JP3485658B2 - Quantitative method for fluorescent molecules - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION [0001] The present invention relates to the determination of fluorescent molecules in a solution.
Regarding the quantity method. For details, see the emission intensity from fluorescent molecules.
Quantitative methods to quantify more fluorescent molecules
A fluorescent aggregate capable of binding to a molecule and
By irradiating the aggregate with light, more sensitive fluorescence
Is a method for quantifying fluorescent molecules that enables quantification of fluorescent molecules.
You. [0002] 2. Description of the Related Art It is most necessary to determine a specific substance.
One of the important fields is clinical testing. Modern clinician
Science is supported by three pillars: examination, examination, and treatment. When
In particular, laboratory tests have the highest objectivity of the data obtained
To provide other scientific evidence and guide them.
It is. Biochemical tests for electrolytes and enzymes among clinical tests
Testing, antigens, antibodies, and other immune serum tests are a field of analytical chemistry.
Yes, trace amounts in biological fluids have been
It has become possible to quantify physiologically active substances. Also the molecule
Functions of bioactive substances at the molecular level due to the development of biology
Has been elucidated, and the determination of trace bioactive substances
This makes it possible to diagnose and treat diseases that were previously impossible.
It is getting. In the analysis of physiologically active substances, the atomic absorption method
Does not require special separation like metal ion determination
Those that can be directly analyzed are rare. In general,
After separation by some means, it is necessary to determine by appropriate means
There is. Many bioactive substances are recognized in vivo
By doing so, its function is exhibited. In other words, heaven
Naturally, there is a molecule that recognizes a substance.
Thereby, the target component can be specifically measured. Biological origin
As a substance recognition molecule, a substance involved in an immune reaction,
Antigens, antibodies, etc. are used for actual clinical analysis
You. [0004] Regarding the acquisition of antigens, antibodies, etc.,
Useful means are being established with advances in gene manipulation technology.
You. In other words, natural biologically-derived poly
Monoclonal by genetic manipulation instead of nal antibody
Null antibodies can be easily prepared. Use the antibody
Allows a specific analyte to bind to the antibody
Can be In clinical tests, these analytes
It is common practice to label and quantify the conjugate of a substance and an antibody.
Has been done. Various labeling methods have been put into practical use.
However, the method used for high-sensitivity analysis is radioisotope
Radioimmunoassay method (hereinafter abbreviated as RIA method)
Have been used. However, the RIA method is radiation
In recent years, there has been a need for special equipment that uses
The use of dye-based labeling has become an increasingly important immunoassay.
It is getting. [0005] Labeling with dyes requires special facilities.
And it is easy to handle.
However, the dye method is generally less sensitive than the RIA method
The development of a dye method with high sensitivity
It is the present situation that is eagerly desired. As a method for measuring the dye concentration, an absorbance method,
There are three main types, the fluorescence method and the luminescence method.
A dye that can be used for the measurement method is used. Absorbance method
For, the sensitivity depends on the molar absorbance of the dye,
There is a problem that the detection limit is relatively low. About the luminescence method
Use a special light source, so the background is low.
High sensitivity, but the available dyes are limited.
And has the problem that the dye is unstable.
You. The fluorescence method was more sensitive than the absorbance method.
Is widely used as a measurement method because of its sensitivity.
There is a problem that it does not reach the IA method. [0007] To improve the sensitivity of the fluorescence method,
Delay with methods to measure anisotropy or specific metal complexes
A method for measuring fluorescence has been reported, but its sensitivity is still not available.
It is enough. Sigmund et al. Use two types of pigments
Improve sensitivity by causing energy transfer
(Shin Solid Films, 19
1992, 210/211, 480 pages [Thin Solid
 Films, 210/211 (1992), 480]). However, its sensitivity is
It is about three times as much as when energy transfer is not used.
Therefore, further improvement in sensitivity has been desired. [0008] SUMMARY OF THE INVENTION Therefore, a fluorescent dye
Highly sensitive conjugates between the recognized analyte and the recognition molecule
It has been desired to develop a simple and easy measurement method. [0009] Means for Solving the Problems The present inventors have made such a question.
To develop a method for quantifying fluorescent molecules that can solve the problem
I repeated my research. As a result, specific dye molecules were oriented
The coexistence of the aggregate enables the fluorescence to be analyzed
The fluorescence of the dye is significantly enhanced and the detection sensitivity is improved.
And completed the present invention. That is, the present invention provides a fluorescent molecule and a fluorescent component
Can be combined with childAverage molecular weight (weight average) of 10,000 to 100 million
Range ofAbsorbable to fluorescent aggregates in the presence of fluorescent aggregates
Energy to transfer energy from fluorescent aggregates
By measuring the emission from the contributing fluorescent molecules,
Determining the amount of the fluorescent molecule
This is a quantitative method. The detection sensitivity used in the present invention means that
Lower detection limit indicating the minimum concentration, and the signal intensity and concentration during analysis.
