrate 6 used in the present invention is not particularly limited as long as it emits light in the mixed solution by reaction with the oxidant 7, and is a metal complex having a porphyrin skeleton or hemoglobin having a porphyrin metal complex as a subunit. It is preferable. The metal forming the complex is preferably iron, and as the iron porphyrin complex, for example, an iron phthalocyanine complex, an iron chlorophyllin complex, hemin, or the like can be used. As the iron phthalocyanine complex, iron phthalocyanine derivative complexes such as iron phthalocyanine tetrasulfonic acid, iron phthalocyanine octasulfonic acid, iron phthalocyanine tetracarboxylic acid, and iron phthalocyanine octacarboxylic acid can be used. Concentration when using iron phthalocyanine sulfonate as a luminescent substrate, 2.0 × It is preferable to be ^{10 -5 M~6.0} × ¹⁰ -5 in the range of M in the final mixed solution, 3. 0 × ¹⁰ -5 is more preferably a M~5.0 × ^{10 -5} in the range of M, and most preferably about 4.0 × ^{10 -5} M.

The oxidizing agent 7 used in the present invention is not particularly limited as long as it can generate luminescence in the mixed solution by oxidizing the luminescent substrate 6, for example, hydrogen peroxide or 2KHSO ₅ · KHSO ₄ · K _2. Although SO ₄ can be mentioned, hydrogen peroxide can be preferably used. Concentration when using hydrogen peroxide, it is preferable to be 1.0 × ^{10 -3} M~4.0 × ¹⁰ -3 in the range of M in the final mixed solution, 2.0 × 10 ^{- 3 M~3.0} × more preferably made ^{10 -3} in the range of M, about 2.4 × ^{10 -3} M and most preferably.

  According to the above configuration, the organic solvent 2, the pH buffer 3, the reducing agent 4, the sample solution 5, and the luminescent substrate 6 are added into the chemiluminescence cell 1, and after gently shaking, the cell holder Set to. Next, the oxidizing agent 7 is added, and chemiluminescence generated in the mixed solution is measured, and the chemiluminescence induction time is also measured. As used herein, “induction time” refers to the chemistry that occurs in a mixed solution from the point of addition of a final reagent (for example, an oxidizing agent) for generating chemiluminescence in the mixed solution. The time until the emission intensity reaches a peak. In such chemiluminescence time measurement, the conditions such as the luminescent substrate concentration, oxidant concentration, reducing agent concentration, and pH are made constant, depending on the concentration of the specific target substance contained in the reaction system. The induction time increases or decreases. Since the correlation graph between the concentration of the detected substance and the induction time is used as a good calibration curve, by measuring the time of the chemiluminescence reaction of the present invention in the presence of the detected substance, from the calibration curve obtained in advance, It becomes possible to measure the concentration of the substance to be detected.

  In addition, the concentration measuring device using the chemiluminescence time measurement according to the present invention is a chemiluminescence produced by mixing a sample solution containing a substance to be detected, a luminescent substrate, an oxidizing agent, and a reducing agent in a solvent. A chemiluminescence measuring means for measuring the induction time, an induction time measuring means for measuring an induction time from immediately after mixing to a time when the emission intensity of the chemiluminescence reaches a peak, and an induction time and a calibration curve obtained in advance. It is comprised from the density | concentration calculation means which calculates a density | concentration.

  The chemiluminescence measuring means used in the present invention is conventionally used as long as it can measure the emission intensity of chemiluminescence generated in the liquid mixture added in the chemiluminescence cell and the luminescence over time. A chemiluminescence measuring device or the like can be used. What is necessary is just to be able to measure the range of 300-700 nm as a measurement wavelength.

  The time measuring means has a function of calculating the induction time of chemiluminescence based on the chemiluminescence measurement result measured by the chemiluminescence measuring means.

  The concentration calculation means used in the present invention has a function of deriving the concentration of the substance to be detected from the induction time calculated by the time measurement means. The concentration calculation means can also create a calibration curve from the measured chemiluminescence intensity and induction time. Further, information on the calibration curve and information such as the concentration of the detected substance after calculation can be stored in the concentration calculation means or in another It also has a function of storing in a recording means or the like. Such concentration calculation means may be integrated with the chemiluminescence measurement means or the time measurement means, or may exist independently.

