JP2003083893A - Method for measuring fluorescence on submicron-scale grain surface - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for measuring fluorescence on submicron-scale grain surfaces, simple but high in detection accuracy. SOLUTION: The method for use in measuring fluorescence in a suspension of submicron-scale grains retaining a fluorescent substance is characterized in that a difference that is great enough to avoid the effect of scattering is provided between the excitation light wavelength and the fluorescence wave length for the detection of fluorescence.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to a method for measuring a fluorescent substance retained on a surface of a submicroparticle and a sample measurement using the method.

[0002]

2. Description of the Related Art Conventionally, there are various methods for retaining and detecting a target substance on the surface of particles. Among them, the method for measuring using a labeled substance and an immobilized substance is an antigen-antibody reaction or DNA. It has been particularly developed in the field of measuring chemicals in plasma or serum using probes.
As a method using the antigen-antibody reaction, an enzyme immunoassay method using an enzyme (EIA method) and a fluorescent immunoassay method using a fluorescent dye (FIA method) are widely known.

As a method for measuring the fluorescent substance on the particles, there is a method using a flow cytometer, a fluorescence microscope, a fluorescence spectrophotometer, a fluorescence microtiter plate reader, or the like. For example, in the case of fluorescence, in the invention described in Japanese Patent Laid-Open No. 2000-105236 relating to the analysis method and the automatic analyzer, the fluorescent substance recovered on the particles is released and the particles are removed as a pretreatment before the measurement. Is disclosed.

[0004]

Generally, a method of directly measuring a fluorescently labeled substance on a particle has been used. However, when submicro-sized particles are used, it is difficult to quantitatively detect the fluorescent substance because the excitation light causes Mie scattering. Further, in the analysis method and the method of the automatic analyzer (Japanese Patent Laid-Open No. 2000-105236), the fluorescent labeling substance retained in the particles is released and detected. This method has a problem that it is difficult to remove particles in the case of submicron-sized particles, and the scattering of the excitation light by the particles at the time of measurement cannot be eliminated. Therefore, the present invention is simple and
An object of the present invention is to provide a fluorescence measurement method on the surface of submicroparticles with high detection accuracy.

[0005]

According to the present invention, in the fluorescence measurement in a submicroparticle suspension holding a fluorescent substance, the excitation light wavelength and the fluorescence wavelength are separated from each other by a distance which is not affected by scattering of the excitation light. The fluorescence measuring method on the surface of the sub-microparticle is characterized in that the fluorescence on the particle surface is detected by.

