JPH08166349A - Method for determining fluorescent molecule - Google Patents

Abstract

PURPOSE: To simply determine fluorescent molecules with high sensitivity by measuring the emission from the fluorescent molecules which is caused by the movement of energy from a fluorescent association body bonded with the fluorescent molecules. CONSTITUTION: In coexistence of a fluprescent associate containing fluorescent molecules of a fluorescence quantum yield of desirably not smaller than 0.001, more preferably not smaller than 0.01 to achieve highly sensitive measurement and, a stilbene group, a naphthalane group and the like that can be bonded with the fluorescent molecules to be analyzed, an absorbable light is cast to the associate and the emission from the fluorescent molecules caused by the movement of energy from the associate is measured, thereby to determine the amount of fluorescent molecules. According to this method, the fluorescent molecules to be analyzed are excited by the movement of energy from the associate, and therefore the fluorescence of fluorescent coloring matter is remarkably enhanced. The fluorescent molecules can accordingly be determined with high sensitivity.

Description

Detailed Description of the Invention

[0001]

FIELD OF THE INVENTION The present invention relates to a method for quantifying fluorescent molecules in a solution. Specifically, in a quantification method for quantifying a fluorescent molecule from the emission intensity from the fluorescent molecule, by allowing a fluorescent associate capable of binding to the fluorescent molecule to coexist and irradiating the fluorescent associate with light, It is a method for quantifying a fluorescent molecule that enables more sensitive quantification of a fluorescent molecule.

[0002]

2. Description of the Related Art Clinical examination is one of the most important fields in which it is necessary to quantify a specific substance. Modern clinical medicine is supported by three pillars: examination, examination, and treatment. Among other things, clinical tests are the most objective of the data that can be obtained, and therefore provide other scientific evidence and guide them. Among clinical tests, biochemical tests for electrolytes, enzymes, etc., and immunoserum tests for antigens, antibodies, etc. are one field of analytical chemistry, and with recent advances in analytical chemistry, quantification of trace amounts of physiologically active substances in biological fluids has become possible. It is becoming possible. In addition, the function of physiologically active substances at the molecular level has been elucidated by the development of molecular biology, and the quantification of a minute amount of physiologically active substances is making possible the diagnosis and treatment of diseases that were impossible in the past.

In the analysis of physiologically active substances, there are rarely those which can be directly analyzed without requiring special separation such as the determination of metal ions by the atomic absorption method. Generally, it is necessary to separate the target substance by some means and then quantify it by an appropriate means. Many physiologically active substances exhibit their functions by being recognized in the living body. That is, molecules that naturally recognize a substance exist in the living body, and by utilizing this, the target component can be specifically measured. As a recognition molecule for a substance derived from a living body, a substance involved in an immune reaction, that is, an antigen, an antibody or the like is used in an analysis for actual clinical examination.

Regarding the acquisition of antigens, antibodies and the like, useful means are being established due to recent advances in gene manipulation techniques. That is, it is possible to easily produce a monoclonal antibody by genetic manipulation, in place of the naturally used polyclonal antibody derived from a living body which has been conventionally used. By using the antibody, a specific substance to be analyzed can be bound to the antibody. In clinical tests, it is common practice to label and quantify the conjugates of these analytes and antibodies. Although various labeling methods have been put into practical use, a radioimmunoassay method (hereinafter abbreviated as RIA method) using a radioisotope has been used as a method for highly sensitive analysis. However, since the RIA method requires special equipment that uses radiation, in recent years, a labeling method using a dye has been in widespread use as an alternative immunoassay method.

The labeling with a dye is characterized by requiring no special facility and being easy to handle.
However, in general, the dye method is problematic in that it has lower sensitivity than the RIA method, and under the present circumstances, development of a dye method having high sensitivity is earnestly desired.

As a method for measuring the dye concentration, an absorbance method,
The three main types are the fluorescence method and the light emission method, and dyes that can be used in each measurement method are used. For the absorbance method, the sensitivity depends on the molar absorption coefficient of the dye,
There is a problem that the detection limit is relatively low. The luminescence method has a feature that the background is low and the sensitivity is good because no special light source is used, but usable dyes are limited, and there is a problem that the dye is unstable. The fluorescence method is widely used as a measurement method because it has better sensitivity than the absorbance method, but in terms of sensitivity, R
There is a problem that it does not reach the IA method.

For the purpose of improving the sensitivity of the fluorescence method, a method for measuring anisotropy of fluorescence and a method for measuring delayed fluorescence using a specific metal complex have been reported, but the sensitivity is still insufficient. . Sigmund et al. Reported that two types of dyes are used to improve the sensitivity by causing energy transfer (Shin Solid Films, 19
1992, 210/211, 480 pages [Thin Solid
Films, 210/211 (1992), 480]). However, the sensitivity is about three times that in the case where energy transfer is not used, and further improvement in sensitivity has been desired.

[0008]

Therefore, it has been desired to develop a method for easily and highly sensitively measuring a conjugate of an analyte and a recognition molecule labeled with a fluorescent dye.

