JP2005189237A - Optical measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an optical measuring method which enables the sensitive and precise measurement of materials being objects to be measured contained in samples, which are protein, peptide, nucleic acid, nucleic-acid base, its derivatives, nucleotide, hormone, organic compounds such as sugars etc., and the like. <P>SOLUTION: In the optical measuring method, a fluorescent material immobilized on a surface of a substrate is excited by evanescent waves produced by bringing exciting light to enter the inside of the substrate and to perform internal reflections, and intensity of emitted fluorescence is measured directly. The fluorescent material for the optical measuring method is composed of a semiconductor nanoparticle. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

The present invention is an optical that can measure with high sensitivity and high accuracy organic substances such as proteins, peptides, nucleic acids, nucleobases and their derivatives, nucleotides, hormones, saccharides, etc., which are substances to be measured contained in a sample. It relates to a measurement method.

In recent years, as a method for measuring biologically related substances such as proteins and nucleic acids contained in living bodies with high sensitivity, the intensity of fluorescence emitted by binding a fluorescent substance as a marker substance and irradiating the fluorescent substance with excitation light is increased. Many methods are used for measurement.

As an optical measurement method using such a fluorescent material, for example, Patent Document 1 discloses an evanescent wave generated by causing excitation light to enter the optical waveguide and propagating through the optical waveguide while totally reflecting the excitation light. A method is disclosed in which a fluorescent substance fixed on the surface of an optical waveguide is excited by an antigen-antibody reaction or the like, the generated fluorescence is propagated through the optical waveguide and emitted, and the intensity of the emitted fluorescence is measured. According to this method, it is possible to perform relatively high-sensitivity measurement by amplifying the weak fluorescence generated by the optical waveguide. However, Patent Document 1 uses a fluorescent substance having an absorption peak near 650 nm and uses excitation light near 635 nm, but the wavelength difference (Stokes shift) between the excitation light and fluorescence is not sufficient, and is sufficient. Measurement sensitivity and measurement accuracy could not be obtained. In addition, there is a problem in that it is necessary to clean the optical waveguide for each measurement and a plurality of substances cannot be continuously measured.

On the other hand, Patent Document 2 excites a fluorescent dye in a sample by using an evanescent wave generated by making excitation light enter the optical waveguide and propagating through the optical waveguide while totally reflecting the excitation light. A method for directly measuring the intensity of the fluorescent light is disclosed. That is, in this method, the fluorescence generated on the surface of the optical waveguide is directly measured instead of measuring the fluorescence by propagating it through the optical waveguide as in the method described in Patent Document 1. According to this method, it is possible to continuously measure a plurality of samples by dividing the surface of the optical waveguide into various subsections and exciting the fluorescent dye in each subsection. However, this method does not provide sufficient fluorescence intensity because there is no amplification by the optical waveguide. In addition, the problem of wavelength difference (Stokes shift) between the excitation light and fluorescence is not solved. Measurement accuracy could not be obtained. In addition, since a fluorescent dye is used, there is a problem in that the fluorescence intensity decreases or decays in a short time depending on the coexisting substances in the sample.

Japanese Patent No. 3362206 Japanese Patent No. 3187386

In view of the above situation, the present invention measures highly sensitive and highly accurate organic compounds such as proteins, peptides, nucleic acids, nucleobases and their derivatives, nucleotides, hormones, saccharides, etc., which are substances to be measured contained in a sample. It is an object of the present invention to provide an optical measurement method that can be used.

The present invention relates to an optical measurement method in which a fluorescent substance fixed on the surface of the substrate is excited by an evanescent wave generated by injecting and internally reflecting excitation light into the substrate, and the intensity of the generated fluorescence is directly measured. In this optical measurement method, the fluorescent material is made of semiconductor nanoparticles.
The present invention is described in detail below.

