JP6119607B2 - Sample dilution solution, kit using the same, and fluorescence measurement method - Google Patents

Description

  The present invention mainly uses a biological sample as a specimen using a fluorescence measurement method such as surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) or total reflection ATR (Attenuated Total Reflection) fluorescence method. The present invention relates to a specimen dilution solution for diluting a specimen when detecting an extremely small amount of analyte, a kit using the same, and a fluorescence measurement method.

  2. Description of the Related Art Conventionally, when detecting a very small amount of substance, various specimen detection apparatuses that can detect such a substance by applying a physical phenomenon of the substance have been used. One example of such an analyte detection apparatus is an SPFS apparatus that can perform analyte detection with high accuracy based on the principle of surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) applying the surface plasmon resonance phenomenon. .

  In this surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), the surface plasmon light is applied to the surface of the metal thin film under the condition that the excitation light such as laser light irradiated from the light source is attenuated total reflection (ATR) on the surface of the metal thin film. By generating (coherent wave), the amount of photons contained in the excitation light irradiated from the light source is increased to several tens to several hundred times, and the electric field enhancement effect of the surface plasmon light is obtained.

  And by this electric field enhancement effect, the fluorescent substance bound (labeled) with the analyte trapped near the surface of the metal thin film is efficiently excited, and by observing this fluorescence, an extremely small amount of analyte can be obtained. It is a method of detection.

  By the way, in such SPFS measurement, it is conceivable to use carboxymethyldextran (CMD) capable of immobilizing an antibody for capturing an analyte at a high density as a solid support for the ligand. Here, “ligand” refers to a molecule or molecular fragment that can specifically recognize (or be recognized) and bind to an analyte contained in a specimen.

  Although this CMD itself is generally considered to be relatively difficult to cause non-specific adsorption, in fact, CMD having various molecular weights and having a wide molecular weight distribution is used as a solid support. In this case, as shown in the schematic diagram of FIG. 4, various contaminants 1 such as proteins and lipids in the specimen adhere to the solid support 3 and the ligand 4, and the solid support 3 and the ligand In some cases, the fluorescently labeled antibody 5 may adhere to the contaminant 1 attached to 4.

  In this way, using surface plasmon excitation enhanced fluorescence spectroscopy (SPFS), in particular in the fields of medicine, biotechnology, and the like, in particular, biological samples such as serum, plasma, whole blood, nasal fluid, and peritoneal fluid are used as specimens. When detecting a very small amount of the analyte 6, various contaminants 1 such as proteins and lipids in the specimen adhere to the solid support 3 and the ligand 4, and the solid support 3 and the ligand 6 are detected. In some cases, the fluorescently labeled antibody 5 adheres nonspecifically to the contaminant 1 attached to 4.

  Thus, when the fluorescently labeled antibody 5 is attached to the solid support 3 or the ligand 4 even slightly through the contaminants 1, the refractive index changes, and the electric field enhancement effect is affected. There is a problem in that assay blanks, which are background noise of fluorescence measurement, greatly increase, which hinders high-sensitivity and high-accuracy SPFS measurement of a very small amount of analyte 6.

  Moreover, in the SPFS measurement, there is a risk that the effect of enhancing the electric field is greatly reduced due to the adsorption of the contaminant 1 on the surface of the sensor chip. For example, ELISA (Enzyme-Linked Immuno Sorbent Assay), luminescent enzyme immunoassay It is necessary to prevent non-specific adsorption to the surface of the sensor chip more than conventional measurement methods such as (CLEIA (Chemiluminesent Enzyme Immunoassay)).

  Table 1 below is a table showing the above-mentioned problems in conventional methods such as ELISA and CLEIA and SPFS measurement, that is, the degree of influence of specific reactions on the measurement results. Thus, it can be seen that the SPFS measurement has a greater influence on the measurement due to non-specific adsorption of impurities in comparison with the conventional method.

  Conventionally, as a method for preventing such non-specific adsorption of impurities, there is a pretreatment method disclosed in Patent Document 1 (Japanese Patent Laid-Open No. 8-327629).

  That is, in Patent Document 1, as a sample pretreatment method for removing a substance that causes a non-specific reaction, an immunoassay carrier that absorbs a substance that causes a non-specific reaction or a magnetic particle bound with a substance such as an antibody is used as a specimen. A specimen pretreatment method is described in which magnetic particles are mixed and magnetic particles are separated by a magnetic force so as to remove substances that cause non-specific reactions in the specimen.

  Patent Document 2 (Japanese Patent Laid-Open No. 9-089893) discloses an indirect agglutination immunoassay using erythrocytes as a carrier, which removes an adsorbed substance or inhibits adsorption by simply adding it to a measurement solution. Non-specific reaction absorption reagents are disclosed.

  As this non-specific reaction absorption reagent, in Patent Document 2, a dextran compound in which at least 60% of the hydroxyl groups of dextran are substituted with sulfate ester groups, or a salt thereof, is used to cause a non-specific reaction in a sample. A non-specific reaction absorption reagent that has a high effect of removing water has been proposed.

Japanese Patent Laid-Open No. 8-327629 Japanese Patent Laid-Open No. 9-089893

  However, in the sample pretreatment method described in Patent Document 1, the effect of removing contaminants in the sample is not yet sufficient, and as described above, for example, serum, plasma, whole blood, nasal cavity When detecting a very small amount of analyte using a biological sample such as liquid or peritoneal fluid as a specimen, especially in SPFS measurement where there is a risk that the electric field enhancement effect is greatly reduced due to the adsorption of impurities to the sensor chip surface. It was not enough to apply.

  That is, in Patent Document 1, although specific contaminants absorbed by a specific carrier or antibody can be removed, other contaminants cannot be removed. The contaminants that could not be removed are solid phase support, ligand and In some cases, non-specific adsorption to the sensor chip surface or the like occurred.

  In addition, in a specimen in which the analyte to be examined is very small, the analyte is removed together with the magnetic particles by the pretreatment of the specimen, so that a trace amount of the analyte may not be detected.

  On the other hand, in the nonspecific reaction absorption reagent of Patent Document 2, since the nonspecific reaction absorption reagent is simply added to the measurement solution (specimen), it is possible to prevent the analyte from being erroneously removed before measurement. It was not possible to sufficiently prevent the adhesion of foreign substances.

  Therefore, even with the nonspecific reaction absorption reagent of Patent Document 2, as described above, a very small amount of analyte is detected using a biological sample such as serum, plasma, whole blood, nasal fluid, and peritoneal fluid as a specimen. In particular, it is not sufficient for application in SPFS measurement, which has a risk that the effect of enhancing the electric field is greatly reduced due to the adsorption of impurities on the surface of the sensor chip.

