JPH10253633A - Immunity analyzing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve measurement sensitivity and to prolong the life of a measurement cell by irradiating a mixed body of a metallic complex, an oxidizing agent which oxidizes the metallic complex, and a reducing agent which reduces the metallic complex with light and determining the oxidation-reduction reaction between the optically pumped metallic complex and the oxidizing agent. SOLUTION: A sample liquid of a mixed body of e.g. a metallic complex such as a tris 2 and 2'-bipyridyl-ruthenium(II) complex [Ru(bpy)3 <2+> ] which is optically pumped, an oxidizing agent such as Fe<3+> which oxidizes the metallic complex, and a reducing agent such as triethanolamine [(NOCH3 )3 N] which reduces the metallic complex is injected into a measurement cell and irradiated with light. Ru(bpy)3 <2+> is optically pumped to be Ru(bpy)3 <2+> * and then irreversibly reacts with Fe<3+> at a diffusion-controlled rate of reaction to be Ru(bpy)3 <3+> . Here, this Ru(bpy)3 <3+> is returned to Ru(bpy)3 <2+> by reduction to repeat optical pumping reaction. As the concentration of Fe<2+> is increased by this repetition, it is determined by absorptiometry.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for quantifying a substance to be measured using a metal complex ion which generates photoexcitation as a labeling substance, and more particularly to an immunoassay apparatus for measuring the concentration of a substance to be measured labeled with a metal complex ion. Things.

[0002]

2. Description of the Related Art In the field of immunoassay, absorptiometry, fluorescence, chemiluminescence and electrochemiluminescence have been used as techniques for identifying the presence and concentration of an analyte to be measured. . The fluorescence method deals with light emission by a mechanism other than heat generation, and the electrochemiluminescence method is a method in which electrochemistry is integrated with the chemiluminescence method. In principle, it is said that the sensitivity is high in the order of absorption method, fluorescence method, and (electro) chemiluminescence method.
Chemiluminescence immunoassay, edited by Akio Tsuji and Takeshi Kanno,
See Life Sciences (1992).

[0003] In the chemiluminescence method, a method of measuring luminescence generated by causing hydrogen peroxide (H ₂ O ₂ ) to act on luminol, isoluminol, or the like has long been adopted. The chemiluminescence method has advantages such as high sensitivity and a long dynamic range, but has disadvantages such as rapid decay of luminescence, poor accuracy, unstable reagent, and high background. . This drawback is being solved by increasing the purity of the reagent and improving the accuracy by improving the apparatus, but has not been solved in principle. The main factor is that the lifetime from the excitation of a conventionally used labeling substance such as luminol or isoluminol to the return to the ground state after being excited once is as short as several tens nanoseconds on average.

In the electrochemiluminescence method, light emission can be synchronized by using an electrode reaction, and the accuracy can be improved. This electrochemiluminescence method is used as a binding assay.
By applying the method, low concentrations of hormones, drugs, and other biologically useful substances have been quantified. For example, in binding assays, a sample mixture is formed that contains an unknown amount of the analyte of interest to be determined and a known amount of the agent to which the labeled molecule for electrochemiluminescence is bound. This mixture is incubated to bind the labeled molecule to the analyte of interest. After the incubation, this mixed sample mixture contains the binding components (component B; binding com.
ponent) and an unbound component (F component; free component). The binding component is the labeled agent bound to the analyte and the unbound component is the labeled agent not bound to the analyte.

In recent years, a ruthenium (II) trisbipyridyl complex Ru (b) having a ruthenium divalent ion as a complex center has been developed.
py) ₃ ²⁺ electrochemiluminescence method using the labeling substance have been developed. Ru (bpy) ₃ ²⁺ is, in electrochemiluminescence is a labeled molecule which is currently the only practical use. In electrochemical methods, Ru (bpy) ₃ ²⁺ to excite, Ru (bpy) ₃ to obtain a ^{2 + *.} The Ru (bpy) ₃ ^{2 + *} emits a phosphorescent through the fast intersystem crossing ^{_{(Ru (bpy) 3 2 +}} * → Ru (bpy) 3 2 + h
v). This light emission amount is measured by a photomultiplier tube or the like.