As a result of intensive studies, the present inventors have been able to emit extremely high-intensity fluorescence and sufficiently take the wavelength difference (Stokes shift) between excitation light and fluorescence if semiconductor nanoparticles are used as the fluorescent substance. Therefore, even when directly measuring the intensity of the fluorescence emitted by the evanescent wave and emitted from the substrate surface, it was found that high measurement sensitivity and measurement accuracy can be obtained, and the present invention has been completed. .
In this specification, the direct measurement of the intensity of the fluorescence means that the intensity of the generated fluorescence itself is measured as it is, instead of measuring the fluorescence by propagating it through the substrate and emitting it.

In the optical measurement method of the present invention, a fluorescent substance fixed in the vicinity of the surface of the substrate is excited by an evanescent wave generated by the excitation light entering the substrate and internally reflected, and the intensity of the generated fluorescence is directly measured. In this method of directly measuring the fluorescence generated on the surface of the substrate, the surface of the substrate is divided into various subsections, and a plurality of samples are continuously obtained by exciting the fluorescent substance in each subsection. It becomes possible to measure.

The substrate is not particularly limited as long as the laser beam can be totally reflected. For example, a resin plate or a glass plate is preferable. Among these, the optical waveguide is preferable because it is excellent in reflection of laser light and the laser light propagates through the optical waveguide, so that various measurements can be performed on one optical waveguide.
Although it does not specifically limit as glass which comprises the said glass plate, For example, quartz glass etc. are suitable.
Although it does not specifically limit as resin which comprises the said resin plate, For example, an acrylic resin, a polyester resin, a polystyrene resin, a polycarbonate resin, an epoxy resin, a fluororesin etc. are suitable. Among them, it is preferable to select one that does not absorb excitation light.

Although it does not specifically limit as thickness of the said board | substrate, A preferable minimum is 5 micrometers and a preferable upper limit is 1000 micrometers, A more preferable minimum is 10 micrometers and a more preferable upper limit is 150 micrometers.

As the light source of the excitation light, for example, a monochromatic laser or a light emitting diode can be used.
When such excitation light is incident at a certain angle from the end of the substrate, the incident excitation light is totally reflected and propagates through the optical waveguide when the substrate is an optical waveguide. When the excitation light is totally reflected in the substrate, an evanescent wave is generated in the reflecting portion and oozes out to the surface of the substrate. When a fluorescent material is fixed on the surface of the substrate, the fluorescent material is excited by an evanescent wave and emits fluorescence.

In the optical measurement method of the present invention, semiconductor nanoparticles are used as the fluorescent material.
In this specification, the semiconductor nanoparticle means a semiconductor material such as CdS, ZnS, CdSc or the like made into fine particles having a particle diameter of nanometer order.
The semiconductor nanoparticles have a property of emitting fluorescence by photoexcitation when the particle diameter is about 10 nm or less. The wavelength necessary for exciting the semiconductor nanoparticles is broad on the ultraviolet side, and the longer wavelength end of the excitation spectrum shifts to the longer wavelength side as the particle diameter increases. The fluorescence wavelength also shifts to the longer wavelength side as the particle size of the semiconductor nanoparticles increases. As described above, the semiconductor nanoparticles have a feature that the fluorescence wavelength and the excitation light wavelength vary depending on the particle diameter. Therefore, it is possible to measure a plurality of objects simultaneously by binding different reactants to semiconductor nanoparticles having different particle diameters.

The semiconductor nanoparticles emit extremely high-intensity fluorescence when irradiated with excitation light, and thus can be suitably used for highly sensitive measurement of trace components. In addition, since the excitation light wavelength and the fluorescence wavelength can be adjusted by the particle diameter, it is possible to sufficiently take the wavelength difference (Stokes shift) between the excitation light and the fluorescence.