This shows the meaning of the combined analytical sensitivity, which indicates the ratio of degrees. In the method for quantifying a fluorescent molecule of the present invention,
If the target fluorescent molecule has fluorescence,
Known molecules can be used without any particular limitation. firefly
Sensitivity in quantifying light molecules depends on the fluorescence of the fluorescent molecules used.
Since the quantum yield is greatly affected, it is desirable to use a fluorescent quantum
For a yield of 0.001 or more and more sensitive measurement
A fluorescent molecule having a fluorescence quantum yield of 0.01 or more is desired
New The maximum absorption wavelength of a fluorescent molecule is
250 nm or more and 800 nm depending on the fluorescent aggregate
Molecules with the following absorption maxima are desirable: Absorption maximum wavelength
If the sensitivity is less than 250 nm, sufficient sensitivity cannot be obtained
There is. When the wavelength is 800 nm or more,
Is required. The maximum emission wavelength of the fluorescent molecule is 30
The thickness is desirably from 0 nm to 900 nm. Absorption maximum wavelength
If it is less than 300 nm, sufficient sensitivity may not be obtained.
Often. When the wavelength is 900 nm or more, the intensity is measured.
Special equipment is required. Maximum emission wavelength is 400n
If it is more than m, inexpensive glass as a material of the cell for measurement
It is particularly suitable because it can use
is there. The quantification method of the present invention is particularly suitable for fluorescence in aqueous solution.
Suitable for quantification of molecules, suitable for quantification of biological components
is there. Particularly preferred fluorescent molecules derived from biological components
There are the following two types. (1) Fluorescent physiologically active substance or
Is a biological substance. (2) Physiologically active substance labeled with a fluorescent dye
Quality or biological material (1) Physiologically active substance having fluorescence or biological origin
For substances (hereinafter abbreviated as fluorescent biomolecules),
Since the body has fluorescence, complicated procedures such as sample pretreatment
Since the operation is unnecessary, the quantification method of the present invention is particularly preferably used.
Is available. Commonly used fluorescent biomolecules
As reduced nicotinamide adenine dinucleotide
De, reduced nicotinamide adenine dinucleotide phosphorus
Acid, vitamin B2 (riboflavin), chlorophyll, leaf
Acids and the like. (2) Physiological activity labeled with a fluorescent dye
Substances or biological substances (hereinafter abbreviated as fluorescent labeled biomolecules)
Indicates that the biological component to be measured has an appropriate reactive group.
Fluorescent dye (hereinafter abbreviated as fluorescent labeling reagent)
It is obtained by knowing. Quantification of the fluorescent molecule of the present invention
In the method, the fluorescently labeled biomolecule is also a suitable fluorescent molecule
is there. Known fluorescent labeling reagents include:
Although it can be used, amino-reactive fluorescent labeling reagents,
Ruboxyl group reactive fluorescent labeling reagent, aldehyde group reaction
Fluorescent labeling reagents, hydroxyl reactive fluorescent labeling reagents,
Is preferably a thiol group reactive fluorescent labeling reagent.
You. Amino group, carboxyl group, aldehyde group, hydroxyl group
And thiol groups are generally
These labeling reagents are particularly preferred because they are present in the molecule of origin.
Appropriately used. The amino group-reactive fluorescent labeling reagent includes:
Fluorescein-4-isothiocyanate, 3-chloro
Carbonyl-6,7-dimethoxy-1-methyl-2 (1
H) -Quinoxalinone, fluorescein-4-isothio
Cyanate, 4-chloro-7-nitrobenzofurazan,
4-fluoro-7-nitrobenzofurazone, sulfolo
-Damine 101 acid chloride, fluorescamine,
Taraldehyde, dansyl chloride, etc. are preferably used
You. As a carboxyl group reactive fluorescent labeling reagent
Is 3-bromomethyl-6,7-dimethoxy-1-me
Tyl-1,2-dihydroquinoxalin-2-one, 4-
Blumomethyl-7-methoxycoumarin, 1,2-diamido
No-4,5-methylenedioxybenzene dihydrochloride
It is preferably used. As an aldehyde group-reactive fluorescent labeling reagent
Is 1,2-diamino-4,5-dimethoxybenzene.
Dihydrochloride, 2,2'-dithiobis (1-aminonaphthale
), 4-amino-3-penten-2-one and the like are preferred.
used. As the hydroxyl-reactive fluorescent labeling reagent, 3
-Chlorocarbonyl-6,7-dimethoxy-1-methyl
-2 (1H) -quinoxalinone, 2- (5-chlorocar
Bonyl-2-oxazolyl) -5,6-methylenedioxy
Cibenzofuran or the like is preferably used. As a thiol group reactive fluorescent labeling reagent
Is 4-fluoro-7-sulfamoylbenzofuraza
N- [4- (6-dimethylamino-2-benzofura)
Nyl) phenyl] maleimide, 2,2'-dihydroxy
-6,6'-dinaphthyl disulfide, N- [4-
(5,6-methylenedioxy-2-benzofuranyl)
Enyl] maleimide, N- (9-acridinyl) male
Mido, 4-chloro-7-sulfobenzofurazan anne
Monium salt, 4-fluoro-7-sulfobenzofuraza
Ammonium salt, dansyl aziridine, 5- (Y
Docetamidoethyl) aminonaphthalene-1-sulfo
Acid, 5-iodoacetamidofluorescein, N-
(1-anilinonaphthyl-4) maleimide, N- (3-
Pyrene) maleimide, eosin-5-iodoacetamy
And the like are preferably used. Physiological physiology labeled with the above-mentioned fluorescent labeling reagent
Quantified in the field as active substance or biological component
There are no restrictions on the biological components that are commonly performed
Used in the quantification method of the present invention.