  In view of such a current situation, the present invention suppresses nonspecific adsorption of contaminants in a sample to a solid support, a ligand, and the like, and a sample dilution solution that can suppress an increase in assay blank, It is an object to provide a kit and a fluorescence measurement method using the kit.

  That is, the present invention provides a specimen dilution solution shown in the following [1] to [11], a kit using the same, and a fluorescence measurement method.

[1] Carboxymethyldextran having a weight average molecular weight of 200,000 to 1,000,000 is 0 . 1 to 1 % by mass of carboxymethyl dextran having a weight average molecular weight of 500000 to 1000000 is contained in an amount of 0.01 to 2% by mass of the sample dilution liquid. .

[2] 0.1-0.5 mass% of carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 with respect to the sample dilution liquid is contained. liquid.

  [3] The specimen dilution liquid according to [1] or [2], wherein the specimen is a biological sample.

  [4] The specimen dilution liquid according to any one of [1] to [3], wherein the pH is adjusted to 4 to 9.

  [5] The specimen dilution liquid according to any one of [1] to [4], which contains a surfactant.

  [6] The specimen dilution liquid according to [5], wherein the surfactant is contained in an amount of 0.00001 to 1% by mass with respect to the specimen dilution liquid.

  [7] A specimen dilution liquid kit comprising the specimen dilution liquid according to any one of [1] to [6].

  [8] After supplying the mixed solution obtained by mixing the sample with the sample dilution solution according to any one of [1] to [6] onto a sensor chip in which a solid phase support is fixed on a metal film, fluorescence A fluorescence measurement method characterized by performing measurement.

  [9] The fluorescence measurement method according to [8], wherein the fluorescence measurement is performed after supplying the liquid mixture, supplying a fluorescently labeled antibody that binds to the analyte in the sample and washing the mixture. .

  [10] The fluorescence measurement method according to [8] or [9], wherein the solid support is carboxymethyl dextran.

  [11] The fluorescence measurement method according to any one of [8] to [10], wherein the fluorescence measurement is performed by surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) or total reflection ATR fluorescence method.

  According to the present invention, a sample dilution solution that suppresses nonspecific adsorption of contaminants in a sample to a solid support, a ligand, and the like, and can suppress an increase in assay blank, and a kit using the same And a fluorescence measurement method can be provided.

FIG. 1 is a schematic diagram for explaining a state in which a sample-diluting solution according to the present invention and a sample derived from a living body are mixed and carboxymethyldextran adheres to the surface of a contaminant. FIG. 2 is a schematic diagram for explaining the state of the sensor chip surface of the fluorescence measuring apparatus to which the mixed liquid of FIG. 1 has been fed. FIG. 3 is a schematic diagram illustrating a state in which a fluorescently labeled antibody solution conjugated with a fluorescently labeled substance is delivered to the surface of the sensor chip in FIG. It is a figure to do. FIG. 4 is a schematic diagram for explaining the state of the surface of a sensor chip in which a sample is processed using a related-art sample dilution solution and this solution and a fluorescently labeled antibody solution are fed.

Hereinafter, embodiments (examples) of the present invention will be described in more detail with reference to the drawings.
1. Preparation of Sample Dilution Solution FIG. 1 shows a solution obtained by mixing the sample dilution solution according to the present invention with a sample derived from a living body, and illustrates a state in which carboxymethyldextran is attached to the surface of a contaminant. It is a schematic diagram. FIG. 2 is a schematic diagram for explaining the state of the surface of the sensor chip to which the liquid mixture of FIG. 1 has been fed.

  The sample dilution liquid according to the present invention contains carboxymethyldextran having a weight average molecular weight of 200,000 to 1,000,000. For example, a biological sample such as serum, plasma, whole blood, nasal fluid, and peritoneal fluid is used as a sample. When diluted, the carboxymethyl dextran molecule 2 adheres to the surface of the contaminant 1 as shown in FIG.

  That is, in a highly sensitive immunoassay measurement, the surface of the sensor (solid phase) is generally neutral or negatively charged, but carboxymethyl dextran having a predetermined weight average molecular weight having an anionic sugar chain is determined in a predetermined mass%. In this way, carboxymethyl dextran and contaminant 1 are not only adsorbed to each other but also carboxymethyl in a state adsorbed to contaminant 1 as shown in FIG. Since the dextran and the sensor surface are both negatively charged, the Coulomb repulsive force F acts and repels each other, so that the adsorption of the contaminants 1 to the sensor (solid phase) surface is suitably suppressed by the Coulomb repulsive force F. Can do.

  Thereby, as shown in the schematic diagrams of FIGS. 2 and 3, the contaminant 1 having the carboxymethyl dextran molecule 2 attached to the surface is difficult to non-specifically adsorb on the solid support 3, the ligand 4, and the like. Therefore, it is possible to efficiently suppress the non-specific adsorption of the contaminant 1 in the specimen to the ligand 4 or the solid support 3 of the ligand 4.

  For this reason, in the fluorescence measurement, the fluorescence-labeled antibody 5 is suppressed from adhering to the solid support 3 and the ligand 4 through the contaminants 1 (see FIG. 3 and FIG. 4 in comparison). As a result, the change in refractive index due to adhesion is reduced, and it is possible to reduce the adverse effect of the contaminant 1 and the adhesion on the electric field enhancement effect, and the assay blank that is background noise of fluorescence measurement is greatly increased. S / B can be improved. Thereby, in particular, it is possible to realize high-sensitivity and high-precision SPFS measurement of an extremely small amount of the analyte 6.

  On the other hand, when the weight average molecular weight of the carboxymethyl dextran contained in the sample dilution liquid is less than 200,000, or the weight average molecular weight of the carboxymethyl dextran contained in the sample dilution liquid is within the range of 200,000 to 1,000,000. Even if the content is less than 0.001% by mass (the final concentration at the time of sample treatment is less than 0.0005% by mass), the contaminant 1 in the sample is supported on the solid phase of the ligand 4 or the ligand 4. The effect of suppressing nonspecific adsorption to the body 3 or the like cannot be expected, and the assay blank, which is background noise of fluorescence measurement, increases.

  Conversely, when the weight average molecular weight of the carboxymethyl dextran contained in the sample dilution liquid exceeds 1000000, or the weight average molecular weight of the carboxymethyl dextran contained in the sample dilution liquid falls within the range of 200000 to 1000000. Even so, if the content exceeds 5% by mass (the final concentration at the time of sample processing exceeds 2.5% by mass), the viscosity of the sample dilution solution becomes too high and the reactivity decreases. .

  Preferably, the carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 is contained in an amount of 0.01 to 2% by mass with respect to the sample dilution liquid (the sample at a final concentration of 0.005 to 1.00% by mass). It is desirable to be used for the above processing.