The semiconductor nanoparticles used in the present invention include, for example, semiconductor crystals such as a group I-VII compound semiconductor such as CuCl, a group II-VI such as CdS and CdSe, a group III-V compound semiconductor such as InAs, and a group IV semiconductor, metal oxides such as TiO _2, phthalocyanine, made of an organic compound such as azo compounds, or composite materials thereof. As such a composite material, for example, CdS is core-CdSe shell, CdSe is core-CdS shell, CdS is core-ZnS shell, CdSe is core-ZnS shell, CdSe nanocrystal is core-ZnS. a shell, the core -ZnSe shell nanocrystals CdSe, core the core -SiO ₂ shell and Si - such as those having a shell structure.

Moreover, in order to disperse and stabilize the semiconductor nanoparticles in an aqueous solution, various dispersants that coat the surface of the semiconductor nanoparticles can be used.
Examples of such a dispersant include a thiol, an amine, a phosphine, or a phosphine oxide as a bonding group to the semiconductor nanoparticles, and in the outermost layer for dispersion in an aqueous solution and labeling of an antibody or an antigen. Examples include those that form carboxylates, amines, hydroxides, polyethylene glycol and other polyols, sulfonates, phosphates, phosphonates, and the like.

The preferable lower limit of the particle diameter of the semiconductor nanoparticles is 3 nm, and the upper limit is 10 nm.
The shape of the semiconductor nanoparticles is not particularly limited, and examples thereof include a spherical shape, a rod shape, a plate shape, a thin film shape, a fiber shape, and a tube shape. Of these, spherical is preferable.

In the optical measurement method of the present invention, the fluorescent substance composed of the semiconductor nanoparticles is excited and the intensity of the generated fluorescence is directly measured.
The method for measuring the fluorescence intensity is not particularly limited, and a conventionally known method can be used. Among these, the single-molecule fluorescence image analyzer method is particularly suitable because the particle diameter of the semiconductor nanoparticles is on the order of nanometers and the emission intensity is high, so that it can be measured with high sensitivity at the single-molecule level.

In the optical measurement method of the present invention, two or more kinds of substances to be measured can be measured simultaneously. Simultaneous measurement of two or more types of samples includes measurement of a sample in which two or more types of measurement target substances are mixed in the sample, or measurement target substances that are separately present at a plurality of locations on a substrate. Is carried out in one measurement. The method for detecting the two or more types of measurement objects is not particularly limited. For example, as described above, in the optical measurement method of the present invention, the surface of the optical waveguide used as the substrate is divided into various subsections. By using a plurality of semiconductor nanoparticles having different particle diameters in combination, a plurality of samples can be continuously measured.
In the above measurement, the method for measuring the concentration of the measurement target substance contained in each sample from the detection light is not particularly limited. For example, the detection light is split into single light using prism spectroscopy or the like. It becomes possible to measure.

Next, an embodiment of a specific method for measuring an organic compound or the like contained in a sample using the optical measurement method of the present invention will be described in detail. In this embodiment, an antibody is used as a molecular recognition substance, and the concentration of a specific protein contained in a sample is measured using an antigen-antibody reaction.

In this embodiment, first, an antibody corresponding to a protein to be measured is prepared and used as a molecular recognition substance. As said antibody, a polyclonal antibody or a monoclonal antibody is mentioned, for example, Any of these can be used conveniently.
The antibody can be prepared, for example, by fusing spleen cells obtained from the spleen of an immunized mouse immunized with the protein to be measured with antibody-producing cells and culturing the fused cells.

The antibody thus prepared is coated on the surface of the substrate. The method of coating is not particularly limited. For example, the antibody solution is dropped on the surface of the substrate, allowed to stand at a constant temperature for a predetermined time, washed with a buffer solution, and then fixed by adding serum albumin (BSA) or the like. Etc.