As the fluorescent dyes held by the sub-microparticles, it is preferable that the maximum fluorescence wavelengths are apart from each other so as not to be affected by the wavelength fluctuation of the excitation light caused by the scattering of the sub-microparticles. In addition, fluorescence retained on the surface of the sub-microparticles can be detected by utilizing fluorescence resonance energy transfer using a plurality of fluorescent dyes and increasing the excitation light and the fluorescence wavelength. Fluorescent dyes include fluorescein, rhodamine, cyanine, Oregon G
reen (Distributor: Funakoshi Co., Ltd.), BODIPY
(Distributor: Funakoshi Co., Ltd.), AMCA (Distributor: Funakoshi Co., Ltd.), Alex (Distributor: Funakoshi Co., Ltd.), TAMRA (Distributor: Funakoshi Co., Ltd.), FI
TC (Distributor: Funakoshi Co., Ltd.), Rhodamin
e (Distributor: Funakoshi Co., Ltd.), TRITC (Distributor: Funakoshi Co., Ltd.), Coumarin (Distributor:
Daiichi Pure Chemicals Co., Ltd., Diethylaminoc
oumarin (Distributor: Daiichi Pure Chemicals Co., Ltd.), N
Apthofluorescent (Distributor: Daiichi Pure Chemicals Co., Ltd.), Cy (Distributor: Amersham Pharmacia Biotech Co., Ltd.), Marine Blue
(Distributor: Funakoshi Co., Ltd.), MRITC (Distributor:
In addition to chemical substances such as Funakoshi Co., Ltd., Texas Red (distributor: Funakoshi Co., Ltd.), XRITC (distributor: Funakoshi Co., Ltd.), and derivatives thereof, a fluorescent protein such as green fluorescence protein, Furthermore, any fluorescent substance such as a fluorescent peptide can be used. It is also possible to use the fluorescence of the complex of nucleic acid and nucleic acid intercalating agent. As the nucleic acid intercalating agent, those having no insertion site specificity are preferable, but it is also possible to use fluorescently labeled nucleic acid-binding proteins, peptides and the like. In addition, SYPRO Red
(Distributor: Funakoshi Co., Ltd.), SYPRO Ruby
(Distributor: Funakoshi Co., Ltd.) It is also possible to use a fluorescent dye for protein staining. The particles used in the present invention are preferably water-insoluble because they are used in an aqueous solvent. Further, having a binding functional group on the surface is important in carrying out the present invention. Examples of the water-insoluble particles include fine particle polymers such as polystyrene beads, latex beads, polyvinyl chloride, polyallomers, polyethylene, polypropylene, acryl and polyurethane. In addition, examples of the inorganic beads include metal powder such as iron powder and magnetic fine particles. As magnetic particles, F
e3O4 (Magnetite), γ-Fe2O3 (Magnetite), C
o · γ-Fe2O3, (NiCuZn) O · Fe2O
3, composite particles of (CuZn) O.Fe2O3, Fe3O4 coated with SiO2, various polymer materials (nylon, polyacrylamide, protein) and ferrite can be mentioned. Further, the magnetic fine particles separated from the magnetic bacteria are preferable because they are coated with an organic thin film of phospholipid having a binding functional group. As the binding functional group,
Examples thereof include an amino group, a carboxyl group, a hydroxyl group, and the like, and an amino group is particularly preferable. Examples of the phospholipid having such a binding functional group include phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidylu N-methylethanolamine. The ferrite particles as the magnetically sensitive fine particles can be produced by a known method. In addition, particles other than ferrite particles may be magnetic metal particles or a composite thereof. Furthermore, F coated with SiO2
e3O4 particles, γ-Fe2O3 particles and iron as a main component produced by a thermal plasma method in a liquid layer in which carbon coexists,
Fine particles containing Ni and Co are also included. These particles can be easily manufactured by a known method. Furthermore, magnetic bacterial particles obtained from magnetic bacteria can be extracted from microorganisms after culturing the magnetic bacteria. As the extraction method, a method of crushing microorganisms by physical pressure such as a French press, a method of boiling alkali, an enzyme treatment, or an ultrasonic crushing treatment can be adopted. As a method of retaining the fluorescent dye on the particles, a method of directly chemically crosslinking the functional groups on the surface of the particles and a mutual force between the fluorescently labeled substance and the functional groups present on the particles are used. Method, and further proteins, peptides immobilized on the particle surface,
There is a method of using a nucleic acid or the like and a fluorescently labeled substance having mutual force. As a device for measuring the fluorescence intensity,
Examples thereof include a device having a mechanism for wavelength separation of excitation light and fluorescence using a monochromator, bandpass filter, etc., and a device using a laser as excitation light.

[0007]

EXAMPLES Example 1 The present invention will be described below based on examples. FIG. 1 is a diagram comparing the effects of scattering of excitation light depending on the particle diameter used. Figure 2 shows FITC-labeled submicroparticles, FIT
It is the thing which showed the fluorescence spectrum of C solution. As described above, it has been shown that it is difficult to measure the fluorescence due to the influence of the light scattered by the particles when detecting the fluorescence usually existing on the surface of the sub-microparticles. Instead of a commonly used fluorescent dye, AMCA () was immobilized on the surface of the sub-microparticle as a fluorescent dye having a maximum excitation light wavelength and a maximum fluorescence wavelength largely separated, and the fluorescence spectrum was measured (FIG. 3). As a result, as shown in FIG. 3, it was confirmed that a fluorescence peak was obtained in a region where there was no effect of submicroparticle scattering. Further, the AMCA-labeled submicroparticle solution was diluted and the calibration range was examined (FIG. 4). As a result, the fluorescence spectrophotometer obtained linearity in the range of 1 to 1 × 10 5. In addition, even in a microtiter plate using a bandpass filter, 1 to 1 x 1
It was confirmed that linearity was obtained in the range of 04.
From these, it was shown that it is possible to detect fluorescence even on the surface of submicroparticles without being affected by the scattering of excitation light.