The luminescence intensity and time measurement of the chemiluminescence reaction according to the present invention were measured based on the following basic operation. Unless otherwise specified, commercially available special grade reagents were used for the operations.
(1. Basic operation)
A pH buffer solution (sodium tetraborate (Borax) manufactured by Wako Pure Chemical Industries, Ltd.) was used for the chemiluminescence cell. Borax was dissolved in distilled water, and the pH was adjusted by adding an aqueous sodium hydroxide solution. The same was used below. 100 μl, L-ascorbic acid aqueous solution (manufactured by Wako Pure Chemical Industries) 100 μl, DMSO (manufactured by Wako Pure Chemical Industries) 100 μl, Cu ²⁺ standard solution 120 μl, luminescent substrate (iron phthalocyanine sulfonic acid aqueous solution) : Sodium phthalocyanine sulfonate manufactured by Aldrich was used by dissolving in distilled water, the same applies hereinafter.) 40 μl was injected by a microsyringe and mixed. Set in the cell holder of a chemiluminescence measuring device (LUMICOUNTER NU-600 manufactured by Nisshin Medical Science Equipment Manufacturing Co., Ltd. or GENELIGHT GL-200S manufactured by Microtech Nichion Co., Ltd.), and a hydrogen peroxide aqueous solution (30% solution manufactured by Wako Pure Chemical Industries, Ltd.) (Used after diluting with distilled water.) 40 μl was injected by microsyringe. Thereafter, the chemiluminescence signal that appeared after injection was measured.

(2. Deproteinization)
To a 20 ml sample tube was added 200 μl of bovine serum and 100 μl of hydrochloric acid aqueous solution containing 2ML-ascorbic acid (12N hydrochloric acid manufactured by Kanto Chemical Co. was used after diluting to 1N with distilled water), and the mixture was mixed and allowed to stand for 5 minutes. Next, 100 μl of 1.2 M trichloroacetic acid (manufactured by Kanto Chemical Co., Inc.) was added, centrifuged at 2000 rpm for 5 minutes, and then filtered. The filtrate was adjusted for pH with 0.1M Brax Buffer and used as a sample.

(Influence of L-ascorbic acid in chemiluminescence system)
The influence of L-ascorbic acid was examined using a chemiluminescence reaction system of an iron phthalocyanine sulfonic acid complex (Fe-PTS) and hydrogen peroxide. Under alkaline conditions of hydrogen peroxide concentration of 6.4 × 10 ⁻³ M, Fe-PTS concentration of 4.0 × 10 ⁻⁵ M, DMSO of 20% by volume and pH of 10, Fe-PTS As a result, a sharp chemiluminescence signal was observed immediately thereafter (FIG. 2). However, when L-ascorbic acid (1.6 × 10 ⁻³ M), which is a strong reducing agent against chemiluminescence due to the oxidation reaction, is added, a chemiluminescence signal is observed after a certain period of time after mixing the solution. (FIG. 3). Thus, when the reducing agent was added to the reaction system of the oxidizing agent and the iron porphyrin complex, a certain induction time was generated until the peak detection of chemiluminescence.

  When the wavelength change of the light absorbency of Fe-PTS by the presence or absence of L-ascorbic acid addition was examined, the absorption spectrum immediately after adding hydrogen peroxide accompanying addition of L-ascorbic acid shifted upwards. (Data omitted). Further, when L-ascorbic acid was added, the higher absorbance was maintained over time, and it took longer time to decrease to the same level of absorbance as when L-ascorbic acid was not added (data not shown). Thus, L-ascorbic acid suppressed the oxidative degradation reaction of the chemiluminescent system using Fe-PTS as a substrate.

(Examination of catalyst)
In this chemiluminescent reaction system consisting of a chemiluminescent substrate, an oxidizing agent and a reducing agent, the influence of the catalyst was examined. L-ascorbic acid is 1.6 × 10 ⁻³ M, hydrogen peroxide concentration is 6.4 × 10 ⁻⁴ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, and DMSO is 20% by volume. The effect of each catalyst on the induction time of chemiluminescence was observed. The results are shown in Table 1.
Table 1 shows the concentration having an influence of ± 5% or more as the allowable concentration on the induction time without addition of the catalyst. As shown in Table 1, the examined substances are strong in induction time in L-tyrosine, L-tryptophan as a singlet oxygen scavenger, L-cysteine as a reducing substance, and D-glucose as a reducing sugar. The effect was seen. These substances are considered to have an influence on the induction time by an action such as elimination of active oxygen species.