Further in the field of photochemical, came to be used in the above electrochemiluminescence, Ru (bpy) ₃ ²⁺ to have been made many studies with the photosensitive substance. See Chemistry Review 93 (1993) Special Issue on Photochemistry. Ru (b
py) ₃ ²⁺ has the advantages of (1) high excitation efficiency and luminous efficiency, and (2) long average life of the excited state of 0.6 microsecond (inorganic and organometallic photo). Chemistry 168 , 1 (1978)
reference).

[0007] This 0.6 microsecond is sufficiently long compared to the time scale (more than several nanoseconds) of the chemical reaction in the solution, so that it can react with other chemical substances contained in the solution. So Ru (bpy) ₃ ²⁺ and Ru
(bpy) ₃ ^{2 + *} to photoexcitation to, Ru (bpy) ₃ methyl viologen ^{2 + *} to a liquid phase or solid phase (methylviologen, MV
²⁺⁾ is reacted with ^{_{(Ru (bpy) 3 2 +}} * + MV 2+ → Ru
(bpy) ₃ ³⁺ + MV ⁺ ) has been studied to convert light energy into chemical energy (electron transfer) (Journal of American Chemical Society 102 , 5119 (1980)). ), Journal of Physical Chemistry 94 , 874 (1990), Journal of Physical Chemistry 96 , 2879 (19)
92)).

Further, Ru (bpy) ₃ produced as a result of the above reaction is obtained.
When ³⁺ EDTA and (ethylenediaminetetraacetic acid) to act as a reducing agent to return to the Ru (bpy) ₃ ²⁺ are, by continuing the irradiation ^{_{Ru (bpy) 3 2 + *}} + repeating to perform the reaction of MV ²⁺ (Photoinduced Electron Transfer Part A Conceptual Basis, Amsterdam, published by Elsevier (1988), page 409).

Also, a method of separating a bound portion and a non-bound portion between a labeling substance and a substance to be measured by using magnetic beads has been widely used as an immunoassay. By the magnetic bead method, the binding portion between the low-concentration labeling substance and the substance to be measured can be efficiently separated from the non-binding portion.

[0010]

The sample concentration C ₁ (mol / dm ³ ) that can be measured by the fluorescence method is as follows: F is the detectable fluorescence intensity, φ is the fluorescence quantum yield, and I _{0 is} the intensity of the incident light. , The molar absorbance is ε, the cell length is 1 and the ratio of the total amount of fluorescence emitted in all directions to the light receiving portion of the detector is K,
Can be represented by

[0011]

[Image Omitted] C ₁ = F / (φ · I ₀ · ε · l · K) (1) In general, C ₁ is obtained by substituting the detected light amount of the photomultiplier tube into F as 100 photons / second, and φ = 0.5, I ₀ = 1 mW
(= 2 × 10 ¹⁵ photons / sec), ε = 10 ³ , l = 1c
If m and K = 0.01, C ₁ = 10 ⁻¹⁴ mol / dm ³ .
The detection sensitivity can be improved by increasing I ₀ and K from Chemical formula 1.

On the other hand, the sample concentration C, which can be measured by the chemiluminescence method,
₂ (mol / dm ³ ) is the total amount of chemiluminescence, assuming that the detectable chemiluminescence intensity is E, the fluorescence quantum yield is φ, and N ₀ is the Avogadro number, assuming that one photon is generated from one molecule. The ratio of the light entering the light receiving portion of the detector can be represented by K as K.