Example 2 Magnetic bacterial particles (50-100 as submicroparticles)
nm) was used. Su to magnetic bacterial particles (1 mg)
1fo-NHS-LC-LC-Biotin 1 mg / m
The magnetic bacterial particles were labeled with biotin by reacting 1 with 1 in a phosphate buffer. By adding 50 μg of streptavidin to 1 mg of biotin-labeled magnetic bacterial particles and reacting them in 1 ml of a phosphate buffer,
Streptavidin-labeled magnetic bacterial particles were prepared. The prepared streptavidin-immobilized magnetic bacterial particles were labeled with a synthetic oligo (5'-AGCAAC) having a 5'end labeled with biotin.
CTGA-3 ′) DNA and Probe A were prepared and immobilized on magnetic bacterial particles using streptavidin-labeled magnetic bacterial particles and avidin-biotin to obtain DNA-immobilized magnetic bacterial particles. For DNA-immobilized magnetic bacterial particles, it has a sequence complementary to ProbeA and has TR at the 5'end.
ITC label (excitation wavelength 530 nm, fluorescence wavelength 570 n
m) Probe B (5'-TCAGGTTGC)
T-3 ′) was hybridized. After hybridization, DNA intercalator (Cyber
Green (excitation wavelength 490nm, fluorescence wavelength 520n
m)), and the fluorescence spectrum was measured (FIG. 5).
As a result, no fluorescence peak could be confirmed at the TRITC excitation wavelength. However, CyberGre
At the excitation wavelength of en, CyberGreen is excited and TRITC is excited by fluorescence resonance energy transfer, and the resulting fluorescence of TRITC is shown to be able to be measured without being affected by particle scattering. It was By using this method, it was shown that it is possible to measure the nucleic acid existing on the submicroparticles in a suspension system using fluorescence.

Example 3 Magnetic bacterial particles (50-100 as submicroparticles)
nm) was used. Su to magnetic bacterial particles (1 mg)
1fo-NHS-LC-LC-Biotin 1 mg / m
The magnetic bacterial particles were labeled with biotin by reacting 1 with 1 in a phosphate buffer. By adding 50 μg of streptavidin to 1 mg of biotin-labeled magnetic bacterial particles and reacting them in 1 ml of a phosphate buffer,
Streptavidin-labeled magnetic bacterial particles were prepared. The prepared streptavidin-immobilized magnetic bacterial particles were labeled with a biotin-labeled synthetic oligo DNA (Probe).
A) was prepared and immobilized on magnetic bacterial particles using streptavidin-labeled magnetic bacterial particles and avidin-biotin to obtain DNA-immobilized magnetic bacterial particles. A 120 bp gene amplification product having a sequence complementary to Probe A for DNA-immobilized magnetic bacterial particles, and Probe A
Has a complementary region in a region different from
TC label (excitation wavelength 530 nm, fluorescence wavelength 570 nm)
Hybridized with the probe C. After hybridization, it was stained with a DNA intercalator (CyberGreen (excitation wavelength: 490 nm, fluorescence wavelength: 520 nm)) and detected with a fluorescence microtiter plate. It was confirmed that measurement is possible without being affected by particle scattering by measuring the fluorescence of TRITC obtained by exciting CyberGreen and exciting TRITC by fluorescence resonance energy transfer at the excitation wavelength of CyberGreen. It was Also,
Almost no fluorescence was detected in the sample containing no gene amplification product. From the above, it was shown that it is possible to detect a nucleic acid using fluorescence in a suspension system of submicroparticles by using this method.

Example 4 Magnetic bacterial particles (50-100 as submicroparticles)
nm) was used. Su to magnetic bacterial particles (1 mg)
1fo-NHS-LC-LC-Biotin 1 mg / m
Immobilization of biotin on magnetic bacterial particles was performed by reacting 1 with 1 in a phosphate buffer. Streptavidin labeled with AMCA as a fluorescent dye was added to 1 mg of biotin-immobilized magnetic bacterial particles and reacted in 1 ml of a phosphate buffer to introduce the fluorescent dye onto the surface of the magnetic fine particles. When the fluorescence intensity was measured by a fluorescence spectrophotometer after the introduction, a peak derived from AMCA was observed. Further, no AMCA-derived peak was observed in the sample to which AMCA-labeled streptavidin was not added. From the above results, the number of Bioti present on the magnetic bacterial particles
It was shown that n can be specifically detected with AMCA-labeled streptavidin.