Examples of suitable biological components are as follows. Immediately
A non-immune protein such as albumin, IgG, Ig
A, IgM, IgD, IgE and other immunoglobulins;
Non-immunospecific binding pars such as tin, receptor, avidin
Toner, glutamate oxaloacetate transaminase,
Glutamate pyruvate transaminase, lactate dehydration
Enzyme, γ-glutamyltransferase, choline
Enzymes such as sterase, human chorionic gonadotropin,
Hormones such as thyroid stimulating hormone, nuclei such as DNA and RNA
Acids and the like. The method for producing the fluorescent labeled biomolecule is as follows:
A method known in the art can be used without particular limitation.
However, the following is a specific example of a method preferably used in general.
It is as follows. That is, bioactive substances or raw
Dissolve the body-derived components in a buffer such as Tris buffer,
The fluorescent labeling reagent is added thereto. At this time, pyridine,
The presence of a small amount of a catalyst such as dimethylformamide
The yield is further improved. After reacting at room temperature for several hours,
Purify the solution several times by gel chromatography
Thus, a desired fluorescently labeled biomolecule can be obtained. [0027] The quantification method of the present invention comprises the steps of:
Absorbs into the fluorescent aggregate in the presence of the bindable fluorescent aggregate.
Irradiates light that can be collected and transfers energy from the fluorescent aggregate.
By measuring the luminescence from fluorescent molecules attributable to movement
And determining the amount of the fluorescent molecule. here
Fluorescent aggregates are fluorescent amphiphilic compounds
(Hereinafter abbreviated as fluorescent amphiphilic compound)
Is an essential component for highly sensitive quantification.
You. Aggregates are hydrogen bonds and charges between the same molecules.
Relatively weak bonds such as mobile bonds and hydrophobic bonds work,
A relatively well-ordered molecular assembly formed in
is there. In the present invention, a fluorescent molecule and a fluorescent
Coupling with the conjugate means that the interaction between the fluorescent molecule and the fluorescent molecule
That the association and the energy
Express. The interaction referred to here is known in the art.
Interactions occurring between molecules are adopted without particular limitation
It is. To illustrate particularly preferred interactions,
Coulomb force interaction, hydrophobic interaction, hydrogen bonding interaction
Action, coordination interaction, π electron / CH interaction, etc.
is there. The average molecular weight of the fluorescent aggregate used in the present invention
(Weight average) in the range of 10,000 to 100 millionInYou. Fluorescent
When the average molecular weight of the aggregate is 10,000 or less, the sensitivity at the time of quantification becomes
Will be insufficient. If the amount is 100 million or more, the solvent
Stability in the inside is badYou.Also, more preferably, fireflies
When the average molecular weight of the photoassociation is determined to be 50,000 to 10,000,000,
Suitable due to extremely good sensitivity and good solution stability
It is. As the solvent in which the fluorescent aggregate is formed,
A solvent that forms an aggregate composed of amphiphilic compounds
Although not particularly limited, generally, a stable aggregate is formed.
Easy water is preferably used. The fluorescent amphiphilic compound may be in a solvent.
Limited as long as it has the property of forming an aggregate at
No known compound can be used, but it has fluorescence
At least one linear hydrophobic moiety comprising a rigid portion in the chain
Organic compound having a hydrophilic group and a hydrophilic group
(Abbreviated as light linear amphiphilic compound) is preferably used.
You. By using the fluorescent linear amphiphilic compound,
The sensitivity during quantification is significantly improved. In the present invention, the rigid portion is, for example,
(A), (b) and (c). (A) Direct bonding or carbon-carbon multiple bonding
Consisting of at least two aromatic rings connected via a bond
Divalent group Specific examples of such groups include, for example, [0034] Embedded image And other divalent groups. (B) Two or more aromatic rings are bonded.
Or a single bond between multiple atoms and the bond is
-A divalent group that is bound Specific examples of such groups include, for example, [0037] Embedded image And other divalent groups. (C) When the aromatic ring forms a condensed ring
Therefore, when this fused ring is stacked between multiple molecules,
Divalent groups in which the inversions are sterically bound to each other Specific examples of such groups include, for example, [0040] Embedded image And other divalent groups. In the present invention, a linear hydrophobic group is a linear
A alkyl group or a halogen-substituted product thereof. Rigid part
Linear Amphiphilicity with a Linear Hydrophobic Group Containing Fluorine in the Chain
The carbon number of the compound is 8 to 8 except for the carbon number of the rigid portion.