On the other hand, a molecule recognizing fluorescent material is prepared by binding an antibody as a molecular recognizing material and semiconductor nanoparticles as a fluorescent material. The antibody coated on the substrate and the antibody used as the molecular recognition substance may be the same or different.
The method for binding the antibody and the semiconductor nanoparticles is not particularly limited. For example, the semiconductor nanoparticle-avidin complex may be formed by further coating avidin via the functional group of the dispersant coated on the surface of the semiconductor nanoparticle. On the other hand, an antibody-biotin complex in which an antibody and biotin are bound is prepared. The obtained semiconductor nanoparticle-avidin complex and antibody-biotin complex can be easily bound via albumin.
In this way, the method of conjugating semiconductor nanoparticles and molecular recognition substances via avidin-albumin-biotin is a composite of not only proteins such as antibodies but also nucleic acids such as DNA and many organic compounds and semiconductor nanoparticles. It can be used for conversion.

In order to measure a protein that is a measurement target substance in a sample, first, the sample and the molecule-recognized fluorescent substance are mixed. Thereby, the protein in the sample specifically binds to the antibody in the molecule-recognizing fluorescent substance by the antigen-antibody reaction.
Next, when the mixed solution is dropped onto the surface of the substrate coated with the antibody, the protein and the antibody on the surface of the substrate are bound by the antigen-antibody reaction. In this way, semiconductor nanoparticles corresponding to the amount of protein in the sample are fixed on the surface of the substrate. Alternatively, after the antigen is immobilized on the surface of the substrate, semiconductor nanoparticles labeled with an antibody are dropped, so that the semiconductor nanoparticles are immobilized according to the amount of antigen immobilized on the surface of the substrate.
In this state, when excitation light is incident at an angle that totally reflects from the edge of the substrate to generate an evanescent wave, fluorescence is emitted from the semiconductor nanoparticles on the surface of the substrate by the evanescent wave.
If a calibration curve is prepared by measuring a sample having a known protein concentration in advance, the amount of protein contained in the unknown sample can be measured.
In addition, by dropping the sample on the surface of the substrate coated with the antibody and binding the protein in the sample to the antibody on the substrate surface by antigen-antibody reaction, dropping the above-mentioned molecular recognition fluorescent substance on this substrate surface, The protein in the sample bound to the substrate surface by the antigen-antibody reaction may be specifically bound to the antibody in the molecule-recognized fluorescent substance, and the semiconductor nanoparticles may be immobilized on the substrate surface according to the amount of the protein in the sample. .

In this embodiment, the antibody immobilized on the substrate surface and / or the semiconductor nanoparticles is preferably an antibody fragment obtained by excising the Fc portion. Since the intensity of the fluorescence emitted from the semiconductor nanoparticles on the substrate surface by the evanescent wave is attenuated as the distance between the substrate surface and the semiconductor nanoparticles increases, the antibody immobilized on the substrate surface and / or the semiconductor nanoparticles is the antibody fragment. By doing so, the distance between the substrate surface and the semiconductor nanoparticles is shortened, and the fluorescence emitted from the semiconductor nanoparticles by the evanescent wave becomes higher in intensity.
Such an antibody fragment is preferably F (ab ′) ₂ or Fab.

As a method for producing the F (ab ′) ₂ antibody fragment, for example, when the sample is human alphafetoprotein (AFP), the solution in which the sample is dissolved and the pepsin-immobilized gel solution are mixed and incubated. And a method of separating F (ab ′) ₂ using a protein A-immobilized gel or the like.
The AFP Fab antibody fragment can be prepared, for example, by mixing a solution prepared by dissolving a sample and a cysteine / HCl solution into a papain-immobilized gel ( For example, a method may be used in which a papain-immobilized gel solution in which cross-linked agarose is equilibrated is added and incubated, and then Fab is separated using a protein A-immobilized gel or the like.

In this embodiment, the antigen-antibody reaction is used as a method for immobilizing the semiconductor nanoparticles on the surface of the substrate, but any other specific binding reaction such as a hybridization reaction between nucleic acids can be used. . By the optical measurement method of the present invention, proteins, peptides, nucleic acids, nucleobases and their aggregates, nucleotides, hormones, saccharides, and other organic compounds can be analyzed.