  According to such a sample dilution solution, the effect of suppressing the non-specific adsorption of the contaminant 1 in the sample to the ligand 4 or the solid support 3 of the ligand 4 can be expected, and the fluorescence measurement can be performed. The assay blank, which is the background noise, can be further reduced. In addition, the viscosity of the sample dilution liquid can be suppressed from becoming unnecessarily high, the decrease in reactivity can be further reduced, and the S / B can be further improved.

  More preferably, the carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 is 0.1 to 0.5% by mass (0.05 to 0 at the final concentration at the time of sample treatment) with respect to the sample dilution solution. .25 mass%) is desirable.

  According to such a sample dilution solution, the effect of suppressing the non-specific adsorption of the contaminant 1 in the sample to the ligand 4 or the solid support 3 of the ligand 4 can be further expected. The assay blank, which is background noise, can be further reduced, and the viscosity of the sample dilution solution can be further prevented from becoming unnecessarily high, the decrease in reactivity can be further reduced, and the S / B can be further improved. it can.

  In addition, the pH of the sample dilution liquid according to the present invention is preferably 4-9. Thus, when the pH of the sample dilution solution is 4 to 9, the pH is maintained higher than about pH 3.5 where the carboxymethyl dextran molecule 2 is negatively charged, and the carboxymethyl dextran molecules 2 repel each other. In addition, since it does not aggregate, it is excellent in storage stability and can highly suppress nonspecific adsorption of the contaminant 1 in the specimen to the ligand 4 or the solid support 3 of the ligand 4 or the like.

  Furthermore, for example, contaminants such as histones and ribosomes present in the living body among the contaminants in the specimen are positively charged in the vicinity of pH 7, and therefore, as shown in FIG. In the treatment, the contaminants 1 are electrically attracted to and adsorbed to the negatively charged carboxymethyl dextran molecules 2.

  As shown in FIG. 3, in the fluorescence measurement scene, the negatively charged carboxymethyl dextran is present against the negatively charged ligand 4 or the solid support 3 of the ligand 4 that is present near the sensor chip surface. The Coulomb repulsive force F of the molecule 2 acts, and the contaminant 1 is maintained in a state of being separated from the ligand 4 and the solid support 3 of the ligand 4 together with the carboxymethyldextran molecule 2.

  As a result, the non-specific adsorption of the contaminant 1 to the ligand 4 or the solid support 3 of the ligand 4 is efficiently suppressed. And the specific reaction of the analyte 6 and the ligand 4 is ensured.

  When the isoelectric point measurement on the surface of the sensor chip was performed, for example, the isoelectric point of the substrate surface having the solid support 3 of carboxymethyldextran was about pH 2-3.

  Therefore, in such a case, the charged state of the carboxymethyl dextran molecule 2 in the sample diluting solution can be easily maintained anionic and the state can be effectively utilized by setting the sample diluting solution to pH 4 or higher. Therefore, it is preferable. Moreover, it is common to set it as reaction conditions at pH 9 or less in an antigen antibody reaction, and it is preferable that the upper limit shall be pH 9 or less.

  In this case, for example, phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS), etc. can be used as the main component of the sample dilution liquid. .

  Further, the sample dilution liquid according to the present invention preferably contains a surfactant. By containing the surfactant, for example, when the contaminant 1 is a lipid, the surfactant acts to form micelles, etc., so that the aggregation of the analyte 6 and the contaminant 1 in the specimen can be suppressed. it can. As a result, it is possible to suppress nonspecific adsorption of the contaminant 1 in the specimen to the ligand 4 or the solid support 3 of the ligand 4.

  In this case, the surfactant is not particularly limited, but is preferably an anionic surfactant from the viewpoint of utilizing the Coulomb repulsion in the same charged state as CMD, for example, sodium dodecyl sulfate (SDS), Monoalkyl sulfates, alkyl polyoxyethylene sulfates, alkylbenzene sulfonates, monoalkyl phosphates and the like can be used.

  In this case, the surfactant should be contained in an amount of 0.00001 to 1% by mass with respect to the sample dilution liquid (used to treat the sample at a final concentration of 0.000005 to 0.5% by mass). desirable.

  Thereby, aggregation of the analyte 6 and the foreign substance 1 in a sample can be suppressed suitably. Moreover, it can suppress that the foreign substance 1 in a test substance adsorb | sucks to the solid support 3 etc. of the ligand 4 or the ligand 4 nonspecifically. In addition, the decrease in reactivity can be reduced more effectively.

  Moreover, the sample dilution liquid according to the present invention is preferably a sample dilution liquid using a biological sample as a sample.

  Thus, for example, in the case of a specimen dilution liquid using a biological sample such as serum, plasma, whole blood, nasal fluid, and peritoneal fluid as a specimen, the contaminant 1 in the specimen is a solid phase of ligand 4 or ligand 4. Non-specific adsorption to the support 3 or the like can be efficiently suppressed.

  Further, in the fluorescence measurement method according to the present invention, after a mixed solution obtained by mixing a sample with a sample dilution solution is supplied onto a sensor chip in which the solid support 3 is fixed on the metal thin film 7, the fluorescence measurement is performed. It is characterized by that.

In this way, by using a mixed solution in which the sample is mixed with the sample dilution solution, the non-specific adsorption of the contaminant 1 in the sample to the ligand 4 or the solid support 3 of the ligand 4 is efficiently suppressed. As a result, it is possible to suppress the increase in assay blank and perform fluorescence measurement with improved S / B (S / N ratio), which is the ratio of assay signal (S) to assay blank (B) to be detected. Can do.
2. Fluorescence measurement method In the fluorescence measurement method according to the present invention, a mixed liquid obtained by mixing a specimen with the specimen dilution liquid prepared as described above is placed on a sensor chip on which a solid support 3 is fixed on a metal thin film 7. Supply. Thereafter, after the fluorescently labeled antibody 5 that binds to the analyte 6 in the specimen is supplied and washed, the fluorescence measurement may be performed.

In the fluorescence measurement method according to the present invention, it is desirable to perform fluorescence measurement after supplying the mixed solution, supplying and washing the fluorescence-labeled antibody 5 that binds to the analyte 6 in the specimen, and washing. In this way, it is possible to highly suppress the non-specific adsorption of the contaminant 1 in the specimen to the ligand 4 or the solid support 3 of the ligand 4, and as a result, the increase in assay blank is suppressed and S / B (SN ratio) is improved, and a very small amount of analyte 6 can be measured with high sensitivity and high accuracy.
2-1. In this case, the sensor chip is obtained by forming the metal thin film 7 on the upper surface of the dielectric member 8 (see FIGS. 2 and 3). Although it does not specifically limit as the dielectric material member 8, Optically transparent, for example, various inorganic substances, such as glass and ceramics, a natural polymer, a synthetic polymer can be used, chemical stability, manufacture From the viewpoints of stability and optical transparency, silicon dioxide (SiO ₂ ) or titanium dioxide (TiO ₂ ) is preferably included.