[0009]

[Means for Solving the Problems] The present inventors have conducted extensive studies to develop a method for quantifying a fluorescent molecule that can solve the above problems. As a result, they have found that the coexistence of an aggregate in which specific dye molecules are oriented significantly enhances the fluorescence of the fluorescent dye to be analyzed and improves the detection sensitivity, thus completing the present invention.

That is, the present invention irradiates the fluorescent aggregate with light absorbable in the coexistence of the fluorescent molecule and the fluorescent aggregate capable of binding to the fluorescent molecule, and the energy from the fluorescent aggregate is irradiated. A method for quantifying a fluorescent molecule, which comprises determining the amount of the fluorescent molecule by measuring the light emission from the fluorescent molecule due to migration.

The detection sensitivity used in the present invention means the sum of the detection lower limit indicating the minimum detectable concentration and the analysis sensitivity indicating the ratio of signal intensity and concentration during analysis.

In the method for quantifying a fluorescent molecule of the present invention,
As the target fluorescent molecule, any known molecule can be used without particular limitation as long as it has fluorescence. Since the sensitivity in quantifying a fluorescent molecule is greatly influenced by the fluorescence quantum yield of the fluorescent molecule used, the fluorescence quantum yield is preferably 0.001 or more, and 0.01 for more sensitive measurement. A fluorescent molecule having the above fluorescence quantum yield is desirable.

The maximum absorption wavelength of the fluorescent molecule depends on the fluorescent aggregate used, but is 250 nm or more and 800 nm or more.
Molecules having the following absorption maxima are desirable. If the maximum absorption wavelength is 250 nm or less, sufficient sensitivity may not be obtained. If the thickness is 800 nm or more, a special measuring device is required.

The maximum emission wavelength of the fluorescent molecule is 30
It is preferably 0 nm or more and 900 nm or less. When the absorption maximum wavelength is 300 nm or less, sufficient sensitivity is often not obtained. Further, if it is 900 nm or more, a special device is required for measuring the intensity. Maximum emission wavelength is 400n
When it is m or more, inexpensive glass, polystyrene, or the like can be used as the material of the cell for measurement, which is particularly preferable.

The quantification method of the present invention is particularly suitable for quantification of fluorescent molecules in an aqueous solution, and is therefore suitable for quantification of biological components. Two types of fluorescent molecules derived from biological components that are particularly preferably used include the following.

(1) A physiologically active substance or a biogenic substance having fluorescence.

(2) Physiologically active substance or biologically-derived substance labeled with a fluorescent dye (1) In the case of a physiologically active substance or biologically-derived substance having fluorescence (hereinafter abbreviated as fluorescent biomolecule), the molecule itself Since it has fluorescence, complicated operations such as pretreatment of the sample are unnecessary, and thus the quantification method of the present invention can be particularly preferably used. Examples of fluorescent biomolecules that are generally suitably used include reduced nicotinamide adenine dinucleotide, reduced nicotinamide adenine dinucleotide phosphate, vitamin B2 (riboflavin), chlorophyll and folic acid.

The physiologically active substance or bio-derived substance (hereinafter abbreviated as a fluorescence-labeled biomolecule) labeled with a fluorescent dye of (2) is a fluorescent dye having an appropriate reactive group for a biological component to be measured ( Hereinafter, it is obtained by labeling with a fluorescent labeling reagent). In the fluorescent molecule quantification method of the present invention, a fluorescently labeled biomolecule is also a suitable fluorescent molecule.

As the above-mentioned fluorescent labeling reagent, known ones can be used, and amino group-reactive fluorescent labeling reagent, carboxyl group-reactive fluorescent labeling reagent, aldehyde group-reactive fluorescent labeling reagent, hydroxyl group-reactive reagent A fluorescent labeling reagent or a thiol group-reactive fluorescent labeling reagent is preferably used. Since the amino group, the carboxyl group, the aldehyde group, the hydroxyl group and the thiol group are generally present in the physiologically active substance or the biological molecule, these labeling reagents are particularly preferably used.

As the amino group-reactive fluorescent labeling reagent,
Fluorescein-4-isothiocyanate, 3-chlorocarbonyl-6,7-dimethoxy-1-methyl-2 (1
H) -quinoxalinone, fluorescein-4-isothiocyanate, 4-chloro-7-nitrobenzofurazan,
4-Fluoro-7-nitrobenzofurazone, sulforudamin 101 acid chloride, fluorescamine, o-phthalaldehyde, dansyl chloride and the like are preferably used.

As the carboxyl group-reactive fluorescent labeling reagent, 3-bromomethyl-6,7-dimethoxy-1-methyl-1,2-dihydroquinoxalin-2-one, 4-
Brumomethyl-7-methoxycoumarin, 1,2-diamino-4,5-methylenedioxybenzene dihydrochloride and the like are preferably used.

Aldehyde group-reactive fluorescent labeling reagents include 1,2-diamino-4,5-dimethoxybenzene.
Dihydrochloride, 2,2'-dithiobis (1-aminonaphthalene), 4-amino-3-penten-2-one and the like are preferably used.