When measuring proteins contained in a sample according to this embodiment, the substrate is coated with a protein or the like as a measurement target substance, and a fluorescent substance is bound to the surface of the substrate by binding a molecular recognition fluorescent substance to the protein. Is fixed. Accordingly, there is a coating layer made of the protein to be measured between the substrate and the fluorescent substance, and in some cases, the thickness of the coating layer may be about several tens of nm. Since the obtained fluorescence intensity is affected by the distance between the substrate and the fluorescent material, if the distance is about several tens of nanometers due to the coating layer, sufficient fluorescence intensity may not be obtained.
In such a case, another substrate is placed on the fluorescent material fixed on the surface of the substrate so as to be in contact with the fluorescent material, and excitation light is incident on the other substrate and internally reflected. Since the other substrate and the fluorescent substance are extremely close to each other, the fluorescent substance is excited by an evanescent wave generated by incident and internally reflecting excitation light into the other substrate, and high intensity fluorescence is generated. By directly measuring this, measurement with extremely high sensitivity becomes possible.
A surface of the first substrate is formed by an evanescent wave generated by placing the second substrate on a fluorescent material fixed on the surface of the first substrate, and entering excitation light into the second substrate and internally reflecting the light. An optical measurement method for directly measuring the intensity of the generated fluorescence by exciting the fluorescent material fixed to the substrate and the fluorescent material is made of semiconductor nanoparticles is also one aspect of the present invention.

According to the present invention, proteins, peptides, nucleic acids, nucleobases and their derivatives, organic compounds such as nucleotides, hormones, and saccharides that are substances to be measured contained in a sample can be measured with high sensitivity and high accuracy. An optical measurement method can be provided.

Hereinafter, the present invention will be described in more detail with reference to examples. However, the present invention is not limited to these examples.

(Example 1)
(1) Preparation of monoclonal antibody against human alphafetoprotein (AFP) A dispersion in which AFP is sufficiently dispersed in Freund's complete adjuvant is prepared, and 100 μL of this dispersion is used to immunize Balb / c mice 4 times every 2 weeks. did.
The spleen was removed the resulting immunized mice to obtain 10 ⁶ splenocytes were digested. A fused cell was prepared by culturing the obtained spleen cell and mouse myeloma cell in the presence of polyethylene glycol, and cultured. The supernatant of each proliferated fusion cell was collected and examined for the presence of anti-AFP antibody by ELISA. AFP positive cells were tested by the limiting dilution method, and cells producing anti-AFP antibodies were confirmed. Anti-AFP antibody-producing cells were cultured in large quantities and injected into the peritoneal cavity of mice. Ascites was collected every 3 days from 2 weeks later, and two types of monoclonal antibodies (IgG) having different AFP recognition sites, anti-AFP-1 antibody and anti-AFP-2 antibody, were obtained.

(2) Preparation of biotinylated anti-AFP antibody-1 Anti-AFP antibody-1 was dissolved in 10 mM HEPES buffer (pH 8.5) to prepare a 0.3 mg / mL solution of 500 μL.
To the obtained anti-AFP antibody-1 solution, 10 μL of a biotinylation reagent (20 mM Biotinamidocapate-NHS, manufactured by Nakarai Tech Task Co., Ltd.) was added and mixed, and then incubated at 25 ° C. for 2 hours. Then, using a PD-10 column (manufactured by Amersham Pharmacia) equilibrated with a phosphate buffer solution (pH 7.2), the unreacted biotinylation reagent was removed and purified to obtain a biotinylated anti-AFP. Antibody-1 was obtained.