[0013]

Embedded image C ₂ = E / (φ · N ₀ · K) (2) For general C ₂ , the detected light amount of the photomultiplier tube is substituted for E as 100 photons / second, and φ = 0.01 , K = 0.01
Then, C ₂ = 10 ⁻¹⁸ mol / dm ³ . It can be seen from the chemical formula 2 that the advantage of the chemiluminescent method is that the Avogadro number can be used as N ₀ in the chemical formula 2 by adding a large number of substances that induce chemiluminescence to the solution. However, the value of φ is generally smaller than that of the fluorescence method.

The electrochemiluminescence method can be basically defined by the same formula as in the above formula (2). However, in the electrochemiluminescence method, the accuracy can be improved by synchronizing the light emission. However, in the measurement by the conventional electrochemiluminescence method, an electrode having a flat plate structure is frequently used due to a principle limitation of having an electrode and a demand to keep the potential distribution on the electrode surface uniform. Therefore, it has been difficult to obtain a cylindrical or spherical measurement cell shape that is optimal for increasing the solid angle when measuring the light emission amount.

[0015] In the electrochemiluminescence using Ru (bpy) ₃ ²⁺ and amine derived reducing agent such as tripropylamine, by leaving the excess tripropylamine concentration,
Although the background light emission is kept constant, this tripropylamine itself emits light. This tripropylamine is a substance that triggers light emission in the electrochemiluminescence method and is indispensable, but has been a source of background light emission. In addition, it is difficult to extend the life of the measurement cell due to generation of heat in the solution sample due to application of an electric field and deterioration of the structural material due to a chemical reaction on the electrode surface.

In the present invention, a measurement method having a measurement sensitivity equivalent to or higher than that of the fluorescence method, the chemiluminescence method and the electrochemiluminescence method is developed, and the above three problems of the electrochemiluminescence method are solved. (1) Improve the measurement sensitivity and (2) extend the life of the measurement cell. Further, by simplifying the device configuration, the device can be downsized.

[0017]

The sample concentration C ₃ (mol / dm ³ ) that can be measured by the present measurement method is as follows: G is the Fe ²⁺ concentration that can be detected by an absorptiometry, φ is the fluorescence quantum yield, The intensity of the incident light is I
₀ , irradiation time of incident light is τ, molar absorbance is ε, cell length is 1, and the number of moles of Fe ³⁺ that actually reacts with Ru (bpy) ₃ ²⁺ is N (= aN ₀ , where α Is a coefficient, N ₀ is Avogadro's number), and if the probability that photo-excited Ru (bpy) ₃ ²⁺ reacts with Fe ³⁺ without emitting light is represented by β, it can be expressed by Formula 3.

[0018]

Embedded image C ₃ = G / (φ · I ₀ · τ · ε · l · N · β) (3) G in the case of using an absorptiometry is 1 nmol / l (10 ⁻⁹ m)
ol / l). In this method, (1) the intensity I _{0 of the} incident light and the irradiation time τ can be increased, and (2) the Fe ³⁺
By increasing the concentration of N, N and β can be increased, so that C ₃ can be reduced in principle.

[0019] said Ru (bpy) ₃ ²⁺ is natural light region (4
52 nm), and has a light absorption maximum at a wavelength of Ru.
(Bpy) ₃ ^{2 + *} to become. The Ru (bpy) ₃ ^{2 + *} is susceptible also to both chemical oxidation and reduction. In particular, Fe ³⁺ reacts with Ru (bpy) ₃ ^{2+ *} at a diffusion-determined rate (reaction rate = 2.7 × 10 ⁹ M ⁻¹ s ⁻¹ ) as shown below to become Fe ²⁺ .
The chemical formulas of the reactions are shown below.