Example 5 Magnetic bacterial particles (50-100 as submicroparticles)
nm) was used. Su to magnetic bacterial particles (1 mg)
1fo-NHS-LC-LC-Biotin 1 mg /
Magnetic bacterial particles were labeled with biotin by reacting with ml in phosphate buffer. By adding 50 μg of streptavidin to 1 mg of biotin-labeled magnetic bacterial particles and reacting them in 1 ml of a phosphate buffer,
Streptavidin-labeled magnetic bacterial particles were prepared. The target gene is prepared by PCR using a primer containing a region containing the nucleotide polymorphism site designed from the sense strand side with biotin labeling at the 5'end and a primer with fluorescent labeling at the 5'end designed from the antisense strand side. Was prepared by. This time, the region containing the polymorphic site of human aldehyde dehydrogenase 2 (ALDH2) was targeted as DWC.
10 (5'-GCCGCGCCCCGCCGCCCCGC
GCCCCCCCCGCCCGCCCCGCGCTCCA
CACTCACAGTTTTCAC-3 '), DWC1
1 (5'-biotin-CAAATTTACAGGGGT
176 bp was amplified by PCR using CAACTGCTATG-3 ′). Fluorescently labeled ALDH2 * 1 (5'-FIT as a polymorphism detection probe for ALDH2
C-CTGAAGTGAAAACTGTGAGT-
3 ') ALDH2 * 2 (5'-FITC-CTAAAG
TGAAAACTGTGAGT-3 ') was prepared. Two
50 μl of * 2, 2 * 1, 1 * 1 type PCR product
After heat denaturing at 5 ° C for 5 minutes, 50 µg of streptavidin-labeled magnetic bacterial particles were added and reacted at 70 ° C for 10 minutes,
The biotin labeled side of the DNA was collected on magnetic bacterial particles. After recovery, an ALDH2 polymorphism detection probe and a DNA intercalator (POPO-3) were added,
Hybridization was performed at 20 ° C. for 10 minutes. After hybridization, excitation light 490 nm, fluorescence 57
The fluorescence intensity was detected at 0 nm. The result is shown in FIG.
Shown in. ALDH2 * 2 for 2 * 2 type sample
Was twice as bright as ALDH2 * 1. 2 *
In the sample of No. 1, the fluorescence intensity of each detection probe was the same. In addition, 1 * 1 is ALDH2 * 1
Detection probe showed the strongest fluorescence intensity. From the above, it was shown that this method can be used to identify a polymorphism in ALDH2.

Example 6 Magnetic bacterial particles (50-100n) as submicroparticles
m) was used. Sul for magnetic bacterial particles (1 mg)
fo-NHS-LC-LC-Biotin 1 mg / ml
The magnetic bacterial particles were labeled with biotin by reacting them with phosphate buffer, and 50 μg of streptavidin was added to 1 mg of biotin-labeled magnetic bacterial particles.
Streptavidin-labeled magnetic bacterial particles were prepared by reacting in 1 ml of phosphate buffer. Human transforming growth as a target gene
Factor beta-1 (TGFβ-1) was used. 139 containing a single nucleotide polymorphism site in TGFβ-1
a primer (5'-GGCCTC) to amplify bp
CCACCACCACCAG-3 ', 5'-GCGG
GCCAGGCGTCAGCACCAGTA-3 ') was designed and amplified by PCR. The amplified fragment is pGEMT
After cloning into E. coli using the easy vector, sequencing was performed to determine the genotype of the single nucleotide polymorphism site. The target gene was prepared by amplification by PCR using the above-mentioned primer set, using the plasmid whose genotype was determined as a template. When PCR was performed, fluorescent labeling was performed using dUTP-Coumarin. As a detection probe for single nucleotide polymorphism (T → C) in TGFβ-1, Tallele specific
probe (5′-CAGCAGCGAG-bioti
n-3 '), Callelle specific
probe (5'-AGCAGCGGC-biotin
-3 ') was produced. 1 mg of Streptavidin-labeled magnetic bacterial particles for each Tallele specific
probe, Callelle specific
2.5 nmol of each probe is added and reacted in 1 ml of a phosphate buffer solution to obtain Specify.
Cprobe labeled magnetic bacterial particles were made. 50 μl of the PCR product whose genotype at the single nucleotide mutation site was determined was heated to 95 ° C.
After denaturing with heat for 5 minutes, quench with an ice bath to single-stranded DNA
Then, 50 μg of the specific probe-labeled magnetic bacterial particles were added, and a hybridization reaction was carried out at 20 ° C. for 10 minutes while being dispersed using ultrasonic waves. After washing and recovery by magnetic separation using a phosphate buffer, fluorescence intensity was detected with excitation light of 402 nm and fluorescence of 443 nm. The result is shown in FIG. For each TGFβ-1 sample, each designed allelespeci
When detection was carried out using a fic probe, different fluorescence intensity patterns were obtained. From the above, it was shown that this method can be used to detect single nucleotide polymorphisms in TGFβ-1.