(3) Preparation of anti-AFP antibody-1 immobilized semiconductor nanoparticles Semiconductor nanoparticles (Qdbt605 streptavidin conjugate, core part: CdSc, shell part: ZnS, particle size 10-15 nm, manufactured by Qdot) 2% bovine serum Dilute 10-fold with albumin (BSA) -50 mM borate buffer (pH 8.3).
500 μL of the obtained semiconductor nanoparticle dispersion and 1 mL of biotinylated anti-AFP antibody-1 solution were mixed and allowed to stand at room temperature for 3 hours. Next, this mixture solution was equilibrated with 2% bovine serum albumin (BSA) -phosphate buffer solution (pH 7.2) using a PD-10 column (manufactured by Amersham Pharmacia), and an unfixed biotinylated anti-antibody. AFP antibody-1 was removed and purified to obtain an anti-AFP antibody-1-fixed semiconductor nanoparticle dispersion.

(4) An optical waveguide made of an acrylic resin having a thickness of 100 μm was used as a covering substrate of the substrate. The optical waveguide was immersed in 30% ethanol for 3 minutes and then washed with distilled water.
The anti-AFP-2 antibody solution was prepared by dissolving the anti-AFP-2 antibody in 0.1 M Tris-HCl buffer (pH 9.0) to a concentration of 1 mg / mL. The optical waveguide was immersed in the obtained anti-AFP-2 antibody solution and stirred at 30 ° C. for 1 hour. The optical waveguide was washed by immersing it in 0.1 M Tris-HCl buffer (pH 9.0), and then immersed in 1% bovine serum albumin (BSA) -phosphate buffer (pH 7.2). After standing for a period of time, the surface was washed with a phosphate buffer solution (pH 7.2) to prepare an anti-AFP-2 antibody-coated optical waveguide.
The obtained anti-AFP-2 antibody-coated optical waveguide was stored at 4 ° C. in a phosphate buffer solution (pH 7.2) until use.

(5) Preparation of calibration curve AFP was dissolved in a phosphate buffer solution (pH 7.2) to prepare 0.5, 2, 5 and 10 ng / mL solutions.
After mixing 10 μL of each concentration of AFP solution and 100 μL of anti-AFP antibody-1 immobilized semiconductor nanoparticle dispersion, this mixture was dropped onto the surface of the anti-AFP-1 antibody-coated optical waveguide.
The anti-AFP-1 antibody-coated optical waveguide was incident from the side through a lens at an angle that totally reflected laser light having a wavelength of 530 nm, and an evanescence wave was generated at a portion where the mixed solution was dropped. The resulting fluorescence near the wavelength of 610 nm was detected with a CCD camera, and the fluorescence intensity was measured.
The results are shown in Table 1.
A calibration curve was prepared based on the results shown in Table 1. This is shown in FIG.

(Example 2)
A sample IgG solution of human alphafetoprotein (AFP) was dialyzed against 20 mM acetate buffer (pH 4.5), and the IgG concentration was adjusted to 10 mg / mL to prepare a sample solution.
A pepsin-immobilized gel solution was prepared by equilibrating 0.125 mL of pepsin-immobilized gel (crosslinked agarose) in 0.5 mL of buffer (20 mM acetate buffer, pH 4.5).
1 mL of the prepared sample solution was put into a pepsin-immobilized gel solution and incubated on a shaker at 37 ° C. for 4 hours.
Thereafter, the pepsin-immobilized gel is separated from IgG and its degradation product (Fc, F (ab ′) ₂ ) by centrifugation, and further, the F (ab ′) ₂ antibody fragment against AFP using the protein A-immobilized gel. Only separated.

Instead of anti-AFP antibody-1 and anti-AFP antibody-2, biotinylated anti-AFP (F (ab ') was used in the same manner as in Example 1 except that the F (ab') ₂ antibody fragment against AFP thus obtained was used. ₂ ) Antibody and anti-AFP (F (ab ′) ₂ ) antibody-immobilized semiconductor nanoparticles were prepared, and an anti-AFP (F (ab ′) ₂ ) elementary antibody-coated optical waveguide was prepared.
The obtained anti-AFP (F (ab ′) ₂ ) antibody-coated optical waveguide was stored at 4 ° C. in a phosphate buffer solution (pH 7.2) until use.