Among metal ions used as catalysts in chemiluminescence, Cu ²⁺ , Al ³⁺ , Fe ³⁺ , Zn ²⁺ , and Ni ²⁺ showed a strong effect, but Cu ²⁺ showed a significant effect. . When Cu ²⁺ was added, the induction time until chemiluminescence was observed was shortened. Ascorbic acid is oxidized to turn into dehydroascorbic acid, but this oxidation is promoted by the presence of heavy metals such as copper and light irradiation. Therefore, it is presumed that ascorbic acid is oxidized by coexistence of Cu ²⁺ , the influence on Fe-PTS and hydrogen peroxide is reduced, and the induction time is shortened. As described above, the concentration of the substance having an influence on the induction time of the chemiluminescence reaction system can be measured by the present invention.

In the following, this chemiluminescent system is further analyzed using Cu ²⁺ as a catalyst.
When L-ascorbic acid is used as the reducing agent, Fe-PTS is used as the chemiluminescent substrate, and hydrogen peroxide is used as the oxidizing agent, each condition (L-ascorbic acid concentration, hydrogen peroxide concentration, pH, DMSO (solvent) Optimization of volume and Fe-PTS concentration was performed.

(Influence of L-ascorbic acid concentration)
The effect of L-ascorbic acid concentration is shown in FIG. Hydrogen peroxide concentration is 4.8 × 10 ⁻³ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, DMSO is 20% by volume, pH is 10, and copper ion concentration is 4.0 × 10 ⁻⁸ M. As a result of adding the L-ascorbic acid concentration in the range of 0 to 2.0 × 10 ⁻³ M under the conditions of, the induction time was prolonged as the L-ascorbic acid concentration increased. This is probably because the L-ascorbic acid concentration affects Fe-PTS or hydrogen peroxide and the induction time is extended. Since the induction time difference between the blank and the Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M was large, the concentration of L-ascorbic acid was set to 1.6 × 10 ⁻³ M.

(Influence of hydrogen peroxide concentration)
The influence of the hydrogen peroxide concentration is shown in FIG. L-ascorbic acid is 1.6 × 10 ⁻³ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, DMSO is 20% by volume, pH is 10, and copper ion concentration is 4.0 × 10 ⁻⁸ M. As a result of investigating the hydrogen peroxide concentration in the range of 1.2 × 10 ^{−3 to} 7.3 × 10 ⁻³ M under the conditions, the induction time was shortened as the hydrogen peroxide concentration increased. The reason for this is thought to be that the chemiluminescence reaction easily occurs and the induction time is shortened by increasing the hydrogen peroxide concentration relative to the coexisting L-ascorbic acid. Since the induction time difference between the blank and the Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M was large, the concentration of hydrogen peroxide was set to 2.4 × 10 ⁻³ M.

(Effect of pH)
The effect of pH is shown in FIG. L-ascorbic acid is 1.6 × 10 ⁻³ M, hydrogen peroxide concentration is 4.8 × 10 ⁻³ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, DMSO is 20% by volume, copper ion As a result of examination in the range of pH 1 to 12 under the condition of the concentration of 4.0 × 10 ⁻⁸ M, no chemiluminescence was observed in the range of pH 1 to 8. In this range, almost no chemiluminescence is observed even in the Fe-PTS chemiluminescence system to which L-ascorbic acid is not added. On the other hand, in the pH range of 9 to 12, the induction time was shortened as the pH increased. The L-ascorbic acid aqueous solution becomes more reducible as the alkalinity becomes stronger, and is easily oxidized by a hydrogen acceptor such as an oxygen molecule (O ₂ ). Therefore, it is considered that L-ascorbic acid, which had suppressed the chemiluminescence reaction, was oxidized, and the oxidative decomposition reaction of Fe-PTS easily occurred. Since the induction time difference between the blank and the Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M was large, the pH was set to 10.

(Effect of volume of DMSO)
The effect of DMSO volume change is shown in FIG. L-ascorbic acid is 1.6 × 10 ⁻³ M, hydrogen peroxide concentration is 4.8 × 10 ⁻³ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, pH 10 and copper ion concentration is 4. Under the condition of 0 × 10 ⁻⁸ M, the volume of DMSO was changed. As the volume of DMSO increased, the induction time was shortened. The reason for this is that superoxide radicals (• O ₂ ⁻ ) are generated from the reaction of DMSO, O ₂ , and OH ⁻ in an alkaline solution, and the rate of generation of • O ₂ ⁻ increases as the volume of DMSO increases. This is considered to be because the oxidative decomposition of Fe-PTS is likely to occur. Since the induction time difference between the blank and Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M is large, the volume of DMSO was set to 20% by volume.