A range of 30 is desirable. If it is 8 or less, it will be associated in the solvent
May not be formed.
Not so desirable in some cases. The above rigid part
In general, the bond to the linear hydrophobic group is a carbon-carbon bond,
Stele bonds and ether bonds are preferred. A linear hydrophobic group containing a rigid portion in the chain is
The fluorescent linear amphiphile in consideration of the ability to form an aggregate in a solvent
Most preferably, one is contained in the sex compound. 2
Does not form an aggregate in the solvent if more than one is included
There are cases. In the fluorescent linear amphiphilic compound of the present invention
Hydrophilic group, the fluorescent linear amphiphilic compound is
It is essential to form a stable aggregate. With hydrophilic groups
In general, a hydrophilic group known as a hydrophilic group of a surfactant is used.
Groups can be used, but ionic groups or polyethylene
The stability of the aggregate is improved by using the glycol group.
Desirable for significant improvement. Here, the ionic group is an acidic group or a basic group.
It is defined as a generic term for a group. Where acidic or basic
Means Bronsted acid or Bronsted base
The acidic groups are generally sulfonic acid groups, carboxyl groups
Group, phosphate group and their salts, basic group
Generally include amino groups, substituted amino groups, and quaternary ammonium
Group, phosphonium group and their salts
Are preferably used. As the polyethylene glycol group,
When the repetition of the ethylene glycol unit is in the range of 6 to 30,
Is desirable. If less than 6, associate in solvent
May not form a body. Also, if more than 30
May not sufficiently improve the sensitivity during quantification. here
Hydrophilicity contained in the fluorescent linear amphiphilic compound of the present invention
The number of groups is one in view of the stability of the resulting fluorescent aggregate.
Or it is preferable that there are two. The fluorescent linear amphiphilic compound of the present invention
Any known materials can be used as long as they satisfy the following conditions.
You can. The typical ones that are generally preferably used are:
The details are shown below. [0048] Embedded image In the structural formulas (a) to (e),
n is an integer of 6 to 12, m is an integer of 2 to 12, Z is [0050] Embedded image (Where X is a halogen ion or a stable ion
An atomic group forming an anion, M is an alkali metal, hydrogen,
Or an atomic group forming a stable cation, and 1 is 6
Shows an integer of ~ 30. ). ｝ In the preferred fluorescent linear amphiphilic compounds described above,
Examples of the alkali metal to be used include lithium and natrium.
Thorium and potassium, forming stable cations
The atomic group is ammonium ion, tetramethylan
Monium ion. With a halogen ion represented by X
Specifically, fluorine, chlorine, bromine, and iodine
The atomic groups that form stable anions are nitrate ions and lithium ions.
Acid ions, sulfate ions, and carbonate ions. The method for producing the fluorescent aggregate of the present invention includes
There is no particular limitation as long as it is a generally known method for producing an aggregate.
Instead, any method may be used. Generally, fluorescent
Morpholinopropane sulfone
Ultrasonic irradiation in a buffer solution such as acid is preferably used.
Can be The formation of the fluorescent aggregate is caused by the light scattering of the solution.
Increase, broadening of peaks during proton NMR measurement of solution
Of the solution stained with uranyl acetate
By direct observation of the aggregate by electron microscope
You can check. The molecular weight of the aggregate is determined by low-angle light scattering analysis,
It is measured by gel chromatography or the like. As a solvent for forming a fluorescent aggregate,
Solvents known in the art can be used, but the fluorescent association
Considering body stability and detection sensitivity during quantification, water
Preferably. In addition, the use of aqueous buffers
In particular, since the effects of pH changes can be ignored,
An impact liquid is preferably used. As the buffer component in the buffer,
But not limited to phosphoric acid, trishydroxyamino
Tan, acetic acid, citric acid, triethanolamine, borane
Acid, maleic acid, carbonic acid, N- (2-acetamido) -2
-Aminoethanesulfonic acid, N- (2-acetamide)
Iminodiacetic acid, N, N-bis (2-hydroxyethyl)
-2-aminoethanesulfonic acid, N, N-bis (2-
Droxyethyl) glycine, bis (2-hydroxyethyl)
L) iminotris (hydroxymethyl) methane, N-cy
Clohexyl-3-aminopropanesulfonic acid, N-cy
Clohexyl-2-hydroxy-3-aminopropanes
Sulfonic acid, N-cyclohexyl-2-aminoethanesul
Honic acid, 3- [N, N-bis (2-hydroxyethyl)
Amino] -2-hydroxypropanesulfonic acid, 3-
[4- (2-hydroxyethyl) -1-piperazinyl]
Propanesulfonic acid, 2- [4- (2-hydroxyethyl
L) -1-Piperazinyl] ethanesulfonic acid, 2-hydrido
Roxy-3- [4- (2-hydroxyethyl) -1-pi
Perazinyl] propanesulfonic acid, 2-morpholinoe
Tansulfonic acid, 3-morpholinopropanesulfone
Acid, 2-hydroxyethyl-3-morpholinopropane
Sulfonic acid, piperazine-1,4-bis (2-ethanes
Sulfonic acid), piperazine-1,4-bis (2-hydroxy
C-3-propanesulfonic acid), N-tris (hydroxy
B) Methyl-3-aminopropanesulfonic acid, 2-hydr
Roxy-N-tris (hydroxymethyl) methyl-3-
Aminopropanesulfonic acid, N-tris (hydroxy
Tyl) methyl-2-aminoethanesulfonic acid, N- [g
Lis (hydroxymethyl) methyl] glycine and the like are preferred
used. The concentration of the fluorescent aggregate is determined by
When the concentration at which the criticality of the amphiphilic compound is
Is necessary for quantification with high sensitivity. The criticality
The aggregate formation concentration is generally 10^-6-10^-3In the range
It is desirable to use this concentration or more. Concentration of the aggregate
Indicates the presence of an insoluble fluorescent linear amphipathic compound in the solvent
Concentration, ie, below the saturation concentration for the solvent used
It is desirable to use it. In the quantification method of the present invention, the fluorescent molecule
And a fluorescent aggregate capable of binding to the fluorescent molecule
Irradiate the fluorescent aggregate with light that can be absorbed and determine whether it is a fluorescent aggregate.