[0020]

## STR00004 ## ^{_{Ru (bpy) 3 2+ + hν}} → Ru (bpy) 3 2 + * (4)

[0021]

[Of 5] ^{_{Ru (bpy) 3 2 + *}} + Fe 3+ → Ru (bpy) 3 3+ + Fe 2+ (5) (k = 2.7 × 10 9 M -1 s -1)

[0022]

Embedded image Ru (bpy) ₃ ³⁺ + Fe ²⁺ → Ru (bpy) ₃ ²⁺ + Fe ³⁺ (6) (k〜5 × 10 ⁶ M ⁻¹ s ⁻¹ ) Does not occur)

[0023]

## STR00008 ## ^{_{Ru (bpy) 3 2 + *}} + Fe 2+ → Ru (bpy) 3 + + Fe 3+ (7) (k 2 / k 4 ~10 5) ( of 5 hardly occurs as compared to)

[0024]

Embedded image Ru (bpy) ₃ ³⁺ + Red → Red ⁺ + Ru (bpy) ₃ ²⁺ (8) Red of the above reaction 8 is triethanolamine, triphenylamine, dimethylaniline, tetramethylphenylenediamine, dimethoxy. Refers to reducing agents such as benzene, trimethoxybenzene, and dimethoxynaphthalene.
The reaction rate constants of the above reaction formulas 5 and 6 are based on the journal of the American Chemical Society 9
8 , 6536 (1976). The reaction of Chemical formula 7 is described in Inorganic and Organometallic Photochemistry 168 , 1 (19)
78). By repeating the above-mentioned series of reactions 4, 4, and 8, Fe ³⁺ changes almost irreversibly to Fe ²⁺ . The amount of change is Fe
²⁺ concentration of 1, 10 phenanthroline method, bipyridyl method,
It can be quantified by determining by an absorption spectrophotometry such as the nitroso R salt method. It can also be determined using chelate titration, polarography, etc. (edited by The Chemical Society of Japan, 4th edition Experimental Chemistry Lecture 15 “Analysis”, Maruzen (199)
1)).

On the other hand, the reaction between Fe ³⁺ and Ru (bpy) ₃ ^{2 + *} is greatly affected by the oxygen content of the solution. This is because Ru (bpy) ₃ ^{2 + *} is oxidized by Chemical formula 9.

[0026]

Embedded image Ru (bpy) ₃ ^{2 + *} + O ₂ → Ru (bpy) ₃ ³⁺ + O ₂ ⁻ (k = 3.3 × 10 ⁹ M ⁻¹ s ⁻¹ ) (9) , CT. Lin
See other Journals of the American Chemical Society 98 , 6536 (1976). However, it is possible to sufficiently reduce the influence of oxygen by making the Fe ³⁺ concentration excessive in advance.

When Ru (bpy) ₃ ²⁺ is used as a photocatalyst for the oxidation reaction by peroxodisulphide ion (S ₂ O ₈ ²⁻ ), the conversion of Ru (bpy) ₃ ²⁺ Ru generated by photoexcitation reaction (bpy) ₃ ^{2 + *} acts on S ^₂ O ₈ ^2-, as shown in the following formula 10.

[0028]

Embedded image Ru (bpy) ₃ ^{2 + *} + S ₂ O ₈ ²⁻ → SO ₂ ⁻ + SO ₄ ²⁻ (10) The reaction rate of this reaction may increase in proportion to the absorption light intensity and the irradiation light intensity. Known (see Journal of the Chemical Society Dalton Transaction 355 (1985)). Therefore, it is expected that the reaction 5 with Fe ³⁺ used in the present measurement device is also proportional to the irradiation light intensity.

In the present invention, in order to solve the above-mentioned problem, R
u a (bpy) ₃ ²⁺ photoexcitation metal complexes such as by binding with the target substance as a labeling substance, after the coupling component and non-binding components were isolated by magnetic bead method, the sample liquid containing the analyte by light irradiation the Fe ³⁺ added during Fe ²⁺
Change to As a result, the irreversibly generated Fe ²⁺ is quantified by a spectrophotometric method, thereby quantifying the substance to be measured in the sample liquid.