[0013]

As described above, it has been confirmed that the use of the present method makes it possible to quantify the fluorescence amount without releasing the fluorescent dye from the sub-microparticles. It is expected that the fluorescence detection in the particle system will be facilitated by using this method.

[Brief description of drawings]

FIG. 1 is a diagram comparing effects of scattering of excitation light due to particle diameter used.

FIG. 2 FITC-labeled submicroparticles, FITC
It is a fluorescence spectrum figure of a solution.

FIG. 3 is a fluorescence spectrum diagram in which AMCA () is immobilized as a fluorescent dye on the surface of submicroparticles and measured.

FIG. 4 is a calibration curve obtained by diluting an AMCA-labeled submicroparticle solution.

FIG. 5 is a fluorescence spectrum diagram measured by dyeing with a DNA intercalator (CyberGreen (excitation wavelength 490 nm, fluorescence wavelength 520 nm)) after hybridization.

FIG. 6 is a diagram showing the result of detection of an ALDH2 gene polymorphism.

FIG. 7 is a diagram showing detection of TGFβ-1 gene polymorphisms.

Claims (15)

[Claims] 1. In fluorescence measurement in a sub-microparticle suspension containing a fluorescent substance, the fluorescence on the particle surface is detected by separating the excitation light wavelength and the fluorescence wavelength from a distance that is not affected by scattering of the excitation light. A method for measuring fluorescence on the surface of submicroparticles, which comprises: 2. A method for measuring fluorescence on the surface of submicroparticles, which uses a fluorescent substance having a difference between the maximum excitation light and the maximum fluorescence wavelength of the fluorescent substance used in claim 1 of 50 nm or more. 3. The excitation light wavelength and the fluorescence wavelength are set to 50 by using two or more kinds of the fluorescent substances used in claim 1.
A method for measuring fluorescence on the surface of submicroparticles, characterized by giving a difference of nm or more. 4. The method for measuring fluorescence on the surface of sub-microparticles according to claim 1, wherein the measurement device uses a fluorescence spectrophotometer having a monochromator. 5. The fluorescence measuring device using a bandpass filter as the measuring device, wherein the measuring device is a fluorescence measuring device.
The method for measuring fluorescence on the surface of submicroparticles according to any one of 1. 6. As a mode of retaining the fluorescent substance on the particles, the substance A is immobilized on the particles, the fluorescent substance is immobilized on the substance B having mutual force with the substance A, and the mutual interaction between the substance A and the substance B is achieved. The method for measuring fluorescence on the surface of sub-microparticles according to any one of claims 1 to 5, wherein the amount of the fluorescent substance of substance B existing on the surface of the particles is measured by an inter-force. 7. A substance A-immobilized particle having a substance A (having a strong mutual force on the substance C) immobilized on the particle and a substance B (fluorescein-labeled substance) as a mode of being retained on the particle. C has a strong mutual force) and the substance C to be measured
To react the substance C on the substance A-immobilized particles.
And the fluorescent labeling substance B are further bound to form a complex of the substance ACB on the particles, and the substance-B
Substance C by measuring the fluorescent substance labeled on
The method for measuring fluorescence on the surface of sub-microparticles according to any one of claims 1 to 5, characterized in that 8. The substance A or B according to claim 6 or 7,
Alternatively, any one of C and C is a protein or peptide, and a fluorometric method on the surface of submicroparticles. 9. The substance A, B according to claim 6 or 7,
Alternatively, either C or C is a nucleic acid, and a fluorometric method on the surface of submicroparticles. 10. The substance A according to claim 6 or 7,
A fluorescence measurement method on the surface of submicroparticles, wherein either B or C is a nucleic acid, protein or peptide. 11. The method for measuring fluorescence on the surface of submicroparticles according to claim 8, wherein streptavidin is used in place of the crosslinking agent as a method for immobilizing the substance A on the particle surface. . 12. The sub-microparticle according to claim 1, which has a particle size of 50 nm to 50 nm.
A method for measuring fluorescence on the surface of submicroparticles, characterized by using particles of 1 μm. 13. The method for measuring fluorescence on the surface of sub-microparticles according to claim 1, wherein the particles used are fine particle polymer, glass, or magnetic particles. 14. A method for measuring fluorescence on the surface of sub-microparticles, wherein magnetic particles are used as the magnetic particles used in claim 13. 15. A method for measuring fluorescence on the surface of sub-microparticles, wherein magnetic bacterial particles are used as the magnetic particles used in claim 13.