(Measurement of fluorescence intensity)
AFP was dissolved in phosphate buffer (pH 7.2) to prepare 0.5, 2, 5 and 10 ng / mL solutions.
After mixing 10 μL of each concentration of AFP solution and 100 μL of anti-AFP (F (ab ′) ₂ ) antibody-immobilized semiconductor nanoparticle dispersion, this mixture was mixed with anti-AFP (F (ab ′) ₂ ) antibody-coated optical waveguide. It was dripped on the surface.
The anti-AFP (F (ab ′) ₂ ) antibody-coated optical waveguide is incident through a lens from the side at an angle that totally reflects a laser beam having a wavelength of 530 nm, and an evanescence wave is generated at a portion where the mixed solution is dropped. . The resulting fluorescence near the wavelength of 610 nm was detected with a CCD camera, and the fluorescence intensity was measured.
As a result, the fluorescence intensity increased by about 50% compared to Example 1.

(Example 3)
A sample IgG solution of human alphafetoprotein (AFP) is dialyzed against 20 mM sodium phosphate solution (pH 7.0) containing 10 mM EDTA, the IgG concentration is adjusted to 20 mg / mL, and the cysteine / HCl solution is adjusted to 20 mM. A sample solution was prepared by addition.
A papain-immobilized gel solution (crosslinked agarose) was equilibrated in a 20 mM cysteine / HCl solution to prepare a papain-immobilized gel solution.
The sample solution was added to the prepared papain-immobilized gel solution and incubated on a shaker at 37 ° C. for 5 hours to overnight.
Thereafter, the papain-immobilized gel and IgG and their degradation products (Fc, Fab) are separated by centrifugation, a 10 mM Tris-HCl (pH 7.5) solution is added to the supernatant, and a protein A-immobilized gel is used. Only Fab antibody fragments against AFP were isolated.

Instead of anti-AFP antibody-1 and anti-AFP antibody-2, a biotinylated anti-AFP (Fab) antibody and anti-AFP (Fab) were used in the same manner as in Example 1 except that the Fab antibody fragment against the obtained AFP was used. Fixed semiconductor nanoparticles were prepared, and an anti-AFP (Fab) antibody-coated optical waveguide was further produced.
The obtained anti-AFP (Fab) antibody-coated optical waveguide was stored at 4 ° C. in a phosphate buffer solution (pH 7.2) until use.

(Measurement of fluorescence intensity)
AFP was dissolved in phosphate buffer (pH 7.2) to prepare 0.5, 2, 5 and 10 ng / mL solutions.
After mixing 10 μL of the AFP solution of each concentration and 100 μL of the anti-AFP (Fab) antibody-immobilized semiconductor nanoparticle dispersion, this mixture was dropped onto the surface of the anti-AFP (Fab) antibody-coated optical waveguide.
The anti-AFP (Fab) antibody-coated optical waveguide was incident from the side through a lens at an angle that totally reflected a laser beam having a wavelength of 530 nm, and an evanescence wave was generated at a portion where the mixed solution was dropped. The resulting fluorescence near the wavelength of 610 nm was detected with a CCD camera, and the fluorescence intensity was measured.
As a result, the fluorescence intensity increased by about 50% compared to Example 1.