(Effect of Fe-PTS concentration)
FIG. 8 shows the effect of changing the Fe-PTS concentration. L-ascorbic acid concentration is 1.6 × 10 ⁻³ M, hydrogen peroxide concentration is 4.8 × 10 ⁻³ M, DMSO is 20% by volume, pH is 10, and copper ion concentration is 4.0 × 10 ⁻⁸ M. Under the conditions, the Fe-PTS concentration was examined in the range of 2.0 × 10 ^{−5 to} 6.0 × 10 ⁻⁵ M. As a result, the induction time was shortened as the Fe-PTS concentration was increased. The reason for this is considered to be that the oxidative decomposition of Fe-PTS occurs faster because the influence of L-ascorbic acid, which suppresses the chemiluminescence reaction, becomes smaller as the concentration of Fe-PTS increases. Since the induction time difference between the blank and the Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M was large, the concentration of Fe—PTS was set to 4.0 × 10 ⁻⁵ M.

(Cu ²⁺ calibration curve)
Iron phthalocyanine sulfonic acid concentration is 4.0 × 10 ⁻⁵ M, hydrogen peroxide concentration is 2.4 × 10 ⁻³ M, L-ascorbic acid concentration is 1.6 × 10 ⁻³ M, DMSO is 20% by volume, Under the condition of pH 10, a correlation graph (calibration curve) between the concentration of Cu ²⁺ and the induction time was prepared with LUMICOUNTER NU-600 (FIG. 9). In addition, FIG. 10 shows a calibration curve created with GENELIGHT GL-200S under the same conditions. As a result, in FIG. ^{9 1.2 × 10 -10 ~1.2 × 10} -5 M, a good linearity in the concentration range of 10 at ^{1.2 × 10 -10 ~6.0 × 10 -6} M Obtained. The correlation coefficient of the calibration curve was 0.9871 in FIG. 9 and 0.9153 in FIG. At a Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M, the coefficient of variation after five measurements was 2.02% in FIG. 9 and 2.27% in FIG. Moreover, the detection limit (3σ) was 1.66 × 10 ⁻¹¹ M in FIG. 9 and 9.52 × 10 ⁻¹¹ M in FIG. These luminol _{^{/ OH - / H 2 O 2}} / Cu Other detection limit and is 6 × ^{10 -6} M of the chemiluminescent _{^{system, 1,10-phen / OH - /}} H 2 O 2 / Cu 2+ / hexadecyl ethyl Quantitative determination of Cu ²⁺ using dimethylammonium bromide (HEDAB) chemiluminescence system 2 × 10 ⁻¹⁰ M, H ₂ O ₂ / flavin mononucleotide (FMN) / phosphate buffer solution of Cu ²⁺ using chemiluminescence system It is more sensitive than any of the 2 × 10 ⁻⁸ M quantitative methods.

(Peroxidase calibration curve)
Iron phthalocyanine sulfonic acid concentration is 4.0 × 10 ⁻⁵ M, hydrogen peroxide concentration is 2.4 × 10 ⁻³ M, L-ascorbic acid concentration is 1.6 × 10 ⁻³ M, DMSO is 20% by volume, Under the condition of pH 10, a correlation graph (calibration curve) between the concentration of peroxidase and the induction time was prepared by LUMICOUNTER NU-600 (FIG. 10). As a result, as shown in FIG. 11, a good straight line could be obtained in the peroxidase concentration range of 0.1 ppm to 30 ppm. The correlation coefficient of the calibration curve was 0.947 in FIG. When the peroxidase concentration was 10 ppm, the coefficient of variation when measured 5 times was 1.07%, and the standard deviation was 1.15. The detection limit was 4.30 × 10 ⁻² ppm (43.0 ppb).

(Examination of obstacles)
The effect on induction time when metals, saccharides, amino acids, vitamins and the like coexist with a Cu ²⁺ concentration of 4.0 × 10 ⁻⁸ M was examined. The results are shown in Tables 4 to 7, respectively. The concentration of the interfering substance giving an error of ± 5% with respect to the induction time when no interfering substance was added was compared by the molar ratio with the Cu ²⁺ concentration (4.0 × 10 ⁻⁸ M).

In Table 2, Fe ³⁺ promotes the oxidation of L-ascorbic acid in the same manner as Cu ²⁺ . Therefore, it seems that the induction time was shortened.

  The saccharides used in Table 3 were all reducing sugars, and all of them had a long induction time. This is thought to be because the induction time was prolonged because each sugar reduced hydrogen peroxide to reduce the oxidizing power.