  Further, the material of the dielectric member 8 is not particularly limited as long as it is made of a material that is optically transparent at least with respect to the excitation light. For example, it is preferably formed from a resin material.

When the dielectric member 8 is formed from a resin material, for example,
-Polyesters such as polyethylene terephthalate (PET) and polyethylene naphthalate,
-Polyolefins such as polyethylene (PE) and polypropylene (PP),
-Polycyclic olefins such as cyclic olefin copolymer (COC) and cyclic olefin polymer (COP),
・ Vinyl resins such as polyvinyl chloride and polyvinylidene chloride,
・ Polystyrene, polyetheretherketone (PEEK), polysulfone (PSF),
Polyethersulfone (PES), polycarbonate (PC), polyamide, polyimide, acrylic resin, triacetylcellulose (TAC), and the like can be used.

  The material of the metal thin film 7 is not particularly limited, but is preferably made of at least one metal selected from the group consisting of gold, silver, aluminum, copper, and platinum, more preferably It is made of gold and may be made of an alloy of these metals.

  That is, such a metal is suitable for the metal thin film 7 because it is stable against oxidation and, as will be described later, the electric field enhancement due to surface plasmon light (coherent wave) increases. Further, the method for forming the metal thin film 7 is not particularly limited, and examples thereof include a sputtering method, a vapor deposition method (resistance heating vapor deposition method, electron beam vapor deposition method, etc.), electrolytic plating, electroless plating method, and the like. . It is preferable to use a sputtering method or a vapor deposition method because it is easy to adjust the thin film formation conditions.

  Further, the thickness of the metal thin film 7 is not particularly limited, but preferably, gold: 5 to 500 nm, silver: 5 to 500 nm, aluminum: 5 to 500 nm, copper: 5 to 500 nm, platinum: 5 ˜500 nm and their alloys: preferably in the range of 5 to 500 nm.

  From the viewpoint of the electric field enhancement effect, the thickness of the metal thin film 7 is more preferably gold: 20-70 nm, silver: 20-70 nm, aluminum: 10-50 nm, copper: 20-70 nm, platinum: 20- It is desirable that the range is 70 nm and alloys thereof: 10 to 70 nm.

If the thickness of the metal thin film 7 is within the above range, it is preferable that surface plasmon light (coherent wave) is easily generated. In addition, as long as the metal thin film 7 has such a thickness, the size (vertical x horizontal) size and shape are not particularly limited.
2-2. Ligand solid-phase support fixation 2-2-1 SAM formation The solid-phase support 3 of the ligand 4 is fixed on the upper surface of the metal thin film 7 of the dielectric member 8 (see FIGS. 2 and 3).

  As a scaffold for immobilizing the solid support 3, it is desirable that a SAM (Self-Assembled Monolayer) is formed on the metal thin film 7 of the dielectric member 8 (not shown).

  The SAM is used as a scaffold for immobilizing the solid support 3, and when the SPFS sensor chip is used in the fluorescence measurement method, the metal thin film 7 of the dielectric member 8 is used for the purpose of preventing metal quenching of the fluorescent molecules. Is formed on the other surface of the sensor chip that is not in contact with the metal chip, that is, on the surface of the metal thin film 7 opposite to the dielectric member 8 with respect to the metal thin film 7.

  As a single molecule contained in SAM, usually a carboxyalkanethiol having about 4 to 20 carbon atoms (for example, available from Dojindo Laboratories Co., Ltd., Sigma Aldrich Japan Co., Ltd.), particularly preferably 10- Carboxy-1-decanethiol is used.

  The carboxyalkanethiol having 4 to 20 carbon atoms has properties such as high transparency, low refractive index, thin film thickness, etc., because the SAM formed using the carboxyalkanethiol has optical properties. It is preferable because it is small.

  A method for forming such a SAM is not particularly limited, and a conventionally known method can be used. For example, a material in which a layer made of a mask material is formed on the surface of the metal thin film 7 of the dielectric member 8 is immersed in an ethanol solution containing 10-carboxy-1-decanethiol (manufactured by Dojindo Laboratories). The method of doing is mentioned.

  In this way, the thiol group of 10-carboxy-1-decanethiol binds to the metal and is immobilized, and self-assembles on the surface of the thin gold film 7 to form a SAM. Further, a “spacer layer made of a dielectric” may be formed instead of forming the SAM.

In this case, the spacer layer is preferably a compound composed of an organic substance and silicon, and is formed using a silane coupling agent having two or more different reactive groups in the molecule. As the silane coupling agent, a molecule having an ethoxy group or a methoxy group that gives a silanol group [Si—OH] by hydrolysis and a reactive group such as an amino group, a glycidyl group, or a carboxyl group at the other end can be particularly used. .
2-2-2 Fixing of Solid-phase Support After the SAM is formed on the metal thin film 7 of the dielectric member 8 in this way, the ligand 4 is immobilized on the other surface not in contact with the metal thin film 7 of the SAM. The phase support 3 is fixed (see FIG. 2 and FIG. 3, partially not shown).

  The solid support 3 preferably has a three-dimensional structure as shown in FIGS. In this case, the “three-dimensional structure” means that the fixation of the ligand 4 described later is not limited to two dimensions on the sensor chip surface (and its vicinity), but can be expanded to a three-dimensional space free from the sensor chip surface. This refers to the structure of the solid support 3.

Such a solid support 3 is not particularly limited, but for example,
Glucose, carboxymethylated glucose,
・ Vinyl esters, acrylic acid esters, methacrylic acid esters, crotonic acid esters, itaconic acid diesters, maleic acid diesters, fumaric acid diesters,
・ Olefins, styrenes, allyl compounds, vinyl ethers, vinyl ketones,
-It is preferable to include a polymer composed of at least one monomer selected from the group consisting of monomers included in each.

In addition, for example,
・ Hydrophilic polymers such as dextran and dextran derivatives,
・ Vinyl esters, acrylic acid esters, methacrylic acid esters, crotonic acid esters, itaconic acid diesters, maleic acid diesters, fumaric acid diesters,
・ Olefins, styrenes, allyl compounds, vinyl ethers, vinyl ketones,
-Hydrophobic polymers composed of hydrophobic monomers included in each,
It is more preferable to contain.

  In addition, carboxymethyldextran (CMD) is particularly preferable from the viewpoint of biocompatibility, suppression of nonspecific adsorption reaction, and high hydrophilicity. In this case, the molecular weight of CMD is preferably 1 kDa or more and 5000 kDa or less, and more preferably 4 kDa or more and 1000 kDa.

  Thus, if the solid support 3 is carboxymethyldextran, the antibody for capturing the analyte 6 can be immobilized at a high density, and a very small amount of the analyte 6 can be highly sensitive and accurate. It can be measured. Further, it is possible to effectively utilize the interaction in the charging characteristics with carboxymethyldextran contained in the sample dilution liquid.