As the hydroxyl-reactive fluorescent labeling reagent, 3
-Chlorocarbonyl-6,7-dimethoxy-1-methyl-2 (1H) -quinoxalinone, 2- (5-chlorocarbonyl-2-oxazolyl) -5,6-methylenedioxybenzofuran and the like are preferably used.

As the thiol group-reactive fluorescent labeling reagent, 4-fluoro-7-sulfamoylbenzofurazan, N- [4- (6-dimethylamino-2-benzofuranyl) phenyl] maleimide, 2,2 '-Dihydroxy-6,6'-dinaphthyl disulfide, N- [4-
(5,6-Methylenedioxy-2-benzofuranyl) phenyl] maleimide, N- (9-acridinyl) maleimide, 4-chloro-7-sulfobenzofurazan ammonium salt, 4-fluoro-7-sulfobenzo Frazan ammonium salt, dansyl aziridine, 5- (iodoacetamidoethyl) aminonaphthalene-1-sulfonic acid, 5-iodoacetamidofluorescein, N-
(1-anilinonaphthyl-4) maleimide, N- (3-
Pyrene) maleimide, eosin-5-iodoacetamide and the like are preferably used.

As the physiologically active substance or biological component labeled with the above-mentioned fluorescent labeling reagent, biological components generally quantified in the field are used without any limitation. Examples of suitable biological components used in the quantification method of the present invention are as follows. That is, non-immunity protein such as albumin, IgG, Ig
A, IgM, IgD, IgE and other immunoglobulins, lectins, receptors, avidin and other non-immunospecific binding partners, glutamate oxaloacetate transaminase,
Examples include enzymes such as glutamate pyruvate transaminase, lactate dehydrogenase, γ-glutamyl transferase, and cholinesterase, human chorionic gonadotropin, hormones such as thyroid stimulating hormone, and nucleic acids such as DNA and RNA.

The method for producing the fluorescently labeled biomolecule is as follows:
Although methods known in the art can be used without particular limitation, specific examples of methods generally suitably used are as follows. That is, a physiologically active substance or a biologically-derived component is dissolved in a buffer such as Tris buffer,
A fluorescent labeling reagent is added there. At this time, pyridine,
The yield is improved by the presence of a small amount of a catalyst such as dimethylformamide. After reacting for several hours at room temperature, the reaction solution is purified several times by gel chromatography to obtain the target fluorescently labeled biomolecule.

The quantification method of the present invention comprises irradiating a fluorescent aggregate with light which can be absorbed in the presence of a fluorescent aggregate capable of binding to a target fluorescent molecule to transfer energy from the fluorescent aggregate. It is characterized in that the amount of fluorescent molecule is determined by measuring the emission from the attributed fluorescent molecule. Here, the fluorescent associate is an associate composed of an amphipathic compound having fluorescence (hereinafter abbreviated as a fluorescent amphipathic compound), and is an essential component for highly sensitive quantification.

The associate is a relatively regular molecular aggregate formed in a solvent by relatively weak bonds such as hydrogen bond, charge transfer bond and hydrophobic bond acting between the same molecules. Further, in the present invention, the binding of the fluorescent molecule and the fluorescent association means that the fluorescent molecule and the fluorescent association are close to each other by the interaction so that the energy transfer is possible. As the interaction here, an interaction occurring between molecules known in the art is employed without particular limitation. As an example of a particularly preferable interaction,
Coulomb force interaction, hydrophobic interaction, hydrogen bonding interaction, coordination bonding interaction, π electron / CH interaction and the like.

The average molecular weight (weight average) of the fluorescent aggregate used in the present invention is preferably in the range of 10,000 to 100 million. If the average molecular weight of the fluorescent aggregate is 10,000 or less, the sensitivity during quantification becomes insufficient. If it is 100 million or more,
It is not preferable because the stability of the aggregate in the solvent becomes poor. Further, more preferably, the average molecular weight of the fluorescent aggregate is 50,000 to 10 million, because the sensitivity at the time of quantification is remarkably good and the stability of the solution is good.

As the solvent for forming the fluorescent aggregate,
The solvent is not particularly limited as long as it is a solvent capable of forming an aggregate composed of an amphipathic compound, but generally water that easily forms a stable aggregate is preferably used.

The above-mentioned fluorescent amphipathic compound is not particularly limited as long as it has a property of forming an associate in a solvent, and known compounds can be used, but a rigid portion having fluorescence is linked. An organic compound having at least one linear hydrophobic group contained therein and having a hydrophilic group (hereinafter, abbreviated as a fluorescent linear amphipathic compound) is preferably used. By using the fluorescent linear amphipathic compound,
The sensitivity at the time of quantification is significantly improved.

In the present invention, the rigid portion means, for example, the following groups (a), (b) and (c).