Emission from fluorescent molecules due to their energy transfer
The amount of the fluorescent molecule is determined by the measurement. Known
Fluorescence analysis can absorb fluorescent molecules to be analyzed
Irradiate the fluorescent molecules and measure the luminescence from the fluorescent molecules. I
However, in the method of the present invention, the fluorescent
Irradiating efficient light. In the present invention, a fluorescent molecule and a fluorescent association
The binding with the body means that the fluorescent molecule
The fluorescent aggregate approaches the distance where energy transfer is possible
Indicates that. The binding between the fluorescent molecule and the fluorescent aggregate is one
Generally spontaneous by mixing each in a suitable solvent
Is formed. As the solvent, the stability of the aggregate is considered.
Water is preferably adopted when envisaged. In the present invention, a generally preferable quantification method is
Specific examples are as follows. That is, (A) (1) A solution of a known concentration of the fluorescent molecule to be measured is placed in a fluorescent cell.
Prepare several types inside. (2) Add a solution of the fluorescent aggregate to the above solution.
Add a fixed amount. (3) Stir the solution. (4) The light having the maximum absorption wavelength of the fluorescent aggregate is
Excitation wavelength, maximum emission wavelength of fluorescent molecule as emission wavelength
Measure the fluorescence intensity. (5) A calibration curve is prepared from the above results. (6) Known concentration solution for unknown concentration sample
Calculate the fluorescence intensity by performing the same operation as in. (7) The above fluorescence intensity is extrapolated to a calibration curve and is unknown.
Determine the concentration of the fluorescent molecule at the concentration. (B) (1) A solid to which a known amount of the fluorescent molecule to be measured is adsorbed
Prepare several types. (2) Fluorescent association of the above solid in a fluorescent cell
Soak in body solution. (3) Stir the solution. (4) The light having the maximum absorption wavelength of the fluorescent aggregate is
Excitation wavelength, maximum emission wavelength of fluorescent molecule as emission wavelength
Measure the fluorescence intensity. (5) A calibration curve is prepared from the above results. (6) Known concentration solution for unknown concentration sample
Calculate the fluorescence intensity by performing the same operation as in. (7) The fluorescence intensity is extrapolated to a calibration curve and is unknown.
Determine the concentration of the fluorescent molecule at the concentration. By the above operation, fluorescent molecules can be quantified.
Yes, compared to the absence of fluorescent aggregates
The detection sensitivity is improved 10 to 100 times. The mechanism by which the detection sensitivity is improved is clear.
However, in the aggregate of the present invention, the fluorescent chromophore
Irradiates fluorescent aggregates with light because
Between excitons generated by the formation of an aggregate
Is known to be freely movable. This result
As a result, energy transfer from the fluorescent aggregate to the fluorescent molecule
It is thought that efficient occurrence is involved in improving sensitivity
Can be The measurement method described in the above-mentioned preferred quantification method (B)
As a solid to which the elephant fluorescent molecule is adsorbed,
Glass, 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, a fluorescent molecule and a fluorescent
The combination of photo-associates is a combination of
Especially limited if it is a combination that causes energy transfer to
Not done. Generally, the energy between two fluorescent dyes
For transfer, the donor dye emission spectrum and the acceptor
That the absorption spectra of the
It is known, and in the present invention, the emission of the fluorescent aggregate
Spectra and the absorption spectra of fluorescent molecules overlap
Is essential for high sensitivity measurements. Each spectrum
The presence or absence of overlap is determined by the fluorescence spectrum of the fluorescent
Confirmed by measuring the absorption spectrum of the light-sensitive molecule
It is. In the above combination, emission of fluorescent molecules
The difference between the maximum wavelength and the absorption maximum wavelength of the fluorescent aggregate is 100 n
m or more, the effect of the scattered light of the excitation light is
It is preferable because the degree is improved. Furthermore, a difference of 200 nm or more
Is preferable because there is less influence of scattered light.