Moreover, it is preferable that the solid support 3 (for example, one made of dextran or a dextran derivative) has a density of less than ² ng / mm ² . In this case, the density of the solid support 3 can be appropriately adjusted according to the type of polymer used.

When such a polymer is immobilized on the SAM within the density range, the assay signal is stabilized and increased when the SPFS sensor chip is used in the fluorescence measurement method. Therefore, it is preferable.
2-3 Ligand Fixation After forming the SAM and the solid support 3 on the metal thin film 7 of the dielectric member 8 in this way, the ligand 4 is fixed to the solid support 3 (FIGS. 2 and 2). 3).

  That is, the ligand 4 is dispersed and immobilized in the three-dimensional structure of the solid support 3. For example, when the SPFS sensor chip is used in the fluorescence measurement method, the analyte 6 in the specimen is immobilized ( (See FIGS. 2 and 3).

Such “molecule” or “molecular fragment” is not particularly limited, and for example,
-Nucleic acids (DNA, RNA, polynucleotide, oligonucleotide, PNA (peptide nucleic acid), etc., which may be single-stranded or double-stranded, nucleosides, nucleotides and their modified molecules),
・ Proteins (polypeptides, oligopeptides, etc.),
・ Amino acids (including modified amino acids),
・ Sugar (oligosaccharides, polysaccharides, sugar chains, etc.),
・ Lipids,
-These modifying molecules, complexes, etc. are mentioned.

  In this case, examples of the protein include an antibody. Specifically, an anti-α fetoprotein (AFP) monoclonal antibody (available from Japan Medical Laboratory) or an anti-carcinoembryonic antigen ( CEA) monoclonal antibody, anti-CA19-9 monoclonal antibody, anti-PSA monoclonal antibody and the like.

In the present invention, the term “antibody” includes polyclonal antibodies or monoclonal antibodies, antibodies obtained by gene recombination, and antibody fragments. As a method for immobilizing the ligand 4, for example, the following method can be used.
-A carboxyl group possessed by a polymer having a reactive functional group such as carboxymethyldextran (CMD) is converted into a water-soluble carbodiimide (WSC) (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC). And the like) and N-hydroxysuccinimide (NHS).

And, a method of dehydrating and immobilizing the carboxyl group thus active esterified and the amino group of the ligand 4 using water-soluble carbodiimide,
A method in which the carboxyl group of SAM is dehydrated and immobilized with the amino group of ligand 4 as described above,
Etc.

The density of the ligand 4 immobilized on the outer surface of the solid support 3 is preferably 1 femto mol / cm ² or more and 1 nano mol / cm ² or less, preferably 10 femto mol / cm ² or more and 100 pico mol /. More preferred is cm ² or less. That is, it is desirable that the density of the ligand 4 is within the above range because the signal intensity increases.
2-4. Preparation of Sample Solution A sample solution (mixed solution) is prepared by mixing the sample with the sample dilution solution prepared as described above.

  The sample solution used here is a solution prepared using a specimen. For example, a process for mixing a specimen and a reagent to bind a fluorescent substance to the analyte 6 contained in the specimen. The one that has been.

  Examples of such specimens include blood (serum / plasma), urine, nasal fluid, saliva, stool, body cavity fluid (spinal fluid, ascites, pleural effusion, etc.), etc. May be used. Of these samples, blood, serum, plasma, urine, nasal fluid and saliva are preferred.

The analyte 6 contained in the specimen is, for example,
-Nucleic acids (DNA, RNA, polynucleotides, oligonucleotides, PNA (peptide nucleic acids) etc., or nucleosides, nucleotides and their modified molecules, which may be single-stranded or double-stranded),
・ Proteins (polypeptides, oligopeptides, etc.),
・ Amino acids (including modified amino acids),
・ Sugar (oligosaccharides, polysaccharides, sugar chains, etc.),
・ Lipids,
-These modifying molecules, complexes and the like can be mentioned, and specifically, carcinoembryonic antigens such as AFP (α-fetoprotein), tumor markers, signaling substances, hormones and the like may be used, and there is no particular limitation.
2-5. Contact between sample solution and sensor chip The sample solution thus prepared is brought into contact with the sensor chip on which the solid support 3 of SAM and ligand 4 is fixed on the metal thin film 7 of the dielectric member 8 as described above. (See FIG. 2).

  In this case, the contact method is not particularly limited. For example, a method of forming a flow path on the sensor chip and feeding the sample solution to contact, or a method of forming a well on the sensor chip A method of supplying a sample solution into the well can be employed.

In the case of liquid feeding, the initial concentration of the analyte 6 contained in the specimen during liquid feeding may be 100 μg / ml to 0.001 pg / ml. Further, the total amount of liquid feeding, that is, the volume of the flow path is usually 0.001 to 20 ml, preferably 0.1 to 1 ml. Further, the flow rate of the liquid feeding is usually 1 to 2,000 μl / min, preferably 5 to 500 μl / min.
2-6 Cleaning Step After contacting the sample solution with the sensor chip on which the solid support 3 of SAM and ligand 4 is fixed on the metal thin film 7 of the dielectric member 8 (see FIG. 2), the sensor Clean the tip surface.

As the cleaning liquid used in the cleaning process, for example,
-Tween 20 (mono-9-octadecanoate poly (oxy-1,2-ethanediyl) in phosphate buffered saline (PBS), Tris buffered saline (TBS), HEPES buffered saline (HBS) A surfactant such as Triton X100, preferably containing 0.00001 to 1% by weight of a surfactant,
-What contains 10-500 mM of salts, such as sodium chloride and potassium chloride, is desirable.

Alternatively, a low pH buffer solution such as 10 mM Glycine HCl having a pH of 1.5 to 4.0 may be used. When the cleaning liquid is fed, the temperature and flow rate to circulate are preferably the same as the temperature and flow rate to circulate the sample solution. In addition, the time which circulates a washing | cleaning liquid is 0.5 to 180 minutes normally, Preferably it is 5 to 60 minutes.
2-7. Contact of Fluorescent Labeled Antibody Solution with Sensor Chip 2-7-1 Preparation of Fluorescent Labeled Antibody Fluorescent labeled antibody 5 is an antibody conjugated with a fluorescent substance (see FIG. 3).
As a production method of the fluorescent labeled antibody 5,
For example, a carboxyl group is imparted to a fluorescent substance, and the carboxyl group is converted into water-soluble carbodiimide [WSC] (for example, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride [EDC]) and N- A method in which a carboxyl group formed by active esterification with hydroxysuccinimide [NHS] and then an amino group possessed by the active ester is dehydrated and immobilized using water-soluble carbodiimide,
A method of reacting and immobilizing an antibody having an isothiocyanate and an amino group with a fluorescent substance,
A method of reacting and immobilizing a secondary antibody having a sulfonyl halide and an amino group, respectively, and a fluorescent substance;
A method of reacting and immobilizing an antibody having an iodoacetamide and a thiol group and a fluorescent substance,
-Alternatively, there may be mentioned a method in which a biotinylated fluorescent substance and a streptavidinated antibody (or a streptavidinated fluorescent substance and a biotinylated antibody) are reacted and immobilized.