(A) Divalent group composed of at least two aromatic rings connected directly or through carbon-carbon multiple bonds. Specific examples of such a group include, for example,

[0034]

Embedded image

And divalent groups such as

(B) A divalent group in which two aromatic rings have a plurality of bonds or a single bond between a plurality of atoms and the bonds are energetically bound to each other. If you show it like this, for example,

[0037]

Embedded image

And divalent groups such as

(C) A divalent group in which aromatic rings form a condensed ring, and when the condensed rings are laminated between multiple molecules, their rotations are sterically constrained to each other. Specific examples of such groups include, for example,

[0040]

Embedded image

And divalent groups such as

In the present invention, the linear hydrophobic group is a linear alkyl group or a halogen-substituted product thereof. The number of carbon atoms of the fluorescent linear amphipathic compound having a linear hydrophobic group containing a rigid portion in the chain is 8 to 8 excluding the carbon number of the rigid portion.
A range of 30 is desirable. When it is 8 or less, an aggregate may not be formed in the solvent, and when it is 30 or more, it may not be dispersed in the solvent, which is not desirable. Generally, a carbon-carbon bond, an ester bond, or an ether bond is suitable for the binding part between the rigid part and the linear hydrophobic group.

A linear hydrophobic group containing a rigid portion in the chain is
It is most preferable that one is contained in the fluorescent linear amphipathic compound in consideration of the ability to form an aggregate in a solvent. Two
When one or more are contained, an aggregate may not be formed in the solvent.

The hydrophilic group in the fluorescent linear amphipathic compound of the present invention is essential for the fluorescent linear amphipathic compound to form a stable aggregate in a solvent. As the hydrophilic group, a hydrophilic group generally known as a hydrophilic group of a surfactant can be used, but by using an ionic group or a polyethylene glycol group, the stability of the aggregate is remarkably improved. desirable.

Here, the ionic group is defined as a general term for acidic groups or basic groups. Here, acidic or basic means a Bronsted acid or a Bronsted base. The acidic group is generally a sulfonic acid group, a carboxyl group, a phosphoric acid group and a salt thereof, and the basic group is generally an amino group. A group, a substituted amino group, a quaternary ammonium group, a phosphonium group, and a salt thereof are preferably used.

The polyethylene glycol group preferably has a repeating ethylene glycol unit in the range of 6 to 30. When it is less than 6, an aggregate may not be formed in the solvent. On the other hand, if the amount is more than 30, the sensitivity during quantification may not be sufficiently improved. Here, the number of hydrophilic groups contained in the fluorescent linear amphipathic compound of the present invention is preferably 1 or 2 from the viewpoint of the stability of the obtained fluorescent aggregate.

The fluorescent linear amphipathic compound of the present invention is not particularly limited as long as it satisfies the above, and known compounds can be used. Typical examples that are generally preferably used are specifically shown below.

[0048]

[Chemical 4]

{However, in the structural formulas (a) to (e),
n is an integer of 6 to 12, m is an integer of 2 to 12, and Z is

[0050]

Embedded image

(However, X is an atomic group forming a halogen ion or a stable anion, M is an alkali metal, hydrogen,
Or represents an atomic group forming a stable cation, and l is 6
Indicates an integer of -30. ) Indicates either. In the above preferred fluorescent linear amphipathic compound, the alkali metal represented by M is specifically lithium, sodium or potassium, and the atomic group forming a stable cation is ammonium ion or tetra. It is a methyl ammonium ion. The halogen ion represented by X is specifically fluorine, chlorine, bromine or iodine, and the atomic groups forming a stable anion are nitrate ion, phosphate ion, sulfate ion and carbonate ion.

The method for producing the fluorescent aggregate of the present invention is not particularly limited as long as it is a generally known method for producing an aggregate, and any method may be used. In general, a method in which an appropriate amount of a fluorescent linear amphipathic compound is placed in a buffer solution such as morpholinopropanesulfonic acid and ultrasonic irradiation is used is suitably used.

The formation of the fluorescent aggregates includes the increase of the light scattering of the solution, the broadening of the peak in the measurement of proton NMR of the solution, or the direct observation of the aggregates by the electron microscope of the cast film of the solution stained with uranyl acetate. It can be confirmed by observation. In addition, the molecular weight of the aggregate is low-angle light scattering analysis,
It is measured by gel chromatography or the like.

As the solvent for forming the fluorescent aggregate,
Although a solvent known in the art can be used, water is preferable in consideration of the stability of the fluorescent associate and the detection sensitivity at the time of quantification. In addition, since the influence of pH change can be ignored by using an aqueous buffer solution, an aqueous buffer solution is particularly preferably used.