You. Irradiation of Light to Fluorescent Aggregate and Fluorescence
To measure the amount of light (intensity) emitted from a molecule, use commercially available fluorescence spectroscopy.
A photometer is conveniently employed. In the quantification method of the present invention, the fluorescence
There is no particular restriction on the combination of a fluorescent molecule and a fluorescent aggregate.
However, if a specific example of a particularly preferable combination is specifically illustrated,
It is as follows. That is, as the fluorescent linear amphiphilic compound,
The following compounds are mentioned. [0081] Embedded image As the fluorescent molecule, a physiologically active substance or
Refers to fluorescein-4-isothiocyane
Fluorescently labeled biomolecules obtained by reacting with
Quantitatively sensitive fluorescently labeled biomolecules
It becomes possible. [0083] The method for quantifying a fluorescent molecule of the present invention comprises
Using fluorescent molecules and energy transferable fluorescent aggregates
Quantification of fluorescent molecules with extremely high sensitivity
Is possible. In addition, fluorescent molecules are labeled with fluorescent dyes.
Identified physiologically active substances or biological molecules can be used.
High-sensitivity measurement, especially in the field of clinical testing
Available to In view of the above, the fluorescent molecule of the present invention
The industrial value of the quantification method is extremely large. [0084] EXAMPLES The present invention will be described in more detail below.
Examples are given below, but the present invention is limited to these Examples.
Not something. In this example, the values were determined by low-angle light scattering analysis.
The molecular weight of the aggregates
This is a value obtained in terms of fonic acid. Embodiment 1 The following compounds [0087] Embedded image50 mg was added to 10 mM 3-morpholinop
Lopansulfonic acid (hereinafter abbreviated as MOPS) buffer
(The pH was adjusted to 7.2 by adding an aqueous sodium hydroxide solution.
Prepared) and put in 10 ml,
A single, transparent dispersion was prepared. The prepared fluorescent aggregate solution
Using the liquid, the following fluorescent molecules were quantified. [0089] Embedded image The maximum absorption wavelength of the fluorescent molecule is 491n.
m, emission maximum wavelength is 525 nm, and fluorescence quantum yield is
0.75. 1 picomol / l of the fluorescent molecule (hereinafter referred to as pM
From 100 nmol / l (hereinafter abbreviated as nM)
Of a known concentration (MOPS buffer, pH 7.0).
2) was produced. First, add 1 ml of the fluorescent dye solution to the fluorescent cell
At an excitation wavelength of 490 nm using a fluorescence spectrophotometer.
The emission intensity at 25 nm was measured. Subsequently, the fluorescent aggregate
Is 50 micromol / l (hereinafter abbreviated as μM).
525 nm at an excitation wavelength of 280 nm.
The emission intensity was measured. At this time, proton NMR of the obtained solution
Was observed in deuterated chloroform solution of the above compound
6.7 to 8.7 ppm peak (proton origin of aromatic ring)
Has been broadening to form an association
It was shown. Furthermore, low-angle light scattering analysis of this solution
The molecular weight of the aggregate was 5 × 10⁶Dalton
Met. For a buffer containing no fluorescent dye,
Emission intensity at 525 nm at 490 nm was measured 10 times
And the average value of the fluorescence intensity at concentration 0 (hereinafter abbreviated as Y)
The standard deviation (hereinafter abbreviated as σ1) was determined. In the same way
Buffer containing fluorescent aggregates but no fluorescent molecules
The emission intensity at 525 nm at an excitation wavelength of 280 nm.
The measurement was performed to determine the fluorescence intensity Y and σ at the concentration of 0. The Act
Sparsest limit where the emission intensity is Y + 2σ or more
Solution concentration. Furthermore, for each concentration above the detection limit
Plotted the fluorescence intensity against the concentration
The slope was determined by regression, and this was used as the analytical sensitivity in this method.
I asked. Table 1 shows the presence and absence of the fluorescent aggregate.
The detection limit and analytical sensitivity of fluorescent molecules
Was. [0095] [Table 1] As can be seen from Table 1, the quantification method of the present invention
50 times or more both in detection limit and analysis sensitivity
The usefulness of the quantification method of the present invention is clear.
You. Embodiment 2 Example 1 was performed using the fluorescent molecules (quantification targets) shown in Table 2.
Similarly, detection sensitivity and analysis sensitivity were determined. Table of results
2 collectively. [0098] [Table 2]Embodiment 3 The following compounds [0100] Embedded image 50 mg was added to a 10 mM MOPS buffer (water).
The pH was adjusted to 7.2 by adding aqueous sodium oxide solution.
In 10 ml and homogenize with ultrasonic irradiator
A transparent dispersion was made. 1 g of human serum albumin was added to 100 ml of M
Dissolve in OPS buffer (pH 7.3)
Resin-4-isothiocyanate (hereinafter abbreviated as FITC)
1 g of acetone solution was added dropwise and left at room temperature for 12 hours.