  The fluorescent substance is a general term for substances that emit fluorescence by irradiating predetermined excitation light or exciting by using an electric field effect, and the fluorescence includes various kinds of light emission such as phosphorescence. The fluorescent material used in the present invention is not particularly limited as long as it is not quenched due to light absorption by the metal thin film 7, and may be any known fluorescent material.

  In general, phosphors with large Stokes shifts that allow the use of a fluorometer with a filter rather than a monochromometer and increase the efficiency of detection are preferred.

As such a fluorescent substance,
-For example, a fluorescent substance of the fluorescein family (manufactured by Integrated DNA Technologies),
-Polyhalofluorescein family fluorescent material (Applied Biosystems Japan Co., Ltd.),
・ Fluorescent substance of hexachlorofluorescein family (Applied Biosystems Japan Co., Ltd.),
-Coumarin family fluorescent material (Invitrogen),
・ Rhodamine family fluorescent substance (manufactured by GE Healthcare Biosciences)
・ Cyanine family fluorescent material,
・ Indocarbocyanine family fluorescent material, Oxazine family fluorescent material,
・ Thioazine family fluorescent material, squarain family fluorescent material,
• BODIPY®, a chelating lanthanide family fluorescent material
・ Family fluorescent material (manufactured by Invitrogen),
・ Naphthalene sulfonic acid ・ Fluorescent materials in the family
・ Pyrene family fluorescent materials,
・ Fluorescent substances of the triphenylmethane family,
-Alexa Fluor (registered trademark) dye series (manufactured by Invitrogen Corporation)
Etc.

  Furthermore, U.S. Patent Nos. 6,406,297, 6,221,604, 5,994,063, 5,808,044, 5,880,287, The fluorescent materials described in US Pat. Nos. 5,556,959 and 5,135,717 can also be used in the present invention.

Moreover, the fluorescent substance that can be used in the present invention is not limited to the organic fluorescent substance. For example, rare earth complex fluorescent materials such as Eu and Tb can also be used as fluorescent materials in the present invention. In general, rare earth complexes have a large wavelength difference between an excitation wavelength (about 310 to 340 nm) and an emission wavelength (about 615 nm for an Eu complex and 545 nm for a Tb complex), and a long fluorescence lifetime of several hundred microseconds or more. is there. An example of a commercially available rare earth complex-based fluorescent material is ATBTA-Eu ³⁺ .

  In the present invention, it is desirable to use a fluorescent material having a maximum fluorescence wavelength in a wavelength region where light absorption by the metal contained in the metal thin film 7 is small when performing fluorescence measurement described later.

  For example, when gold is used as the metal thin film 7, it is desirable to use a fluorescent material having a maximum fluorescence wavelength of 600 nm or more in order to minimize the influence of light absorption by the metal thin film 7.

  Therefore, in this case, it is particularly desirable to use a fluorescent material having a maximum fluorescence wavelength in the near-infrared region, such as Cy5, Alexa Fluor (registered trademark) 647. The use of a fluorescent substance having the maximum fluorescence wavelength in the near-infrared region can minimize the influence of light absorption by iron derived from blood cell components in the blood. Is also useful.

On the other hand, when silver is used as the metal thin film 7, it is desirable to use a fluorescent material having a maximum fluorescence wavelength of 400 nm or more. These fluorescent materials may be used alone or in combination of two or more.
2-7-2 Contact between Fluorescent Labeled Antibody Solution and Sensor Chip Contact the sample solution with the sensor chip to bind the analyte 6 to the ligand 4 on the sensor chip (see FIG. 2). Then, the sensor chip is brought into contact with a solution containing the fluorescently labeled antibody 5 to bind the fluorescently labeled antibody 5 to the analyte 6 (see FIG. 3). This step is preferably performed after the washing step. Moreover, it is more preferable to perform the said washing | cleaning process further after this process.

  When the fluorescent labeled antibody solution is sent to the sensor chip and the fluorescent labeled antibody 5 is bound to the analyte 6, the concentration of the fluorescent labeled antibody solution to be sent is 0.001 μg / ml or more and 10,000 μg / ml or less. It is preferably 1 μg / ml or more and 1,000 μg / ml or less.

Moreover, the preferable flow rate at the time of sending the fluorescence labeled antibody solution is the same as that at the time of sending the sample solution.
2-8. Fluorescence measurement The sensor chip thus obtained is excited via the prism from the other surface of the dielectric member 8 where the metal thin film 7 is not formed (the lower surface of the dielectric member 8 in FIG. 2). Laser light 9 as light is irradiated (see FIG. 3), and the amount of fluorescence emitted from the excited fluorescent material is measured.

  The light source used in the fluorescence measurement method according to the present invention is not particularly limited as long as it can cause plasmon excitation in the metal thin film 7, but in terms of unity of wavelength distribution and intensity of light energy. It is preferable to use laser light as a light source.

  It is desirable to adjust the energy and photon amount immediately before the laser light 9 enters the prism through an optical filter, a condenser lens, and the like. Irradiation with the laser beam 9 generates surface plasmons on the surface of the metal thin film 7 under the condition of attenuated total reflection (ATR). Due to the electric field enhancement effect of the surface plasmon, the fluorescent material is excited by photons which are increased by several tens to several hundred times the amount of photons irradiated.

  In addition, although the photon increase amount by the electric field enhancement effect depends on the refractive index of the dielectric member 8, the metal species of the metal thin film 7, and the film thickness thereof, the increase amount is usually about 10 to 20 times for gold. In the fluorescent substance, electrons in the molecule are excited by light absorption, move to the first electron excited state in a short time, and when returning from this state (level) to the ground state, the wavelength of the wavelength corresponding to the energy difference Fluoresce.

  Moreover, as the laser beam 9 to be used, for example, an LD having a wavelength of 200 to 900 nm, 0.001 to 1,000 mW; a wavelength of 230 to 800 nm (resonance wavelength is determined by the metal species used for the metal thin film 7), 0.01 to A 100 mW semiconductor laser can be used.

  The “prism” is intended to allow the laser light 9 through various filters to efficiently enter the SPFS sensor chip, and preferably has the same refractive index as that of the dielectric member 8.

  In the present invention, various prisms that can set the conditions of attenuated total reflection can be appropriately selected. Therefore, the angle and the shape are not particularly limited, and may be, for example, a 60-degree dispersion prism. Examples of such commercially available prisms include those similar to the above-described commercially available “glass dielectric member”.