  In Table 4, the induction time became longer with L-cysteine, L-histidine, L-tryptophan and the like. L-cysteine is a reducing substance and is presumed to affect the oxidation reaction with hydrogen peroxide. L-histidine is contained in hemoglobin protein and is bound to heme iron. Also in this chemiluminescence system, it is considered that the coordination activity is coordinated at the axial coordination site of Fe-PTS, the catalytic activity is reduced, and the induction time is prolonged. Since L-tryptophan is used as a singlet oxygen scavenger, it is considered that reactive oxygen species are affected.

In Table 5, thiamine hydrochloride increased the induction time. Like L-ascorbic acid, this has the property of being unstable and easily decomposed under alkaline conditions, and is considered to affect the oxidation reaction with hydrogen peroxide.
From these results, it was speculated that Cu ²⁺ acts selectively because no substance having a particularly large effect was confirmed in this system.

以上より、算出された計算値と原子吸光法により得られた値を表６に示す。その相対誤差は８．７０％であり、ほぼ近似した結果が得られた。 (Quantification of Cu ^{2+ in} bovine serum)
As an application to actual samples, quantification of Cu ^{2+ in} bovine serum was performed. First, bovine serum albumin subjected to protein removal treatment was used as a sample and applied to a chemiluminescence system of Fe-PTS added with L-ascorbic acid, and the induction time until chemiluminescence was observed was measured. In addition, L-ascorbic acid is 1.6 × 10 ⁻³ M, hydrogen peroxide concentration is 2.4 × 10 ⁻³ M, Fe-PTS concentration is 4.0 × 10 ⁻⁵ M, DMSO is 20% by volume, This was performed under the condition of pH10. Next, the measured induction time (139 sec) = Y was substituted into the equation of the calibration curve, and the concentration of Cu ²⁺ was calculated. In addition, the following formula | equation is a thing of the calibration curve of GENELIGHT GL-200S.

From the above, the calculated values and the values obtained by the atomic absorption method are shown in Table 6. The relative error was 8.70%, and an approximate result was obtained.

(Comparison with other methods)
A comparison of this chemiluminescent system with other methods for the determination of Cu ²⁺ is shown in Table 7. This system is a system that can quantify Cu ²⁺ at a low concentration and in a wide concentration range as compared with other methods.

1 Cell for Chemiluminescence 2 Organic Solvent 3 pH Buffer 4 Reducing Agent 5 Sample Solution 6 Luminescent Substrate 7 Oxidizing Agent

Claims (12)

A method for measuring the concentration of a substance to be detected contained in a sample solution,
A step in which chemiluminescence is generated by mixing the sample solution, a luminescent substrate, an oxidizing agent, and a reducing agent in a solvent;
Measuring the time from immediately after the mixing until the emission intensity of the chemiluminescence reaches a peak; and
Calculating the concentration of the substance to be detected from the measured time.   The method according to claim 1, wherein the luminescent substrate is an iron porphyrin complex or hemoglobin.   The method according to claim 2, wherein the iron porphyrin complex is an iron phthalocyanine complex or an iron chlorophyllin complex.   The method according to claim 1, wherein the oxidizing agent is hydrogen peroxide.   The method according to any one of claims 1 to 4, wherein the reducing agent is L-ascorbic acid or L-cysteine.   The substance to be detected is one metal ion selected from the group consisting of copper ions, aluminum ions, iron ions, zinc ions, and nickel ions, L-tyrosine, L-tryptophan, L-cysteine, D-glucose, L -The method according to any one of claims 1 to 5, which is ascorbic acid, hydrogen peroxide, peroxidase, or an enzyme-labeled antibody. A kit for measuring the concentration of a substance to be detected contained in a sample,
The kit includes a luminescent substrate, an oxidizing agent, and a reducing agent,
A kit in which chemiluminescence is generated after a lapse of time according to the concentration of the detected substance when the detected substance, the luminescent substrate, the oxidizing agent, and the reducing agent are mixed in a solvent.   The kit according to claim 7, wherein the luminescent substrate is an iron porphyrin complex or hemoglobin.   The kit according to claim 8, wherein the iron porphyrin complex is an iron phthalocyanine complex or an iron chlorophyllin complex.   The kit according to any one of claims 7 to 9, wherein the oxidizing agent is hydrogen peroxide.   The kit according to any one of claims 7 to 10, wherein the reducing agent is L-ascorbic acid or L-cysteine.   The substance to be detected is one metal ion selected from the group consisting of copper ions, aluminum ions, iron ions, zinc ions, and nickel ions, L-tyrosine, L-tryptophan, L-cysteine, D-glucose, L The kit according to any one of claims 7 to 11, which is ascorbic acid, hydrogen peroxide, peroxidase, or an enzyme-labeled antibody.

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