Reaction. After the reaction, high molecular weight components are removed by gel filtration.
By fractionation, FITC-labeled human serum albumin was obtained. Absorption maximum wavelength of the obtained fluorescently labeled biomolecule
Is 500 nm, the emission maximum wavelength is 520 nm, and the fluorescence is
The quantum yield was 0.31. One picosecond of FITC-labeled human serum albumin
mol / l (hereinafter abbreviated as pM) to 100 nanomo
1 / l (hereinafter abbreviated as nM) at a known concentration (MO
PS buffer, pH 7.2). First fluorescent color
Take 1 ml of the solution into a fluorescent cell and excite with a fluorescence spectrophotometer.
The emission intensity at 520 nm at an emission wavelength of 490 nm was measured.
Subsequently, the fluorescent aggregate was added to a concentration of 50 μM.
And measure the emission intensity at 520 nm at an excitation wavelength of 280 nm
did. Further, the solution was analyzed by low-angle light scattering analysis.
When the molecular weight of the coalescence was measured, it was 10⁷Dalton
Was. For a buffer containing no fluorescent dye,
Measures the emission intensity at 520 nm at an emission wavelength of 500 nm 10 times
Then, Y and σ of the fluorescence intensity at a concentration of 0 were obtained. Fireflies in the same way
Buffers containing photoaggregates and no fluorescent molecules
The emission intensity at 520 nm at an excitation wavelength of 280 nm.
Then, Y and σ of the fluorescence intensity at a concentration of 0 were determined. In this law
The lowest detection limit where the emission intensity is Y + 2σ or more.
It was determined as the solution concentration. In addition, for each concentration above the detection limit,
Plots the fluorescence intensity versus concentration for the first-order linear
The slope is determined by the regression, and this is used as the analytical sensitivity in this method.
I asked. Table 3 shows the presence and absence of the fluorescent aggregate.
The detection limit and analytical sensitivity of fluorescent molecules
Was. [0108] [Table 3] Embodiment 4 Shown in Tables 4 and 5 in place of FITC as a fluorescent labeling reagent
The detection limit and the min.
The analysis sensitivity was determined. The results are summarized in Tables 4 and 5. [0110] [Table 4][0111] [Table 5] Embodiment 5 Table 6 replaces human serum albumin as a biological substance
Using the indicated compound, the detection limit was determined in the same manner as in Example 3.
Analytical sensitivity was determined. The results are summarized in Table 6. [0113] [Table 6] Embodiment 6 30 mg of the amphiphilic fluorescent compound shown in Table 7 was added to 10 mM 3
-Morpholinopropanesulfonic acid (hereinafter abbreviated as MOPS)
Buffer solution (not shown)
H adjusted to 7.2) Put into 10 ml, ultrasonic
A uniform and transparent dispersion was prepared using an irradiator. Made
The following fluorescent molecules were quantified using the fluorescent aggregate solution.
Was. [0115] Embedded image The maximum absorption wavelength of the fluorescent molecule is 491n.
m, emission maximum wavelength is 525 nm, and fluorescence quantum yield is
0.75. Fluorescent molecules from 1 pM to 100 nM are known.
Make a solution of concentration (MOPS buffer, pH 7.2)
Was. First, take 1 ml of the fluorescent dye solution into the fluorescent cell,
And 50 μmol / l (hereinafter referred to as μ)
M) (abbreviated as M).
The emission intensity at a wavelength of 525 nm was measured. Also obtained
The molecular weight of the aggregate by low-angle light scattering analysis of the solution
Was. The results are shown in Table 7. The same applies to the buffer containing no fluorescent dye.
The emission intensity at 525 nm was measured 10 times at the same excitation wavelength and the concentration was measured.
The fluorescence intensity Y and σ at 0 were determined. Detection limit in this method
The dilute solution concentration at which the emission intensity becomes Y + 2σ or more in the field
Asked. Fluorescence at each concentration above the detection limit
Intensity is plotted against concentration and by linear regression of first order
Calculate the slope and determine this as the analytical sensitivity in this method.
Was. Table 7 shows the fluorescent molecules in the presence of the fluorescent aggregate.
The detection limit and analytical sensitivity are summarized. Also, with the comparative example
In the presence of an aggregate having no fluorescence (No. 11)
And a fluorescent amphiphilic compound that does not form an aggregate
Table 7 also shows the detection limit and analytical sensitivity in the case of
It is shown together. [0120] [Table 7]As can be seen from Table 7, the fluorescent association of the present invention
The detection limit and analytical sensitivity of fluorescent molecules
The degree is significantly improved, and the effect is obvious. Embodiment 7 60 mg of the amphiphilic fluorescent compound shown in Table 8 was added to 10 mM 2
-[4- (2-hydroxyethyl) -1-piperazini
] Propanesulfonic acid (hereinafter abbreviated as HEPES)
Buffer (pH 7.0 by adding aqueous sodium hydroxide solution).
2) Adjusted to 10ml, put in ultrasonic irradiator
A more uniform and transparent dispersion was made. Fluorescent society created
Using the coalescence solution, the following fluorescent molecules were quantified.