The buffer component in the buffer solution is not particularly limited, but phosphoric acid, trishydroxyaminomethane, acetic acid, citric acid, triethanolamine, boric acid, maleic acid, carbonic acid, N- (2-acetamido)- Two
-Aminoethanesulfonic acid, N- (2-acetamide)
Iminodiacetic acid, N, N-bis (2-hydroxyethyl)
2-Aminoethanesulfonic acid, N, N-bis (2-hydroxyethyl) glycine, bis (2-hydroxyethyl) iminotris (hydroxymethyl) methane, N-cyclohexyl-3-aminopropanesulfonic acid, N-cyclohexyl- 2-hydroxy-3-aminopropanesulfonic acid, N-cyclohexyl-2-aminoethanesulfonic acid, 3- [N, N-bis (2-hydroxyethyl)
Amino] -2-hydroxypropanesulfonic acid, 3-
[4- (2-hydroxyethyl) -1-piperazinyl]
Propanesulfonic acid, 2- [4- (2-hydroxyethyl) -1-piperazinyl] ethanesulfonic acid, 2-hydroxy-3- [4- (2-hydroxyethyl) -1-piperazinyl] propanesulfonic acid, 2- Morpholinoethanesulfonic acid, 3-morpholinopropanesulfonic acid, 2-hydroxyethyl-3-morpholinopropanesulfonic acid, piperazine-1,4-bis (2-ethanesulfonic acid), piperazine-1,4-bis ( 2-hydroxy-3-propanesulfonic acid), N-tris (hydroxy) methyl-3-aminopropanesulfonic acid, 2-hydroxy-N-tris (hydroxymethyl) methyl-3-
Aminopropanesulfonic acid, N-tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid, N- [tris (hydroxymethyl) methyl] glycine and the like are preferably used.

The concentration of the fluorescent aggregate is required to be higher than the critical aggregate-forming concentration of the amphipathic compound for investigating fluorescence used in order to quantify with high sensitivity. The critical aggregate formation concentration is generally in the range of 10 ⁻⁶ to 10 ⁻³ ,
It is desirable to use above this concentration. It is desirable that the concentration of the aggregate is such that the insoluble matter of the fluorescent linear amphipathic compound does not exist in the solvent, that is, the concentration is equal to or lower than the saturation concentration for the solvent used.

In the quantification method of the present invention, the fluorescent aggregate is irradiated with light which can be absorbed in the presence of the fluorescent molecule and the fluorescent aggregate capable of binding to the fluorescent molecule. The amount of fluorescent molecule is determined by measuring the emission from the fluorescent molecule due to the energy transfer of the fluorescent molecule. In a known fluorescence analysis method, light that can be absorbed by the fluorescent molecule to be analyzed is irradiated, and the light emission from the fluorescent molecule is measured. However, the method of the present invention is characterized in that the fluorescent aggregate is irradiated with light that can be absorbed.

In the present invention, the binding of the fluorescent molecule and the fluorescent association means that the fluorescent molecule and the fluorescent association come close to the energy transferable distance by the above-mentioned interaction. The bond between the fluorescent molecule and the fluorescent aggregate is generally formed spontaneously by mixing each in a suitable solvent. Water is preferably used as the solvent in consideration of the stability of the aggregate.

In the present invention, the following is a concrete example of a generally suitable quantification method. That is, (A) (1) Several kinds of solutions of fluorescent molecules to be measured having known concentrations are prepared in a fluorescent cell.

(2) A certain amount of the fluorescent association solution is added to the above solution.

(3) Stir the solution.

(4) Fluorescence intensity is measured with the light having the absorption maximum wavelength of the fluorescent aggregate as the excitation wavelength and the emission maximum wavelength of the fluorescent molecule as the emission wavelength.

(5) A calibration curve is prepared from the above results.

(6) The fluorescence intensity is obtained by performing the same operation on the unknown concentration sample as for the known concentration solution.

(7) The fluorescence intensity is extrapolated to the calibration curve to obtain the concentration of the fluorescent molecule of unknown concentration.

(B) (1) Several kinds of solids to which a known amount of fluorescent molecules to be measured are adsorbed are prepared.

(2) The above solid is immersed in a solution of the fluorescent association material in the fluorescent cell.

(3) Stir the solution.

(4) The fluorescence intensity is measured with the light having the absorption maximum wavelength of the fluorescent aggregate as the excitation wavelength and the emission maximum wavelength of the fluorescent molecule as the emission wavelength.

(5) A calibration curve is prepared from the above results.

(6) The fluorescence intensity is obtained by performing the same operation on the unknown concentration sample as for the known concentration solution.

(7) The fluorescence intensity is extrapolated to the calibration curve to obtain the concentration of the fluorescent molecule of unknown concentration.

By the above operation, the fluorescent molecule can be quantified, and the detection sensitivity thereof is improved by 10 to 100 times as compared with the case where the fluorescent aggregate is not present.

Although the mechanism for improving the detection sensitivity is not clear, since the fluorescent chromophores are densely oriented in the aggregate of the present invention, the excitation generated by irradiating the fluorescent aggregate with light. It is known that a child can freely move between molecules forming an aggregate. As a result, it is considered that efficient energy transfer from the fluorescent aggregate to the fluorescent molecule is involved in the sensitivity improvement.

The solid to which the fluorescent molecule to be measured described in the above-mentioned suitable quantification method (B) is adsorbed is glass beads, glass plate, quartz plate, magnetic particles, porous cellulose film, porous polycarbonate film, polystyrene. A film or the like is used.

In the quantification method of the present invention, the combination of the fluorescent molecule and the fluorescent associate is not particularly limited as long as the energy transfer from the fluorescent associate to the fluorescent molecule occurs. It is generally known that the donor dye emission spectrum and the acceptor dye absorption spectrum need to overlap each other for energy transfer between two types of fluorescent dyes. It is essential for highly sensitive measurement that the emission spectrum and the absorption spectrum of the fluorescent molecule overlap. The presence or absence of overlapping of the spectra is confirmed by measuring the fluorescence spectrum of the fluorescent association and the absorption spectrum of the fluorescent molecule.