Example 4
(1) Preparation of antibody-immobilized semiconductor nanoparticle dispersion solution Two types of semiconductor nanoparticles having different particle diameters with CdS as a core and ZnS as a shell are mixed with a phosphate buffer (pH 7) using mercaptoundecanoic acid as a particle dispersant. 2) dispersed in 1) and coupled with cysteine using 1-ethyl-3 (3-diethylaminopropyl) -carbodiimide crosslinking reagent (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide cross-linking reagents (EDC, Pierce)) I let you. Further, semiconductor nano-crystals having a light emission wavelength peak at 520 nm using succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (cyclohexane-1-carboxylate (SMCC, Pierce Biotech)). The particles (QD520) include human-derived anti-diphtheria toxin antibodies, and the semiconductor nanoparticles (QD640) having an emission wavelength peak at 640 nm include rabbit-derived anti-tetanus toxin antibodies in the amount of phosphate buffer. It was made to bind to 1 μg / 100 μL.

(2) Two-item simultaneous measurement of fluorescence intensity using a single-molecule fluorescence image analyzer 100 μL of a mixed solution of diphtheria toxin and tetanus toxin at a predetermined concentration is dropped on a cover glass, and 100 μL of the antibody-immobilized semiconductor nanoparticle solution prepared thereon is added thereto. It was dripped.
Two items of diphtheria toxin and tetanus toxin were simultaneously measured using a condenser-type total reflection fluorescence microscope system BX2W1-TIRFM (manufactured by Olympus).
In addition, the concentration ratio (diphtheria toxin (ng / mL): tetanus toxin (ng / mL)) of the mixed solution of diphtheria toxin and tetanus toxin dropped on the cover glass is (100: 100) and (100: 10), respectively. ), (10: 100), (10:10), (1:10), (10: 1), (1: 1), (0: 0), and adjusted to 5 μm × 5 μm for each. The number of fluorescence was counted in the visual field, and the average value and standard deviation of the fluorescence intensity were measured.
The results are shown in FIG. In FIG. 2, the upper photo shows the state of simultaneous detection of single-molecule fluorescence of diphtheria toxin, the lower photo shows the state of simultaneous detection of single-molecule fluorescence of tetanus toxin, and the numbers below each photo are The average value and standard deviation of fluorescence intensity are shown.

According to the present invention, proteins, peptides, nucleic acids, nucleobases and their derivatives, organic compounds such as nucleotides, hormones, and saccharides that are substances to be measured contained in a sample can be measured with high sensitivity and high accuracy. An optical measurement method can be provided.

2 is a calibration curve of AFP concentration prepared in Example 1. FIG. 4 is a measurement result of fluorescence intensity by a single molecule fluorescence image analyzer measured in Example 4. FIG. 4 is a measurement result of fluorescence intensity by a single molecule fluorescence image analyzer measured in Example 4. FIG.

Claims (8)

An optical measurement method for directly measuring the intensity of generated fluorescence by exciting a fluorescent substance fixed on the surface of the substrate by evanescent waves generated by entering and internally reflecting excitation light into the substrate. An optical measurement method, wherein the substance comprises semiconductor nanoparticles. A surface of the first substrate is formed by an evanescent wave generated by placing a second substrate on a fluorescent material fixed on the surface of the first substrate, and entering excitation light into the second substrate and internally reflecting the excitation light. An optical measurement method for exciting a fluorescent substance fixed on a substrate and directly measuring the intensity of the generated fluorescence, wherein the fluorescent substance is made of semiconductor nanoparticles. The optical measurement method according to claim 1 or 2, wherein two or more kinds of substances to be measured are measured simultaneously. The optical measurement method according to claim 1, wherein the substrate is an optical waveguide. The optical measurement method according to claim 1, wherein the semiconductor nanoparticles have a particle diameter of 3 to 10 nm. 6. The optical measurement method according to claim 1, wherein the fluorescence intensity is measured by a single molecule fluorescence image analyzer method. 7. The optical measurement method according to claim 1, 2, 3, 4, 5 or 6, wherein an antibody fragment obtained by cleaving the Fc portion is immobilized on the substrate surface and / or the semiconductor nanoparticles. The optical measurement method according to claim 7, wherein the antibody fragment is F (ab ') ₂ or Fab.