  Examples of the “optical filter” include a neutral density (ND) filter, a polarizing filter, a cut filter, and a diaphragm lens. In this case, the “darkening (ND) filter” (or neutral density filter) is intended to adjust the amount of incident laser light. In particular, when a detector with a narrow dynamic range is used, it is preferable to use it for carrying out a highly accurate measurement.

  The “polarizing filter” is used to make the laser light P-polarized light that efficiently generates surface plasmons. In addition, the “cut filter” includes external light (illumination light outside the device), excitation light (excitation light transmission component), stray light (excitation light scattering component in various places), and plasmon scattering light (excitation light originates from the excitation light). , A filter that removes optical noise such as scattered light generated by the influence of structures or deposits on the surface of a sensor chip for SPFS, and autofluorescence of a fluorescent substance, such as an interference filter, a color filter, etc. Can be mentioned.

  The “condensing lens” is intended to efficiently collect the fluorescent signal on the detector, and may be an arbitrary condensing system. As a simple condensing system, a commercially available objective lens (for example, manufactured by Nikon Corporation or Olympus Corporation) used in a microscope or the like may be used. The magnification of the objective lens is preferably 10 to 100 times.

  As the “SPFS detection unit”, a photomultiplier (a photomultiplier manufactured by Hamamatsu Photonics Co., Ltd.) is preferable from the viewpoint of ultrahigh sensitivity. Also, although the sensitivity is lower than these, a CCD image sensor capable of multipoint measurement is also suitable because it can be viewed as an image and noise light can be easily removed.

  From the measurement result thus obtained, the amount of analyte contained in the sample is calculated. More specifically, a calibration curve is created by performing measurement with a target antigen of a known concentration or a target antibody, and an analyte (target antigen level) in the sample to be measured is created based on the created calibration curve. Alternatively, the target antibody) amount is calculated from the measurement signal.

[Example 1]
1. Fabrication of sensor chip A glass dielectric member 8 (“S-LAL 10” manufactured by OHARA INC.) Having a refractive index (n _d ) of 1.72 and a thickness of 1 mm is plasma-cleaned, and one surface of this support is formed. A chromium thin film was formed by a sputtering method.

  Thereafter, a gold thin film was further formed on the surface by a sputtering method. The chromium thin film had a thickness of 1 to 3 nm, and the gold thin film had a thickness of 42 to 47 nm. The substrate thus obtained was immersed in 10 ml of an ethanol solution of 10-amino-1-decanethiol prepared to 1 mM for 24 hours to form a SAM on one side of the gold thin film. The substrate was taken out from the ethanol solution, washed with ethanol and isopropanol, and then dried using an air gun.

  Subsequently, pH 7.4 MES containing 1 mg / ml of carboxymethyldextran (CMD) having a molecular weight of 500,000, 0.5 mM of N-hydroxysuccinimide (NHS), and 1 mM of water-soluble carbodiimide (WSC). An unreacted succinic acid ester is obtained by immersing a support in which SAM is formed in buffered saline (MES) (ionic strength: 10 mM) for 1 hour, immobilizing CMD in SAM, and immersing in 1N NaOH aqueous solution for 30 minutes. Was hydrolyzed.

Next, after immersing in MES containing NHS 50 mM and WSC 100 mM for 1 hour, by immersing in an anti-cTnI IgG1 monoclonal antibody (MF4; 2.5 μg / ml, Hytest) solution for 30 minutes, The primary antibody was immobilized on CMD.
2. Preparation of sample solution For serum containing no cardiac troponin (cTn), the final concentration of cardiac troponin I (cTnI), which is a standard antigen, after dilution with a sample dilution solution (diluted 2-fold) is 100 pg / ml. This was added as an evaluation sample (sample solution). As the assay signal, the standard antigen cTnI was added, and the final concentration was 100 pg / ml.

  In addition, a surfactant (Tween 20) in the sample dilution solution is added in an amount of 0.05% by mass (relative to the sample dilution solution). Tris buffered saline (TBS) is used as the main solution for the sample dilution solution. ) Was used.

Furthermore, for the sample solution prepared in this way, the weight average molecular weight is respectively
・ 10000 (“CMD-L (trade name)” manufactured by Meito Sangyo Co., Ltd.),
・ 40000 (“CMD-D40 (trade name)” manufactured by Meisei Sangyo Co., Ltd.),
・ 200000 (“CMD-GH (trade name)” manufactured by Meito Sangyo Co., Ltd.),
・ 500,000 (“CMD-500 (trade name)” manufactured by Meisei Sangyo Co., Ltd.),
・ 1000000 (“CMD (trade name)” manufactured by Meisei Sangyo Co., Ltd.),
A sample solution was prepared by changing the mass% between 0 and 10 for the sample dilution solution containing carboxymethyldextran (CMD).

Moreover, about the sample dilution liquid containing the dextran whose weight average molecular weight is 100,000, the sample solution which changed mass% between 0-10 was prepared as a comparison.
3. Preparation of fluorescently labeled antibody solution As fluorescently labeled antibody 5, a solution of anti-cTnI IgG2a monoclonal antibody (4C2; 2.5 mg / ml, Hytest) and Alexa Fluor (registered trademark) 647 (Molecular Probes) labeling kit were used. The reaction was carried out by stirring and mixing at room temperature (25 ° C.) for 60 minutes according to the procedure for using the kit. Then, Alexa Fluor (registered trademark) 647-labeled anti-cTnI IgG2a monoclonal antibody was obtained by purification using a molecular weight cut filter (manufactured by Nippon Millipore). In this way, a solution containing an antibody labeled with a fluorescent substance (PBS solution prepared to 2 μg / ml) was prepared.
4). Fluorescence measurement Each sample solution and fluorescence-labeled antibody solution obtained as described above were sent to individual sensor chips, and fluorescence measurement was performed on each.

  The measurement procedure common to the individual fluorescence measurements was performed as follows. First, 0.1 ml of the sample solution was circulated for 25 minutes through the flow path of the sensor chip, and then Tris buffered saline [TBS] containing 0.05% by mass of Tween 20 was circulated and washed for 10 minutes. .

  Next, 0.1 ml of the fluorescence labeled antibody solution was circulated for 5 minutes, and then TBS containing 0.05% by mass of Tween 20 was circulated for 10 minutes for washing. Thereafter, laser light 9 (640 nm, 640 nm, from the other surface of the dielectric member 8 made of glass, on which the metal thin film 7 is not formed, passes through the prism (manufactured by Sigma Koki Co., Ltd.). 40 μW), and the amount of light (signal value) detected by the photomultiplier tube (PMT) of the amount of fluorescence emitted from the excited fluorescent substance was measured and used as an assay signal for each sample solution containing the standard antigen.

  Further, a sample solution not containing myocardial troponin I (cTnI), which is a standard antigen, was measured in the same manner, and an assay blank was measured. The results thus obtained are shown in Table 2 below.