In the above combination, the difference between the emission maximum wavelength of the fluorescent molecule and the absorption maximum wavelength of the fluorescent aggregate is 100 n.
When it is at least m, the influence of scattered light of excitation light is reduced and the sensitivity is improved, which is preferable. Further, if there is a difference of 200 nm or more, the influence of scattered light becomes smaller, which is preferable.

A commercially available fluorescence spectrophotometer is simply adopted for irradiating the fluorescent aggregate with light and measuring the amount of light emission (intensity) from the fluorescent molecule.

In the quantification method of the present invention, it is not particularly limited as long as it is a combination of the above-mentioned fluorescent molecule and a fluorescent association, but a particularly preferable combination is as follows.

That is, the following compounds can be mentioned as the fluorescent linear amphipathic compound.

[0081]

[Chemical 6]

As the fluorescent molecule, when a fluorescently labeled biomolecule obtained by reacting a physiologically active substance or a biologically-derived substance with fluorescein-4-isothiocyanate is used, a particularly highly sensitive determination of the fluorescently labeled biomolecule can be performed. It will be possible.

[0083]

INDUSTRIAL APPLICABILITY Since the fluorescent molecule quantification method of the present invention uses a fluorescent association capable of energy transfer with the fluorescent molecule, it is possible to quantify the fluorescent molecule with extremely high sensitivity. . Furthermore, since a physiologically active substance labeled with a fluorescent dye or a biomolecule can be used as the fluorescent molecule, it can be used for high-sensitivity measurement required particularly in the field of clinical examination. From the above points, the industrial value of the fluorescent molecule quantification method of the present invention is extremely large.

[0084]

EXAMPLES Examples will be given below to more specifically describe the present invention, but the present invention is not limited to these examples.

The molecular weight of the aggregate obtained by the low-angle light scattering analysis in this example is the weight average molecular weight in terms of polystyrene sulfonic acid.

Example 1 The following compound

[0087]

[Chemical 7]

50 mg was placed in 10 ml of 10 mM 3-morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS) buffer solution (the pH of which was adjusted to 7.2 by adding an aqueous sodium hydroxide solution), and ultrasonic irradiation was carried out. A uniform and transparent dispersion was prepared by a machine. The following fluorescent molecules were quantified using the prepared fluorescent association solution.

[0089]

Embedded image

The absorption maximum wavelength of the fluorescent molecule is 491n.
m, the maximum emission wavelength was 525 nm, and the fluorescence quantum yield was 0.75.

1 pico mol / l of the fluorescent molecule (hereinafter referred to as pM
Abbreviated to 100 nanomol / l (hereinafter abbreviated as nM) at a known concentration (MOPS buffer, pH 7.
2) was produced. First, 1 ml of the fluorescent dye solution is placed in a fluorescent cell, and the fluorescence wavelength is set at 490 nm with a fluorescence spectrophotometer.
The emission intensity at 25 nm was measured. Subsequently, the fluorescent aggregate was added so as to be 50 μmol / l (hereinafter abbreviated as μM), and the emission intensity at 525 nm was measured at an excitation wavelength of 280 nm.

At this time, proton NMR of the obtained solution
It was shown that the peak of 6.7 to 8.7 ppm (derived from the proton of the aromatic ring) observed in the deuterated chloroform solution of the above compound is broadened and forms an aggregate. Furthermore, when the molecular weight of the aggregate was measured by low-angle light scattering analysis of this solution, it was 5 × 10 ⁶ daltons.

With respect to the buffer solution containing no fluorescent dye, the emission intensity at 525 nm at an excitation wavelength of 490 nm was measured 10 times, and the average value of fluorescence intensity at a concentration of 0 (hereinafter abbreviated as Y) and standard deviation (hereinafter abbreviated as σ1). To do). Similarly, the emission intensity at 525 nm at an excitation wavelength of 280 nm was measured for a buffer solution containing a fluorescent associate and not containing a fluorescent molecule, and Y and σ of the fluorescence intensity at a concentration of 0 were determined. The detection limit in this method was determined as the most dilute solution concentration at which the emission intensity was Y + 2σ or more. Further, the fluorescence intensity at each concentration above the detection limit was plotted against the concentration, the slope was determined by linear regression of the first order, and this was determined as the analytical sensitivity in this method.

Table 1 summarizes the detection limits and analytical sensitivities of fluorescent molecules in the presence and absence of fluorescent aggregates.

[0095]

[Table 1]

As can be seen from Table 1, both the detection limit and the analytical sensitivity are improved by 50 times or more by using the quantification method of the present invention, and the usefulness of the quantification method of the present invention is clear.

Example 2 Detection sensitivity and analytical sensitivity were determined in the same manner as in Example 1 using the fluorescent molecules (quantification target) shown in Table 2. The results are summarized in Table 2.