[0030]

FIG. 1 shows a chemical reaction cycle diagram used in the present measurement. Ru (bpy) ₃ ²⁺ is Ru by photoexcitation
(bpy) ₃ ^{2 + *,} and then Fe ³⁺ at a diffusion-controlled rate
Irreversibly react with a Ru (bpy) ₃ ^3+. This Ru
By reducing (bpy) ₃ ³⁺ with a reducing agent substance, Ru
(Bpy) by returning to ^_32+, repeats the above photoexcitation reaction. By repeating this chemical reaction cycle, Fe
^{Although the 2+} concentration increases, this Fe ²⁺ concentration is quantified by an absorption spectrophotometric method.

FIG. 2 shows a basic structural diagram of the present measuring apparatus. 2 is a light source for absorbance analysis, 2 is a lens, 3 is a filter, and 4 is a shutter. The measurement cell 8 is irradiated with light according to these steps 1 to 4. Electromagnets for capturing magnetic beads 5 and 6 in FIG. 2 perform B / F separation by the magnetic bead method.
Reference numeral 7 denotes a light source for photoexcitation, which is provided in a measuring cell 8 in which a sample solution labeled with a metal complex ion enters, as shown in FIG.
The reaction of e ³⁺ → Fe ²⁺ is advanced. In the case of the 1,10 phenanthroline method, the light source 1 has a wavelength of 510 nm. In FIG. 2, reference numeral 9 denotes a monochromator, 10 denotes a photomultiplier tube, 11 denotes a controller / A / D converter, and 12 denotes a personal computer, which constitute a measurement circuit for analysis.

FIG. 3 shows an example of a measurement procedure for continuous measurement. The substance to be measured is measured cell 8
And discharged out of the measuring cell 8 from the vertical depth direction in the figure. In FIG. 3A, the electromagnet for capturing magnetic beads and the light source 15 for light excitation are switched on, and the reaction of Fe ³⁺ → Fe ²⁺ proceeds in the measurement cell 8. By extending the light irradiation time, Fe ²⁺ is accumulated, and the measurement sensitivity is improved. Since the magnetic beads 13 are distributed along the wall surface, the light source 15 for light excitation can irradiate the magnetic beads 13 uniformly.

In FIG. 3A, since the light absorption analysis is not performed, the light source 14 for the light absorption analysis is switched off, and the shutter 1 extending to the monochromator 9 and the photomultiplier tube 10 is turned off.
6 is also closed.

FIG. 3 (b) shows how the Fe ²⁺ ions generated in FIG. 3 (a) are measured by the absorption spectroscopy. Since a large amount of the generated Fe ²⁺ ions are present in the vicinity of the labeling molecule bound to the magnetic beads 13, the electromagnet 18 for capturing magnetic beads is turned off, and the measuring cell 8 is rotated by 90 ° to obtain the light for the absorption spectrophotometry. F on the optical path of the light source 17
e ²⁺ is distributed, and a spectrophotometric reagent is added,
After performing operations such as agitation, the light source 14 for absorptiometry is turned on, and the spectrum of Fe ²⁺ is output as a signal by the 9 to 10 detection circuits.

FIG. 4 shows a three-dimensional structure diagram of the present measuring apparatus when performing continuous measurement. A measurement circuit for the absorptiometry is arranged before this figure (not shown in this figure). A sample solution 21 labeled with metal complex ions is injected from above the measurement cell 8 and discharged from below. Since biomolecules such as DNA may be damaged by Fe ²⁺ and Fe ³⁺ ions, it is desirable to consider adding Fe ³⁺ ions to the sample solution immediately before light irradiation. .

One embodiment of the present invention is to accurately and reproducibly measure low concentrations of hormones, drugs and other biologically useful substances.

Further, by rapidly oxidizing and quenching the light emitted when the labeling substance such as a metal complex ion excited by the light absorption returns to the ground state, the electric energy is converted into chemical energy and is contained in the solution in advance. The method of measuring the concentration of a target substance labeled with a metal complex ion by changing the concentration of a metal ion, etc., and measuring the change in the concentration of the metal ion by absorptiometry, chelation titration, polarography, etc. Irrespective of the invention. If this label is used in any of the embodiments according to the present invention, other molecules, such as analytes and the like, reactive components capable of binding to the binding partners mentioned above. Are included in the scope of the present invention.