Was. [0123] Embedded image The maximum absorption wavelength of the fluorescent molecule is 425 n.
m, the emission maximum wavelength is 500 nm, and the fluorescence quantum yield is
0.11. Fluorescent molecules from 1 pM to 100 nM are known.
Make a solution (HEPS buffer, pH 7.2)
Was. First, take 1 ml of the fluorescent dye solution into the fluorescent cell,
And added so that the fluorescent aggregate becomes 100 μM.
And measured the emission intensity at 500 nm with the excitation wavelengths shown in Table 8.
did. The low-angle light scattering analysis of the obtained solution
The child size was measured. The results are shown in Table 8. The same applies to the buffer containing no fluorescent dye.
The emission intensity at 500 nm was measured 10 times at the same excitation wavelength and the concentration was measured.
The fluorescence intensity Y and σ at 0 were determined. Detection limit in this method
The dilute solution concentration at which the emission intensity becomes Y + 2σ or more in the field
Asked. Fluorescence at each concentration above the detection limit
Intensity is plotted against concentration and by linear regression of first order
Calculate the slope and determine this as the analytical sensitivity in this method.
Was. Table 8 shows fluorescent molecules in the presence of fluorescent aggregates.
The detection limit and analytical sensitivity are summarized. Also, with the comparative example
In the presence of an aggregate having no fluorescence (No. 5, 6)
Table 8 also summarizes the detection limits and analytical sensitivities for. [0128] [Table 8] As can be seen from Table 8, it has no fluorescence
For the aggregate composed of amphiphilic compounds, the detection sensitivity and
The sensitivity of the present invention is not improved
By using the aggregate, the detection limit and analytical sensitivity are remarkable.
The effect is clear. Embodiment 8 The following compounds [0131] Embedded image 50 mg was added to 10 mM 3-morpholinop
Lopansulfonic acid (hereinafter abbreviated as MOPS) buffer
(The pH was adjusted to 7.2 by adding an aqueous sodium hydroxide solution.
Prepared) and put in 10 ml,
A single, transparent dispersion was prepared. Then, the following fluorescent molecule [0133] Embedded imageThe stone was obtained by the Langmuir-Blodgett method.
10 on English board^-15mol-10^-13Fixed in mol range
It has become. The maximum absorption wavelength of the fluorescent molecule is 493 nm,
The emission maximum wavelength is 525 nm, and the fluorescence quantum yield is 0.5.
33. Fill with 2 ml of MOPS buffer (pH 7.2)
A quartz plate with fluorescent molecules immobilized in the quartz cell
525 n at an excitation wavelength of 490 nm using a fluorescence spectrophotometer.
m was measured. Subsequently, the fluorescent association was 50
Micromol / l (hereinafter abbreviated as μM)
At a wavelength of 525 nm at an excitation wavelength of 280 nm.
The degree was measured. At this time, low-angle light scattering analysis of the obtained solution was performed.
The molecular weight of the aggregate was measured to be 5 × 10⁶Dalt
Was. [0137] A quartz plate not immobilized with fluorescent molecules
525 at an excitation wavelength of 493 nm in MOPS buffer.
The emission intensity at 10 nm was measured 10 times, and the fluorescence intensity at 0 concentration was measured.
Average (hereinafter abbreviated as Y) and standard deviation (hereinafter abbreviated as σ1)
To). Fluorescence in a fluorescent aggregate solution in the same manner
A quartz plate without immobilized reactive molecules is placed, and excitation wavelength 28
Measure the emission intensity at 525 nm at 0 nm and measure the fluorescence at 0 concentration
The strengths Y and σ were determined. The detection limit in this method
Calculated as the dilute concentration of the solution with a degree of Y + 2σ or more
Was. Furthermore, the fluorescence intensity at each concentration above the detection limit
And the slope is calculated by first-order linear regression.
Therefore, this was determined as the analytical sensitivity in this method. Table 9 shows the presence and absence of the fluorescent aggregate.
The detection limit and analytical sensitivity of fluorescent molecules
Was. [0139] [Table 9] As can be seen from Table 9, the quantification method of the present invention
Is also effective for fluorescent molecules immobilized on the substrate surface.
Yes, the detection limit and analysis sensitivity were improved by 50 times or more.

──────────────────────────────────────────────────続 き Continued on the front page (58) Fields investigated (Int. Cl. ⁷ , DB name) G01N 21/75-21/83 G01N 21/62-21/74 JICST file (JOIS) Practical file (PATOLIS) Patent File (PATOLIS)

Claims (1)

(57) [Claim 1] Fluorescent molecule and capable of binding to the fluorescent molecule
Irradiating the fluorescent aggregate with light that can be absorbed in the presence of the fluorescent aggregate having an average molecular weight (weight average) in the range of 10,000 to 100 million, and the fluorescence attributed to energy transfer from the fluorescent aggregate. A method for quantifying a fluorescent molecule, comprising determining the amount of a fluorescent molecule by measuring luminescence from the fluorescent molecule.