[0098]

[Table 2]

Example 3 The following compound

[0100]

[Chemical 9]

50 mg was put into 10 ml of 10 mM MOPS buffer solution (the pH of which was adjusted to 7.2 by adding an aqueous sodium hydroxide solution), and a uniform and transparent dispersion liquid was prepared by an ultrasonic wave irradiator.

1 g of human serum albumin was added to 100 ml of M
It was dissolved in an OPS buffer (pH 7.3), and an acetone solution of 1 g of fluorescein-4-isothiocyanate (hereinafter abbreviated as FITC) was added dropwise thereto and reacted at room temperature for 12 hours. After the reaction, high molecular weight components were collected by gel filtration to obtain FITC-labeled human serum albumin.

The absorption maximum wavelength of the obtained fluorescence-labeled biomolecule was 500 nm, the emission maximum wavelength was 520 nm, and the fluorescence quantum yield was 0.31.

FITC-labeled human serum albumin from 1 pico mol / l (hereinafter abbreviated as pM) to 100 nanomo
1 / l (hereinafter abbreviated as nM) solution of known concentration (MO
PS buffer, pH 7.2) was prepared. First, 1 ml of the fluorescent dye solution was placed in a fluorescent cell, and the emission intensity at 520 nm was measured at an excitation wavelength of 490 nm with a fluorescence spectrophotometer.
Subsequently, the fluorescent aggregate was added so as to be 50 μM, and the emission intensity at 520 nm was measured at an excitation wavelength of 280 nm.

Further, the molecular weight of the aggregate was measured by low-angle light scattering analysis of this solution, and it was 10 ⁷ Daltons.

With respect to the buffer solution containing no fluorescent dye, the emission intensity at 520 nm was measured 10 times at an excitation wavelength of 500 nm, and Y and σ of the fluorescence intensity at a concentration of 0 were determined. Similarly, the emission intensity at 520 nm at an excitation wavelength of 280 nm was measured for a buffer solution containing a fluorescent associate and not containing a fluorescent molecule, and Y and σ of the fluorescence intensity at a concentration of 0 were determined. The detection limit in this method was determined as the most dilute solution concentration at which the emission intensity was Y + 2σ or more. Further, the fluorescence intensity at each concentration above the detection limit was plotted against the concentration, the slope was determined by linear regression of the first order, and this was determined as the analytical sensitivity in this method.

Table 3 shows the detection limits and analytical sensitivities of fluorescent molecules in the presence and absence of the fluorescent aggregate.

[0108]

[Table 3]

Example 4 The detection limits and analytical sensitivities were determined in the same manner as in Example 3 except that the compounds shown in Tables 4 and 5 were used instead of FITC as the fluorescent labeling reagent. The results are summarized in Tables 4 and 5.

[0110]

[Table 4]

[0111]

[Table 5]

Example 5 The detection limit and analytical sensitivity were determined in the same manner as in Example 3 except that the compounds shown in Table 6 were used in place of human serum albumin as the substance of biological origin. The results are summarized in Table 6.

[0113]

[Table 6]

Example 6 30 mg of the amphipathic fluorescent compound shown in Table 7 was added to 10 mM of 3
-Morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS) buffer solution
H (adjusted to 7.2) 10 ml, and a uniform transparent dispersion was prepared by an ultrasonic wave irradiator. The following fluorescent molecules were quantified using the prepared fluorescent association solution.

[0115]

[Chemical 10]

The absorption maximum wavelength of the fluorescent molecule is 491n.
m, the maximum emission wavelength was 525 nm, and the fluorescence quantum yield was 0.75.

A solution of a fluorescent molecule having a known concentration of 1 pM to 100 nM (MOPS buffer, pH 7.2) was prepared. First, 1 ml of the fluorescent dye solution was placed in a fluorescent cell, and then, 50 μmol / l (hereinafter μ
(Abbreviated as M), and the emission intensity at 525 nm was measured at the excitation wavelength shown in Table 7. The molecular weight of the aggregate was measured by low-angle light scattering analysis of the obtained solution. The results are also shown in Table 7.

With respect to the buffer solution containing no fluorescent dye, the emission intensity at 525 nm was measured 10 times at the same excitation wavelength, and Y and σ of the fluorescence intensity at a concentration of 0 were obtained. The detection limit in this method was determined as the most dilute solution concentration at which the emission intensity was Y + 2σ or more. Further, the fluorescence intensity at each concentration above the detection limit was plotted against the concentration, the slope was determined by linear regression of the first order, and this was determined as the analytical sensitivity in this method.

Table 7 summarizes the detection limits and analytical sensitivities of fluorescent molecules in the presence of fluorescent aggregates. In addition, as a comparative example, the detection limit and analytical sensitivity in the presence of an aggregate having no fluorescence (No. 11) and when using a fluorescent amphipathic compound that does not form an aggregate (No. 10) are also shown. It is shown collectively in 7.

[0120]

[Table 7]

As can be seen from Table 7, by using the fluorescent aggregate of the present invention, the detection limit and analytical sensitivity of the fluorescent molecule are remarkably improved, and the effect is obvious.