As an embodiment of the present invention, it is an advantageous feature that the light source for photoexcitation is visible light, but this is an electromagnetic wave other than visible light such as infrared light, ultraviolet light, X-ray, and microwave. Are also included in the present invention.

[0039]

The effect of the present invention is that by repeating the reaction cycle shown in FIG. 1 a plurality of times, a trace amount of a labeled substance can be quantified with high sensitivity and high reproducibility. In the present invention, by combining B / F separation by the magnetic bead method incorporated in enzyme / immunoassay with spectrophotometry, hormones, tumor markers,
Trace amounts of biological substances such as blood drugs, blood enzymes, and cytokines can be measured with good reproducibility.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram of a chemical reaction cycle used in the measurement of the present invention.

FIG. 2 is a block diagram showing a basic structure of a measuring apparatus according to one embodiment of the present invention.

FIG. 3 is an explanatory diagram of a measurement procedure in the case of a continuous measurement method according to one embodiment of the present invention.

FIG. 4 is a perspective view of a main part of a measuring device in the case of continuous measurement according to one embodiment of the present invention.

[Explanation of symbols]

DESCRIPTION OF SYMBOLS 1 ... Light source for absorption spectroscopy, 2 ... Lens, 3 ... Shutter, 4 ... Filter, 5 ... Electromagnet for capturing magnetic beads (shown from the vertical direction), 6 ... Electromagnet for capturing magnetic beads (shown from the horizontal direction), 7 ... Light source for photoexcitation, 8 ... measurement cell, 9 ... monochromator, 10 ... photomultiplier tube, 11 ... controller / A / D converter, 12 ... personal computer,
13 ... magnetic beads, 14 ... light source for absorption spectroscopy, 15 ...
Light excitation light source and electromagnet for capturing magnetic beads, 16 shutter, 20 colored Fe ²⁺ light, 21 sample liquid to be measured.

Claims (5)

[Claims] A metal complex, an oxidizing agent for oxidizing a metal complex, and a metal complex comprising at least one light source, a metal complex for photoexcitation, an oxidizing agent for oxidizing the metal complex, and a reducing agent for reducing the metal complex. A mixture of a reducing agent that reduces the amount of light from the light source and a means for quantifying an oxidation-reduction reaction between the photoexcited metal complex and the oxidizing agent. 2. The photo-excited metal complex according to claim 1 is a tris 2,2'-bipyridyl-ruthenium (II) complex (Ru
(Bpy) ₃ ²⁺ ) or a complex such as Ru, Os, Eu, etc., and a metal ion such as Fe ³⁺ is used as an oxidizing agent for oxidizing the metal complex, and triethanol is used as a reducing agent for reducing the metal complex. An immunoanalyzer using a reducing agent such as amine, triphenylamine, dimethylaniline, tetramethylphenylenediamine, dimethoxybenzene, trimethoxybenzene, dimethoxynaphthalene and the like. 3. An immunoassay apparatus for measuring the concentration of an antigenic substance by binding a molecule having an antigen recognizing ability to the metal complex to be photoexcited according to claim 1 and measuring a change in the concentration of the oxidizing agent according to claim 2. 4. The immunoassay apparatus according to claim 3, wherein the concentration of the antigen substance is measured using a solid phase method such as a sandwich method and a magnetic bead method in the immunoassay. 5. The means for quantifying an oxidation-reduction reaction according to claim 1, wherein the n-valent ions such as Fe ³⁺ previously contained in the solution are converted into (n-1) -valent ions such as Fe ^2+. An immunoassay device characterized in that it is means for measuring the concentration of (n-1) -valent ions such as Fe2 ⁺ resulting from the reduction by an absorption spectrophotometric method or an atomic absorption method.