In Table 2, regarding the CMD addition effect determination,
X: S / B effect% is less than 130,
Δ: S / B effect% is 130 to 210
○: S / B effect% is 210 to 400,
◎… S / B effect% is 400 or more,
Judged by.

  As is apparent from the results of Table 2, when dextran is added, the assay blank, which is the background noise of fluorescence measurement, greatly increases, and high-sensitivity and high-precision SPFS measurement of extremely small amount of analyte 6 occurs. It becomes an obstacle.

  On the other hand, as in the example according to the present invention, the carboxymethyl dextran having a weight average molecular weight of 200000 to 1000000 is contained in an amount of 0.001 to 5% by mass with respect to the sample dilution liquid, that is, the final concentration. It is used for sample processing at 0.0005 to 2.5% by weight, the assay blank which is the background noise of the fluorescence measurement does not increase greatly, and the increase of the assay blank is suppressed and the S / B (SN ratio) ) Can be measured with improved fluorescence (see the range of Δ to ◎ in Table 2).

  In this case, preferably, the carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 is contained in an amount of 0.01 to 2% by mass with respect to the sample dilution liquid, that is, the final concentration is 0.005 to 1 mass. It can be seen that it is desirable to be used in the treatment in% (see the range of ○ to ◎ in Table 2).

  More preferably, the carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 is contained in an amount of 0.1 to 0.5 mass% with respect to the sample dilution liquid, that is, the final concentration is 0.05 to 0.00. It can be seen that it is desirable to use the sample at 25% by mass (see the range of ◎ in Table 2).

[Example 2]
In order to confirm the effect of the surfactant to suppress non-specific adsorption and the effect of suppressing increase in assay blank, the following test was performed. The test was conducted in the same manner as in Example 1 under the condition that 0.025% by mass of Tween 20 was added as a surfactant and no addition.

  In a system in which the weight average molecular weight of carboxymethyldextran (CMD) is 100,000 or more, the CMD concentration was 0.25% by mass. As a result, no significant difference was observed in the assay signal, and it was confirmed that the assay blank had a tendency to decrease under the condition where 0.025 mass% of Tween 20 was added.

  In addition, as shown in Table 3 below, the average of N = 3 in each condition was evaluated.

  The preferred embodiment of the present invention has been described above, but the present invention is not limited to this. For example, in Examples 1 and 2 described above, the SPFS measurement was described as the fluorescence measurement method. Other than this, various modifications can be made without departing from the object of the present invention, for example, a fluorescence measurement method such as an ATR (Attenuated Total Reflection) fluorescence method can be used.

  For example, the reagent dilution solution according to the present invention and another reagent used together with the sample dilution solution (for example, a reagent related to sample dilution or a reagent used in the above fluorescence measurement method) as a set It can be a kit.

  Among the other reagents used together with the sample dilution liquid, examples of the reagent used in the fluorescence measurement method include a fluorescence-labeled antibody solution and a washing solution.

On the other hand, among other reagents used together with the sample dilution liquid, a reagent related to sample dilution includes a surfactant. Examples of the surfactant include surfactants that can be included in the specimen dilution liquid described above, and the following surfactants used for electrophoresis of DNA and proteins, for example. . As a specific example of the latter surfactant,
-Briji 35 (registered trademark, polyethylene glycol monododecyl ether, Polyethylene Glycol Monododecyl Ether),
-Dodecyl-β-D-maltoside,
-Octyl-β-D-glucoside,
Nonidet (registered trademark) P-40 (polyoxyethylene nonylphenyl ether),
Triton X-100 (registered trademark) (polyethylene glycol mono-P-inoctylphenyl ether, Polyoxyethylene (10) Octylphenyl Ether),
・ Tween 20 (registered trademark) (a derivative of mono-9-octadecanoate poly (oxy-1,2-ethanediyl)),
CHAPS (registered trademark) ((3- [3-colamidopropyl] dimethylamino) propanesulfonic acid, 3-[(3-Cholamidopropyl) dimethylammonio] propanesulfonate),
・ Zwittergent 3-12 (n-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonic acid) ),
-SDS (sodium dodecyl sulfate) and those similar to these reagents can be mentioned.

  The present invention uses a biological sample as a specimen using a fluorescence measurement method such as surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) or total reflection ATR (Attenuated Total Reflection) fluorescence. Thus, when detecting a very small amount of the analyte 6, it can be applied to a sample dilution solution for diluting the sample, a kit using the same, and a fluorescence measurement method.

1 Contaminant 2 Carboxymethyldextran molecule 3 Solid support 4 Ligand 5 Fluorescent labeled antibody 6 Analyte 7 Metal thin film (metal film)
8 Dielectric member 9 Laser light (excitation light)

Claims (11)

Carboxymethyl dextran having a weight average molecular weight of 200,000 to 1,000,000 is contained in an amount of 0.1 to 1% by mass with respect to the sample dilution liquid, or carboxymethyl dextran having a weight average molecular weight of 500,000 to 1,000,000 is diluted with the sample. It is contained in 0.01 to 2% by mass with respect to the working solution, and is used in a fluorescence measurement method using the electric field enhancement effect of surface plasmons generated on the surface of the metal film when the excitation light is irradiated onto the metal film. specimen dilution liquid, characterized in that that.   2. The sample dilution liquid according to claim 1, comprising 0.1 to 0.5 mass% of carboxymethyldextran having a weight average molecular weight of 500,000 to 1,000,000 with respect to the sample dilution solution.   The sample dilution liquid according to claim 1 or 2, wherein the sample is a biological sample.   The sample dilution liquid according to any one of claims 1 to 3, wherein the pH is adjusted to 4 to 9.   5. The specimen dilution liquid according to claim 1, further comprising a surfactant.   6. The specimen dilution liquid according to claim 5, wherein the surfactant is contained in an amount of 0.00001 to 1% by mass with respect to the specimen dilution liquid.   The sample dilution liquid according to any one of claims 1 to 6, wherein the fluorescence measurement method is surface plasmon excitation enhanced fluorescence spectroscopy (SPFS) or total reflection ATR fluorescence method. A kit for specimen dilution liquid comprising the specimen dilution liquid according to any one of claims 1 to 7 . After a mixed solution obtained by mixing the specimen in the specimen diluent solution according to any one of claims 1 to 7, were fed to the sensor chip in which the solid support is immobilized on the surface of the metal film, the A fluorescence measurement method characterized by performing fluorescence measurement by a fluorescence measurement method. The fluorescence measurement method according to claim 9 , wherein the fluorescence measurement is performed after supplying the mixed solution , supplying a fluorescently labeled antibody that binds to the analyte in the specimen and washing the sample. The fluorescence measurement method according to claim 9 or 10, wherein the solid support is carboxymethyl dextran.

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