Example 7 60 mg of the amphipathic fluorescent compound shown in Table 8 was added to 10 mM of 2
-[4- (2-Hydroxyethyl) -1-piperazinyl] propanesulfonic acid (hereinafter abbreviated as HEPES)
Buffer solution (sodium hydroxide solution was added to adjust the pH to 7.
(Adjusted to 2) and placed in 10 ml to prepare a uniform and transparent dispersion liquid with an ultrasonic wave irradiator. Using the prepared fluorescent association solution, the following fluorescent molecules were quantified.

[0123]

[Chemical 11]

The maximum absorption wavelength of the fluorescent molecule is 425n.
m, the maximum emission wavelength was 500 nm, and the fluorescence quantum yield was 0.11.

A solution (HEPS buffer, pH 7.2) having a known concentration of 1 pM to 100 nM of a fluorescent molecule was prepared. First, 1 ml of the fluorescent dye solution was placed in a fluorescent cell, and then the fluorescent aggregate was added so as to have a concentration of 100 μM, and the emission intensity at 500 nm was measured at the excitation wavelength shown in Table 8. The molecular weight of the aggregate was measured by low-angle light scattering analysis of the obtained solution. The results are also shown in Table 8.

With respect to the buffer solution containing no fluorescent dye, the emission intensity at 500 nm was measured 10 times at the same excitation wavelength to obtain Y and σ of the fluorescence intensity at a concentration of 0. The detection limit in this method was determined as the most dilute solution concentration at which the emission intensity was Y + 2σ or more. Further, the fluorescence intensity at each concentration above the detection limit was plotted against the concentration, the slope was determined by linear regression of the first order, and this was determined as the analytical sensitivity in this method.

Table 8 summarizes the detection limits and analytical sensitivities of fluorescent molecules in the presence of fluorescent aggregates. In addition, as a comparative example, in the presence of an aggregate without fluorescence (No. 5, 6)
Table 8 also shows the detection limits and analytical sensitivities.

[0128]

[Table 8]

As can be seen from Table 8, the detection sensitivity and the analysis sensitivity are not improved in the aggregate composed of the amphipathic compound having no fluorescence, whereas the use of the fluorescent aggregate of the present invention As a result, the detection limit and the analytical sensitivity are remarkably improved, and the effect is clear.

Example 8 The following compound

[0131]

[Chemical 12]

50 mg was placed in 10 ml of 10 mM 3-morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS) buffer solution (whose pH was adjusted to 7.2 by adding an aqueous sodium hydroxide solution), and ultrasonic irradiation was carried out. A uniform and transparent dispersion was prepared by a machine. Then, the following fluorescent molecule

[0133]

[Chemical 13]

Was immobilized on a quartz substrate in the range of 10 ⁻¹⁵ mol to 10 ⁻¹³ mol by the Langmuir-Blodgett method. The absorption maximum wavelength of the fluorescent molecule is 493 nm,
The maximum emission wavelength is 525 nm, and the fluorescence quantum yield is 0.
It was 33.

A quartz plate on which fluorescent molecules were immobilized was placed in a quartz cell filled with 2 ml of MOPS buffer (pH 7.2), and a fluorescence spectrophotometer was used for 525 n at an excitation wavelength of 490 nm.
The emission intensity of m was measured. Then, 50 fluorescent aggregates are formed.
Micromol / l (hereinafter abbreviated as μM) was added to measure the emission intensity at 525 nm at an excitation wavelength of 280 nm.

At this time, the molecular weight of the aggregate was measured by low-angle light scattering analysis of the obtained solution, and it was 5 × 10 ⁶ daltons.

For a quartz plate on which no fluorescent molecule was immobilized, 525 at an excitation wavelength of 493 nm in a MOPS buffer solution was used.
The emission intensity at nm was measured 10 times, and the average value of fluorescence intensity at a concentration of 0 (hereinafter abbreviated as Y) and the standard deviation (hereinafter abbreviated as σ1) were obtained. Similarly, a quartz plate in which fluorescent molecules are not immobilized is placed in a fluorescent association solution, and an excitation wavelength of 28
The emission intensity at 525 nm was measured at 0 nm, and Y and σ of the fluorescence intensity at a concentration of 0 were obtained. The detection limit in this method was determined as the most dilute solution concentration at which the emission intensity was Y + 2σ or more. Further, the fluorescence intensity at each concentration above the detection limit was plotted against the concentration, the slope was determined by linear regression of the first order, and this was determined as the analytical sensitivity in this method.

Table 9 summarizes the detection limits and analytical sensitivities of fluorescent molecules in the presence and absence of the fluorescent aggregate.

[0139]

[Table 9]

As can be seen from Table 9, the quantification method of the present invention is also effective for the fluorescent molecule immobilized on the substrate surface, and the detection limit and the analytical sensitivity are improved by 50 times or more.

Claims (1)

[Claims] 1. A fluorescent assembly and a fluorescent assembly capable of binding to the fluorescent molecule are coexistent, and the fluorescent assembly is irradiated with light that can be absorbed, and is attributed to energy transfer from the fluorescent assembly. The method for quantifying a fluorescent molecule is characterized in that the amount of the fluorescent molecule is determined by measuring the light emission from the fluorescent molecule.